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          "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Measurement of oxidative stress index&#46; Silencing CASC15 alleviated H&#47;R-induced oxidative stress injury&#44; as shown by decreased production of &#40;A&#41; MDA and increased activity of &#40;B&#41; SOD and &#40;C&#41; GSH-Px&#46; &#42;&#42;&#42;p&#60;0&#46;001 vs&#46; control group&#59; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#46; GSH-Px&#58; glutathione peroxidase&#59; H&#47;R&#58; hypoxia&#47;reoxygenation&#59; MDA&#58; methylenedioxyamphetamine&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#59; SOD&#58; superoxide dismutase&#46;</p>"
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Acute myocardial infarction &#40;AMI&#41; and other types of cardiovascular disease are common entities&#46; With the aging of populations&#44; the incidence and mortality of cardiovascular disease have increased significantly&#46;<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">1</span></a> Therefore&#44; exploring the molecular mechanisms underlying the occurrence and development of cardiovascular disease is of great significance for the prevention and treatment of these conditions&#46; For ischemic heart disease such as AMI&#44; restoring blood flow to ischemic myocardial tissue through revascularization is the most effective method to save life at present&#46;<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">2</span></a> However&#44; studies have confirmed that such treatment can also cause myocardial ischemia&#8211;reperfusion &#40;I&#47;R&#41; injury when coronary blood flow is restored&#44; which significantly reduces the therapeutic effect&#46;<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">3</span></a> Therefore&#44; how to reduce myocardial I&#47;R injury has become a focus of research&#46;</p><p id="par0010" class="elsevierStylePara elsevierViewall">Long non-coding RNA &#40;lncRNA&#41; consists of endogenous non-coding RNA sequences that can regulate the expression of target genes by acting as microRNA sponges&#44; and thus participate in many biological processes such as the development of diseases and tumors&#46;<a class="elsevierStyleCrossRefs" href="#bib0165"><span class="elsevierStyleSup">4&#44;5</span></a> Previous studies have reported that lncRNAs play a crucial role in myocardial I&#47;R injury&#46; For example&#44; a study by Long et al&#46; showed that the expression of lncRNA FTX decreased in injured myocardium and inhibited the apoptosis of cardiomyocytes by targeting miR-29b-1-5p&#46;<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">6</span></a> The mechanism of lncRNAs in myocardial I&#47;R injury remains unknown&#46; Cancer susceptibility 15 &#40;CASC15&#41;&#44; an lncRNA on human chromosome 6p22&#46;3&#44; has been shown to play a regulatory role in the growth&#44; metastasis and drug resistance of breast cancer&#44; colorectal cancer&#44; hepatocellular carcinoma&#44; melanoma and other malignant tumors&#46;<a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">7&#8211;10</span></a> Yu et al&#46; found that CASC15 promoted cell proliferation and metastasis in non-small cell lung carcinoma by sponge adsorption of miR-130b-3p in vitro and in vivo&#46;<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">11</span></a> In recent years&#44; its expression has been found to be abnormal in cardiovascular disease&#46; A study showed that compared to healthy volunteers&#44; serum CASC15 was increased in patients with atherosclerosis and AMI&#46;<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">12</span></a> However&#44; the mechanism of CASC15 in I&#47;R injury has not been fully elucidated&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Objectives</span><p id="par0015" class="elsevierStylePara elsevierViewall">In this study&#44; an AMI cell model was constructed by hypoxia&#47;reoxygenation &#40;H&#47;R&#41; treatment&#44; and the mechanism of CASC15 in I&#47;R injury was explored through this model&#44; which provided a theoretical basis for the prevention and treatment of I&#47;R injury in patients with AMI&#46;</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Methods</span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Cell culture and treatment</span><p id="par0020" class="elsevierStylePara elsevierViewall">H9c2 cells were purchased from the American Type Culture Collection and were grown in Dulbecco&#39;s Modified Eagle Medium &#40;DMEM&#41; &#40;containing 10&#37; fetal bovine serum &#91;FBS&#93; and 1&#37; penicillin&#8211;streptomycin mixture&#41; in an incubator containing 5&#37; CO<span class="elsevierStyleInf">2</span> at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall">The AMI cell model was constructed by inducing H&#47;R&#46; Briefly&#44; H9c2 cells were cultured in serum-free DMEM and then placed in a hypoxic incubator containing 5&#37; CO<span class="elsevierStyleInf">2</span> and 95&#37; N<span class="elsevierStyleInf">2</span> for 30 min&#46; The FBS-free DMEM was then replaced with DMEM containing 10&#37; FBS&#44; and the cells were placed in a conventional incubator containing 5&#37; CO<span class="elsevierStyleInf">2</span> and 95&#37; air for reoxygenation&#46; Cells cultured under conventional conditions were used as a control group&#46; After 24 h of reoxygenation&#44; cells were collected for subsequent experimental operations&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Cell transfection</span><p id="par0030" class="elsevierStylePara elsevierViewall">Small interfering RNA &#40;siRNA&#41; against CASC15 &#40;si-CASC15&#41;&#44; si-negative control &#40;si-NC&#41;&#44; miR-542-3p mimic&#44; miR-542-3p inhibitor&#44; mimic NC and inhibitor negative control &#40;NC&#41; were purchased from GenePharma &#40;Shanghai&#44; China&#41;&#46; H9c2 cells were inoculated in 24-well plates and cultured overnight&#46; The above oligonucleotides were mixed with the transfection reagent Lipofectamine 2000 for 10 min at room temperature and then diluted with serum-free DMEM to a final concentration of 50 nM&#46; Cell transfection procedures were performed according to the instructions for Lipofectamine 2000&#46; After transfection for 24 h&#44; cells were subjected to H&#47;R treatment&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Reverse transcription-quantitative polymerase chain reaction</span><p id="par0035" class="elsevierStylePara elsevierViewall">RNA was extracted using TRIzol reagent&#46; The PrimeScript RT kit was used for cDNA synthesis&#44; and the SYBR Green PCR kit was used for the polymerase chain reaction &#40;PCR&#41; reaction&#46; Glyceraldehyde 3-phosphate dehydrogenase &#40;GAPDH&#41; and U6 were used as an internal reference for CASC15 and U6&#44; respectively&#46; The primers used were as follows&#58; CASC15&#58; forward&#58; 5&#8242;-CTTTGTCTGCTCCGGGACT-3&#8242;&#44; reverse&#58; 5&#8242;-TTAAGGGACATTTCCCCCG-3&#8242;&#59; miR-542-3p&#58; forward&#58; 5&#8242;-TCGGGGATCATCATGTCA-3&#8242;&#44; reverse&#58; 5&#8242;-GAGTGGCTCCCAGACCTT-3&#8242;&#59; GAPDH&#58; forward&#58; 5&#8242;-GTGGCTCCCAGACCTTTC-3&#8242;&#44; reverse&#58; 5&#8242;-AGCTTGACAAAGTGGTCG-3&#8242;&#59; U6&#58; forward&#58; 5&#8242;-CTCGCTTCGGCAGCACA-3&#8242;&#44; reverse&#58; 5&#8242;-AACGCTTCACGAATTTGCGT-3&#8242;&#46; Reaction conditions were initial denaturation at 95<span class="elsevierStyleHsp" style=""></span>&#176;C for 10 min&#44; followed by 40 cycles of denaturation at 92<span class="elsevierStyleHsp" style=""></span>&#176;C for 15 s&#44; annealing at 60<span class="elsevierStyleHsp" style=""></span>&#176;C for 1 min and extension at 60<span class="elsevierStyleHsp" style=""></span>&#176;C for 1 min&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Cell viability assay</span><p id="par0040" class="elsevierStylePara elsevierViewall">Cell viability was assessed with the Cell Counting Kit-8 assay &#40;CCK-8&#41;&#46; H9c2 cells were seeded in 96-well plates at a density of 4000 cells&#47;well and cultured overnight&#46; After cell transfection and H&#47;R treatment according to the experimental procedure&#44; cell viability was monitored every 24 h according to the manufacturer&#39;s scheme&#44; whereby CCK-8 solution at a concentration of 10 nM was added to the cells at a preset time point &#40;0 h&#44; 24 h&#44; 48 h&#44; 72 h&#44; 96 h&#41;&#44; and the cells were incubated in the dark at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 2 h&#44; and then optical density values were measured at 450 nm&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Cell apoptosis assay</span><p id="par0045" class="elsevierStylePara elsevierViewall">Cell apoptosis was detected by flow cytometry&#46; H9c2 cells were seeded in 6-well plates and treated according to the steps of transfection and H&#47;R treatment&#46; They were then harvested and stained with the Annexin V-FITC&#47;PI apoptosis detection kit&#46; After staining&#44; the cell apoptosis rate was immediately determined by flow cytometry&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Assessment of oxidative stress index</span><p id="par0050" class="elsevierStylePara elsevierViewall">Cell supernatants were used to determine superoxide dismutase &#40;SOD&#41;&#44; methylenedioxyamphetamine &#40;MDA&#41;&#44; and glutathione peroxidase &#40;GSH-Px&#41; content&#46; The corresponding kits were purchased from Nanjing Jiancheng Biotechnology Co&#46;&#44; Ltd&#46;&#44; as follows&#58; SOD assay kit &#40;Sigma-Aldrich&#44; cat&#46; CS1000&#41; using the water-soluble tetrazolium method&#59; MDA assay kit &#40;Beyotime&#44; cat&#46; S0131&#41; using the thiobarbituric acid method&#59; and GSH-Px assay kit &#40;Nanjing Jiancheng Bioengineering Institute&#44; cat&#46; A005&#41; using the colorimetric method&#46; Changes in absorbance were detected by spectrophotometry&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Luciferase reporter assay</span><p id="par0055" class="elsevierStylePara elsevierViewall">Starbase v2&#46;0 was used to analyze RNA&#8211;RNA interactions&#44; and predicted that miR-542-3p had a complementary binding site to CASC15&#46; We hypothesized that these two molecules have a targeted binding relationship&#44; and tested this hypothesis by luciferase reporter gene assay&#46; Briefly&#44; the steps are as follows&#58; wild-type CASC15 &#40;WT-CASC15&#41; and mutant CASC15 &#40;mut-CASC15&#41; were inserted into pmirGLO reporter vectors&#44; and these reporter vectors were co-transfected into H9c2 cells with miR-542-3p mimic or miR-542-3p inhibitor using Lipofectamine 2000&#46; After transfection for 48 h&#44; relative luciferase activity was measured on a dual-luciferase reporter assay system&#46; Renilla was used as an internal reference&#44; and data were expressed as the ratio of Renilla luciferase activity to Firefly luciferase activity&#46;</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Statistical analysis</span><p id="par0060" class="elsevierStylePara elsevierViewall">Data are presented as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46; The two-tailed Student&#39;s <span class="elsevierStyleItalic">t</span> test was used to compare the two groups&#44; and multiple groups were compared by one-way analysis of variance&#46; Statistical significance was assumed for p&#60;0&#46;05&#46; Each test was repeated in triplicate&#46;</p></span></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Results</span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Determination of reoxygenation time</span><p id="par0065" class="elsevierStylePara elsevierViewall">Reoxygenation times are presented in <a class="elsevierStyleCrossRef" href="#fig0005">Figure 1</a>&#46; They show that CASC15 expression of cells in the H&#47;R group was significantly higher than in the control group after reoxygenation &#40;except at 0 h&#41; &#40;p&#60;0&#46;05&#41;&#46; In addition&#44; with the time extension&#44; after reoxygenation culture&#44; there were differences between the groups&#46; CASC15 levels in the H&#47;R group at 24 h were significantly higher than at 12 h &#40;p&#60;0&#46;001&#41;&#44; and similarly were significantly higher at 48 h than at 24 h &#40;p&#60;0&#46;01&#41;&#46; Therefore&#44; 24 h was selected as the reoxygenation culture time for this study&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Effect of CASC15 downregulation on cell survival</span><p id="par0070" class="elsevierStylePara elsevierViewall">The intracellular CASC15 expression level was regulated by cell transfection&#46; As shown in <a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>A&#44; CASC15 expression in the H&#47;R model group was significantly increased compared with the control group&#44; however&#44; si-CASC15 transfection significantly downregulated CASC15 levels &#40;p&#60;0&#46;001&#41;&#46; Investigation of cell viability showed that compared with the control group&#44; cell viability in the H&#47;R model group was significantly inhibited&#44; but was significantly promoted after CASC15 was inhibited &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>B&#41;&#46; Concerning cell apoptosis&#44; the proportion of apoptotic cells in the H&#47;R model group was about 20&#37;&#44; which was significantly higher than in the control group&#46; As expected&#44; CASC15 depletion helped alleviate H&#47;R-induced apoptosis &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>C&#41;&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Effect on oxidative stress of inhibiting CASC15 expression</span><p id="par0075" class="elsevierStylePara elsevierViewall">It is well known that H&#47;R induces oxidative damage&#46; In the assessment of oxidative stress indicators&#44; it was found that MDA levels in the H&#47;R model group increased significantly&#44; while SOD and GSH-Px levels decreased&#44; indicating that H&#47;R treatment caused oxidative damage to H9c2 cells &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Figure 3</a>A&#8211;C&#41;&#46; It is worth noting that after CASC15 expression was suppressed&#44; oxidative damage was significantly reduced&#44; mainly manifested by decreased MDA and increased SOD and GSH-Px &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Figure 3</a>A&#8211;C&#41;&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Validation of the targeting relationship between miR-542-3p and CASC15</span><p id="par0080" class="elsevierStylePara elsevierViewall">Bioinformatics predicted that CASC15 and miR-542-3p may have a targeting relationship&#44; and the complementary binding sites of the two molecules are shown in <a class="elsevierStyleCrossRef" href="#fig0020">Figure 4</a>A&#46; The luciferase reporter gene was used to verify this relationship&#44; and the results showed that for the WT-CASC15 group&#44; transfection of miR-542-3p mimic or inhibitor respectively weakened or enhanced luciferase activity&#44; while no similar results were obtained in the mut-CASC15 group &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Figure 4</a>B&#41; &#40;p&#60;0&#46;001&#41;&#46; Further verification results were as follows&#58; in H&#47;R model cells&#44; miR-542-3p expression decreased&#44; and at the same time&#44; inhibition of CASC15 helped to improve the expression of miR-542-3p&#44; which further underlines the relationship between CASC15 and miR-542-3p &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Figure 4</a>C&#41;&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0145">Effect of miR-542-3p on cell survival</span><p id="par0085" class="elsevierStylePara elsevierViewall">As shown in <a class="elsevierStyleCrossRef" href="#fig0025">Figure 5</a>A&#44; miR-542-3p expression&#44; which increased due to the restricted expression of CASC15&#44; was significantly downregulated after transfection with miR-542-3p inhibitor &#40;p&#60;0&#46;001&#41;&#46; The cell viability assay showed that inhibition of miR-542-3p reversed the increase in cell viability resulting from silencing CASC15 &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Figure 5</a>B&#41;&#46; Similarly&#44; assessment of cell apoptosis showed that the loss of miR-542-3p promoted cell apoptosis compared to the inhibition of apoptosis induced by the attenuation of CASC15 &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Figure 5</a>C&#41;&#46;</p><elsevierMultimedia ident="fig0025"></elsevierMultimedia></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0150">Effect of miR-542-3p on oxidative stress in H9c2 cells</span><p id="par0090" class="elsevierStylePara elsevierViewall">As mentioned above&#44; H&#47;R-induced oxidative stress damage was alleviated by the inhibition of CASC15&#46; However&#44; oxidative stress damage was found to be worsened when miR-542-3p in the cells was knocked down&#46; It was observed that after miR-542-3p was inhibited&#44; MDA levels increased and SOD and GSH-Px levels decreased &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Figure 6</a>A&#8211;C&#41;&#46;</p><elsevierMultimedia ident="fig0030"></elsevierMultimedia></span></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0155">Discussion</span><p id="par0095" class="elsevierStylePara elsevierViewall">Abnormal lncRNA expression following I&#47;R after AMI may be involved in the occurrence and development of myocardial injury&#46; However&#44; the regulatory mechanism of some lncRNAs in I&#47;R injury remains unclear&#46; The oncogenic effects of CASC15 have been demonstrated by its upregulation in liver cancer and promotion of hepatic carcinoma&#46;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">13</span></a> Recent reports indicate that CASC15 is upregulated in acute ischemic stroke&#44; and can function as a sponge molecule of miR-338-3p&#44; promoting inflammatory damage to nerve cells&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">14</span></a> The results of our study showed that CASC15 expression in cardiomyocytes treated with H&#47;R was significantly increased compared with normal cultured cardiomyocytes&#44; suggesting that the abnormal increase in CASC15 was closely related to the occurrence of H&#47;R injury&#46; Further experiments showed that CASC15 inhibition significantly improved the H&#47;R-induced dysfunction of H9c2 cells &#40;including inhibition of cell viability and enhancement of cell apoptosis&#41; and oxidative stress response&#46; In addition&#44; dual-luciferase reporter assay revealed that CASC15 targets miR-542-3p and that CASC15 expression was negatively regulated by miR-542-3p&#44; indicating that CASC15 may promote H&#47;R injury in H9c2 cells by suppressing the expression of miR-542-3p&#46;</p><p id="par0100" class="elsevierStylePara elsevierViewall">I&#47;R injury is a common perioperative complication&#44; especially in the reoxygenation phase&#44; in which oxidative stress and a large increase in reactive oxygen species activate the mitochondrial apoptotic program&#44; triggering the caspase cascade and ultimately leading to cardiomyocyte apoptosis&#44;<a class="elsevierStyleCrossRefs" href="#bib0220"><span class="elsevierStyleSup">15&#44;16</span></a> a key factor in the development of heart failure after I&#47;R injury&#46; It is therefore important to inhibit cardiomyocyte apoptosis for prevention and treatment of the consequences of I&#47;R injury&#46;<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">17</span></a> In the present study&#44; cell viability was significantly inhibited after H&#47;R treatment&#44; and the proportion of apoptotic cells was significantly greater than in the control group&#46; Surprisingly&#44; after CASC15 knockout and H&#47;R treatment&#44; the effect on cell viability and apoptosis was no longer serious&#44; implying that excessive CASC15 plays an important role in H&#47;R injury&#46; Induction of apoptosis by CASC15 has been demonstrated in several studies&#46; For example&#44; Qin et al&#46; showed that CASC15 expression increased in diabetes-induced chronic renal failure&#44; and overexpression of CASC15 promoted podocyte apoptosis by sponging miR-34c&#46;<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">18</span></a> The molecular mechanism by which CASC15 promotes apoptosis of cardiomyocytes in our study needs further investigation&#46;</p><p id="par0105" class="elsevierStylePara elsevierViewall">Under normal physiological conditions&#44; there is a dynamic balance between oxidation and antioxidation in the body&#46; In the process of I&#47;R damage&#44; excessive production of oxidants leads to an imbalance between the oxidant and antioxidant systems&#46;<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">19</span></a> This imbalance results in oxidative stress&#44; which leads not only to inflammation&#44; but also to apoptosis&#44; which is a major factor in aging and disease&#46;<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">20</span></a> Presently&#44; the levels of three substances are commonly used as indices to assess the degree of oxidative stress&#58; MDA&#44; SOD and GSH-Px&#46; MDA is an end-product of lipid peroxidation&#44; and when increased indicates oxidative damage to the cell membrane&#46;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">21</span></a> SOD activity is closely associated with oxidative damage caused by free radicals&#44; and may indirectly reflect the body&#39;s ability to scavenge free radicals and prevent peroxidation of low-density lipoprotein&#46;<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">22</span></a> GSH-Px catalyzes the formation of glutathione disulfide &#40;GSSG&#41; from reduced monomeric glutathione &#40;GSH&#41;&#44; and its activity can be used as a biochemical index to measure antioxidant levels in cells&#46;<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">23</span></a> In the current study&#44; after H&#47;R treatment&#44; oxidative stress indicators such as MDA increased while SOD and GSH-Px decreased&#44; indicating that H9c2 cells were damaged by oxidation&#46; However&#44; after downregulation of CASC15&#44; MDA concentrations in cells decreased&#44; while SOD and GSH-Px levels increased&#44; indicating that reduction of CASC15 alleviates the oxidative damage in cells caused by H&#47;R and significantly enhances the antioxidant capacity of cells&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">miRNAs can inhibit the expression of target genes by recognizing the corresponding target site in the 3&#8217;-UTR region of the target gene and binding to it through complementary base pairing&#46;<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">24</span></a> Studies have shown that miRNAs can influence the process of I&#47;R injury by inhibiting oxidative damage and apoptosis of cardiomyocytes&#46; As a tumor inhibitor&#44; miR-542-3p mRNA expression is abnormally downregulated in many human tumors and other diseases&#44; including colon cancer&#44;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">25</span></a> prostate cancer&#44;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">26</span></a> gastric cancer&#44;<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">27</span></a> and ischemic stroke&#46;<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">28</span></a> A previous study showed that miR-542-3p was significantly reduced in mice with middle cerebral artery occlusion&#44; and overexpression of miR-542-3p mitigated apoptosis and activation of inflammatory responses caused by oxygen and glucose deprivation&#46;<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">29</span></a> Another study confirmed that overexpression of miR-542-3p has a protective effect on H&#47;R-induced cardiomyocytes and reduces cell apoptosis&#46;<span class="elsevierStyleSup">30</span> After a literature search&#44; we selected miR-542-3p&#44; which is clearly associated with cardiovascular disease&#44; as the target of CASC15 in this study&#46; Our results showed that the expression of miR-542-3p in cells was reduced after H&#47;R treatment&#44; suggesting that the loss of miR-542-3p may be involved in I&#47;R injury&#46; Furthermore&#44; luciferase reporter assay confirmed that CASC15 targeted miR-542-3p&#44; and that the latter can negatively regulate the expression of CASC15&#46; In this study&#44; H9c2 cells were co-transfected with si-CASC15 and miR-542-3p inhibitor&#44; and then treated with H&#47;R for 24 h&#46; The results showed that CASC15 inhibition promoted H&#47;R-induced cell viability&#44; inhibited cell apoptosis and attenuated oxidative damage&#46; However&#44; the reduction of miR-542-3p significantly inhibited cardiomyocyte viability and promoted oxidative damage&#44; which suggests that CASC15 overexpression may promote H&#47;R-induced myocardial injury by inhibiting the expression of miR-542-3p&#46;</p></span><span id="sec0100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0160">Conclusion</span><p id="par0115" class="elsevierStylePara elsevierViewall">In conclusion&#44; CASC15 expression increased in H&#47;R-induced H9c2 cells&#46; Overexpression of CASC15 aggravated oxidative stress injury in H&#47;R-induced cells&#44; inhibited cardiomyocyte viability&#44; and promoted cardiomyocyte apoptosis&#46; CASC15 can target miR-542-3p&#46; miR-542-3p expression decreased in the H&#47;R group&#44; which aggravated H&#47;R-induced oxidative stress&#44; inhibited cell viability and promoted apoptosis&#46; Therefore&#44; based on the above results&#44; we preliminarily speculate that CASC15 may promote H&#47;R-induced myocardial injury by inhibiting the expression of miR-542-3p in H&#47;R-induced H9c2 cells&#46;</p></span><span id="sec0105" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0165">Funding</span><p id="par0120" class="elsevierStylePara elsevierViewall">No funding was received for conducting this study&#46;</p></span><span id="sec0110" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0170">Conflicts of interest</span><p id="par0125" class="elsevierStylePara elsevierViewall">The authors have no conflicts of interest to declare&#46;</p></span></span>"
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            0 => array:2 [
              "identificador" => "sec0020"
              "titulo" => "Cell culture and treatment"
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            1 => array:2 [
              "identificador" => "sec0025"
              "titulo" => "Cell transfection"
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              "titulo" => "Reverse transcription-quantitative polymerase chain reaction"
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              "titulo" => "Cell viability assay"
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              "titulo" => "Cell apoptosis assay"
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              "titulo" => "Assessment of oxidative stress index"
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              "titulo" => "Luciferase reporter assay"
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          "titulo" => "Results"
          "secciones" => array:6 [
            0 => array:2 [
              "identificador" => "sec0065"
              "titulo" => "Determination of reoxygenation time"
            ]
            1 => array:2 [
              "identificador" => "sec0070"
              "titulo" => "Effect of CASC15 downregulation on cell survival"
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            2 => array:2 [
              "identificador" => "sec0075"
              "titulo" => "Effect on oxidative stress of inhibiting CASC15 expression"
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              "identificador" => "sec0080"
              "titulo" => "Validation of the targeting relationship between miR-542-3p and CASC15"
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              "identificador" => "sec0085"
              "titulo" => "Effect of miR-542-3p on cell survival"
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              "identificador" => "sec0090"
              "titulo" => "Effect of miR-542-3p on oxidative stress in H9c2 cells"
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          "titulo" => "Discussion"
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    "fechaRecibido" => "2023-02-21"
    "fechaAceptado" => "2023-04-24"
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          "clase" => "keyword"
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          "palabras" => array:5 [
            0 => "Hypoxia&#47;reperfusion"
            1 => "Oxidative stress"
            2 => "CASC15"
            3 => "MiR-542-3p"
            4 => "Myocardial infarction"
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          "clase" => "keyword"
          "titulo" => "Palavras-chave"
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          "palabras" => array:5 [
            0 => "Hip&#243;xia&#47;reperfus&#227;o"
            1 => "Tens&#227;o oxidativa"
            2 => "CASC15"
            3 => "MiR-542-3p"
            4 => "Enfarte do mioc&#225;rdio"
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      "en" => array:3 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Introduction and Objectives</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">The increasing incidence of ischemic heart disease is a serious threat to human health&#46; Increased CASC15&#44; a long non-coding RNA&#44; has been shown to adversely affect cardiac muscle&#46; The objective of this paper was to explore the effect of CASC15 on a cell model of myocardial infarction and its possible mechanism&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">H9c2 cells were selected to establish the myocardial infarction model through hypoxia&#47;reoxygenation &#40;H&#47;R&#41; treatment&#46; The expression of CASC15 was attenuated by cell transfection in vitro&#46; The level of CASC15 was detected by RT-qPCR&#46; Cell viability was detected by CCK-8 assay&#44; and cell apoptosis was assessed by flow cytometry&#46; The content of MDA and the activity of SOD and GSH-Px were measured by ELISA&#46; Luciferase reporter gene assay was used to determine the relationship between CASC15 and miRNA&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">CASC15 expression was increased in H&#47;R-treated H9c2 cells&#46; Overexpression of CASC15 adversely affected cell viability and promoted H&#47;R-induced oxidative stress&#46; Inhibition of CASC15 promoted cell viability and suppressed cell apoptosis and oxidative stress damage&#46; Additionally&#44; luciferase reporter gene assay confirmed the targeting relationship between CASC15 and miR-542-3p&#44; and attenuating CASC15 expression enhanced the level of miR-542-3p&#46; Reduction of miR-542-3p weakened the viability of the H&#47;R cell model&#44; increased apoptosis&#44; and enhanced oxidative stress damage&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusion</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">This study suggests that overexpression of CASC15 may inhibit the viability of H9c2 cells&#44; promote apoptosis and induce oxidative stress through targeted regulation of miR-542-3p expression&#46;</p></span>"
        "secciones" => array:4 [
          0 => array:2 [
            "identificador" => "abst0005"
            "titulo" => "Introduction and Objectives"
          ]
          1 => array:2 [
            "identificador" => "abst0010"
            "titulo" => "Methods"
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          2 => array:2 [
            "identificador" => "abst0015"
            "titulo" => "Results"
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            "titulo" => "Conclusion"
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      "pt" => array:3 [
        "titulo" => "Resumo"
        "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Introdu&#231;&#227;o e objetivos</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">O aumento da doen&#231;a isqu&#233;mica card&#237;aca representa uma s&#233;ria amea&#231;a &#224; sa&#250;de humana&#46; Um aumento no CASC15&#44; um RNA n&#227;o codificante longo&#44; est&#225; associado &#224; les&#227;o do m&#250;sculo card&#237;aco&#46; O objetivo deste trabalho foi explorar o mecanismo celular atrav&#233;s do qual a CASC15 contribui para o enfarte do mioc&#225;rdio&#46;</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">M&#233;todos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">C&#233;lulas H9c2 expostas a hip&#243;xia&#47;reoxigena&#231;&#227;o &#40;H&#47;R&#41; foram selecionadas como modelo para estudar os mecanismos envolvidos no enfarto do mioc&#225;rdio&#46; A express&#227;o de CASC15 foi inibida pela transfec&#231;&#227;o com siRNA&#46; Os n&#237;veis de CASC15 foram avaliados por RT-qPCR e a viabilidade celular e apoptose determinadas pelo ensaio CCK-8 e citometria de fluxo&#44; respetivamente&#46; Os teores de MDA&#44; SOD e GSH-Px foram medidos por ELISA&#46; O ensaio do gene rep&#243;rter da luciferase foi utilizado para verificar a rela&#231;&#227;o entre CASC15 e os n&#237;veis do miRNA miR-542-3p&#46;</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">C&#233;lulas H9c2 sujeitas a H&#47;R apresentam n&#237;veis aumentados de CASC15&#46; A sobre-express&#227;o de CASC15 afetou negativamente a fun&#231;&#227;o celular e promoveu a les&#227;o induzida pelo <span class="elsevierStyleItalic">stress</span> associado H&#47;R&#46; Por outro lado&#44; a inibi&#231;&#227;o do CASC15 promoveu viabilidade celular e suprimiu apoptose celular e os danos oxidativos&#46; Al&#233;m disso&#44; o ensaio do gene rep&#243;rter da luciferase confirmou a rela&#231;&#227;o alvo entre CASC15 e miR-542-3p&#44; com uma diminui&#231;&#227;o da express&#227;o de CASC15 a resultar num aumento do n&#237;vel de miR-542-3p&#46; Uma diminui&#231;&#227;o do miR-542-3p comprometeu a viabilidade do modelo de c&#233;lulas H&#47;R e aumentou a apoptose e o dano oxidativo&#46;</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclus&#227;o</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Este estudo sugere que um aumento da express&#227;o de CASC15 durante a H&#47;R pode contribuir para diminuir a viabilidade dos cardiomi&#243;citos&#44; promover apoptose e induzir <span class="elsevierStyleItalic">stress</span> oxidativo&#44; atrav&#233;s da regula&#231;&#227;o da express&#227;o de miR-542-3p&#46;</p></span>"
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          "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Expression level of CASC15 under different reoxygenation times&#46; CASC15 levels increased with prolonged reoxygenation&#46; &#42;&#42;p&#60;0&#46;01&#44; &#42;&#42;&#42;p&#60;0&#46;001 vs&#46; control group&#59; <span class="elsevierStyleSup">&#35;</span>p&#60;0&#46;05&#44; <span class="elsevierStyleSup">&#35;&#35;</span>p&#60;0&#46;01&#44; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; H&#47;R group of the previous group&#46; H&#47;R&#58; hypoxia&#47;reoxygenation&#59; lncRNA&#58; long non-coding RNA&#46;</p>"
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          "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Validation of cell transfection&#44; viability&#44; and apoptosis&#46; &#40;A&#41; Transfection of si-CASC15 effectively inhibited CASC15 expression in the H&#47;R group&#59; &#40;B&#41; inhibition of CASC15 promoted cell viability in the H&#47;R group&#59; &#40;C&#41; loss of CASC15 alleviated cell apoptosis in the H&#47;R group&#46; &#42;&#42;&#42;p&#60;0&#46;001 vs&#46; control group&#59; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#46; H&#47;R&#58; hypoxia&#47;reoxygenation&#59; lncRNA&#58; long non-coding RNA&#59; OD&#58; optical density&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#46;</p>"
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          "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Measurement of oxidative stress index&#46; Silencing CASC15 alleviated H&#47;R-induced oxidative stress injury&#44; as shown by decreased production of &#40;A&#41; MDA and increased activity of &#40;B&#41; SOD and &#40;C&#41; GSH-Px&#46; &#42;&#42;&#42;p&#60;0&#46;001 vs&#46; control group&#59; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#46; GSH-Px&#58; glutathione peroxidase&#59; H&#47;R&#58; hypoxia&#47;reoxygenation&#59; MDA&#58; methylenedioxyamphetamine&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#59; SOD&#58; superoxide dismutase&#46;</p>"
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          "en" => "<p id="spar0060" class="elsevierStyleSimplePara elsevierViewall">Verification of the relationship between CASC15 and miR-542-3p&#46; &#40;A&#41; Binding site of CASC15 and miR-542-3p&#59; &#40;B&#41; luciferase reporter gene assay&#59; &#40;C&#41; miR-542-3p expression increased after inhibition of CASC15 in the H&#47;R group&#46; &#42;&#42;&#42;p&#60;0&#46;001 vs&#46; control group&#59; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#46; H&#47;R&#58; hypoxia&#47;reoxygenation&#59; mut-CASC15&#58; mutant CASC15&#59; NC&#58; negative control&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#59; WT-CASC15&#58; wild-type CASC15&#46;</p>"
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          "en" => "<p id="spar0065" class="elsevierStyleSimplePara elsevierViewall">Effect of miR-542-3p on cell viability and apoptosis&#46; &#40;A&#41; Transfection with miR-542-3p inhibitor counteracted the enhanced effect of CASC15 knockdown on miR-542-3p expression&#59; &#40;B&#41; downregulation of miR-542-3p reduced cell viability enhancement caused by CASC15 silencing&#59; &#40;C&#41; loss of miR-542-3p increased the number of apoptotic cells in the si-CASC15 group&#46; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#59; <span class="elsevierStyleSup">&#38;&#38;&#38;</span>p&#60;0&#46;001 vs&#46; Si-CASC15 group&#46; NC&#58; negative control&#59; OD&#58; optical density&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#46;</p>"
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          "en" => "<p id="spar0070" class="elsevierStyleSimplePara elsevierViewall">Effect of miR-542-3p on oxidative stress injury&#46; Downregulation of CASC15 expression had an inhibitory effect on oxidative stress injury in the H&#47;R group&#44; and this inhibitory effect was abolished by the deletion of miR-542-3p&#46; With the absence of miR-542-3p&#44; &#40;A&#41; MDA concentration and &#40;B&#41; SOD and &#40;C&#41; GSH-Px contents increased&#46; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#59; <span class="elsevierStyleSup">&#38;&#38;&#38;</span>p&#60;0&#46;001 vs&#46; Si-CASC15 group&#46; GSH-Px&#58; glutathione peroxidase&#59; MDA&#58; methylenedioxyamphetamine&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#59; SOD&#58; superoxide dismutase&#46;</p>"
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Original Article
Effect of CASC15 on apoptosis and oxidative stress of cardiomyocytes after hypoxia/reperfusion injury
Efeito da CASC15 na apoptose e stress oxidativo em cardiomiócitos após lesão de hipóxia/reperfusão
Shuai Sun, Xue Mei
Autor para correspondência
meixue_dr@163.com

Corresponding author.
Emergency Medicine Clinical Research Center, Beijing Chao-Yang Hospital, Capital Medical University, Beijing Key Laboratory of Cardiopulmonary Cerebral Resuscitation, Beijing, China
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    "titulo" => "Effect of CASC15 on apoptosis and oxidative stress of cardiomyocytes after hypoxia&#47;reperfusion injury"
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          "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Measurement of oxidative stress index&#46; Silencing CASC15 alleviated H&#47;R-induced oxidative stress injury&#44; as shown by decreased production of &#40;A&#41; MDA and increased activity of &#40;B&#41; SOD and &#40;C&#41; GSH-Px&#46; &#42;&#42;&#42;p&#60;0&#46;001 vs&#46; control group&#59; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#46; GSH-Px&#58; glutathione peroxidase&#59; H&#47;R&#58; hypoxia&#47;reoxygenation&#59; MDA&#58; methylenedioxyamphetamine&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#59; SOD&#58; superoxide dismutase&#46;</p>"
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Acute myocardial infarction &#40;AMI&#41; and other types of cardiovascular disease are common entities&#46; With the aging of populations&#44; the incidence and mortality of cardiovascular disease have increased significantly&#46;<a class="elsevierStyleCrossRef" href="#bib0150"><span class="elsevierStyleSup">1</span></a> Therefore&#44; exploring the molecular mechanisms underlying the occurrence and development of cardiovascular disease is of great significance for the prevention and treatment of these conditions&#46; For ischemic heart disease such as AMI&#44; restoring blood flow to ischemic myocardial tissue through revascularization is the most effective method to save life at present&#46;<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">2</span></a> However&#44; studies have confirmed that such treatment can also cause myocardial ischemia&#8211;reperfusion &#40;I&#47;R&#41; injury when coronary blood flow is restored&#44; which significantly reduces the therapeutic effect&#46;<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">3</span></a> Therefore&#44; how to reduce myocardial I&#47;R injury has become a focus of research&#46;</p><p id="par0010" class="elsevierStylePara elsevierViewall">Long non-coding RNA &#40;lncRNA&#41; consists of endogenous non-coding RNA sequences that can regulate the expression of target genes by acting as microRNA sponges&#44; and thus participate in many biological processes such as the development of diseases and tumors&#46;<a class="elsevierStyleCrossRefs" href="#bib0165"><span class="elsevierStyleSup">4&#44;5</span></a> Previous studies have reported that lncRNAs play a crucial role in myocardial I&#47;R injury&#46; For example&#44; a study by Long et al&#46; showed that the expression of lncRNA FTX decreased in injured myocardium and inhibited the apoptosis of cardiomyocytes by targeting miR-29b-1-5p&#46;<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">6</span></a> The mechanism of lncRNAs in myocardial I&#47;R injury remains unknown&#46; Cancer susceptibility 15 &#40;CASC15&#41;&#44; an lncRNA on human chromosome 6p22&#46;3&#44; has been shown to play a regulatory role in the growth&#44; metastasis and drug resistance of breast cancer&#44; colorectal cancer&#44; hepatocellular carcinoma&#44; melanoma and other malignant tumors&#46;<a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">7&#8211;10</span></a> Yu et al&#46; found that CASC15 promoted cell proliferation and metastasis in non-small cell lung carcinoma by sponge adsorption of miR-130b-3p in vitro and in vivo&#46;<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">11</span></a> In recent years&#44; its expression has been found to be abnormal in cardiovascular disease&#46; A study showed that compared to healthy volunteers&#44; serum CASC15 was increased in patients with atherosclerosis and AMI&#46;<a class="elsevierStyleCrossRef" href="#bib0205"><span class="elsevierStyleSup">12</span></a> However&#44; the mechanism of CASC15 in I&#47;R injury has not been fully elucidated&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Objectives</span><p id="par0015" class="elsevierStylePara elsevierViewall">In this study&#44; an AMI cell model was constructed by hypoxia&#47;reoxygenation &#40;H&#47;R&#41; treatment&#44; and the mechanism of CASC15 in I&#47;R injury was explored through this model&#44; which provided a theoretical basis for the prevention and treatment of I&#47;R injury in patients with AMI&#46;</p></span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Methods</span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Cell culture and treatment</span><p id="par0020" class="elsevierStylePara elsevierViewall">H9c2 cells were purchased from the American Type Culture Collection and were grown in Dulbecco&#39;s Modified Eagle Medium &#40;DMEM&#41; &#40;containing 10&#37; fetal bovine serum &#91;FBS&#93; and 1&#37; penicillin&#8211;streptomycin mixture&#41; in an incubator containing 5&#37; CO<span class="elsevierStyleInf">2</span> at 37<span class="elsevierStyleHsp" style=""></span>&#176;C&#46;</p><p id="par0025" class="elsevierStylePara elsevierViewall">The AMI cell model was constructed by inducing H&#47;R&#46; Briefly&#44; H9c2 cells were cultured in serum-free DMEM and then placed in a hypoxic incubator containing 5&#37; CO<span class="elsevierStyleInf">2</span> and 95&#37; N<span class="elsevierStyleInf">2</span> for 30 min&#46; The FBS-free DMEM was then replaced with DMEM containing 10&#37; FBS&#44; and the cells were placed in a conventional incubator containing 5&#37; CO<span class="elsevierStyleInf">2</span> and 95&#37; air for reoxygenation&#46; Cells cultured under conventional conditions were used as a control group&#46; After 24 h of reoxygenation&#44; cells were collected for subsequent experimental operations&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Cell transfection</span><p id="par0030" class="elsevierStylePara elsevierViewall">Small interfering RNA &#40;siRNA&#41; against CASC15 &#40;si-CASC15&#41;&#44; si-negative control &#40;si-NC&#41;&#44; miR-542-3p mimic&#44; miR-542-3p inhibitor&#44; mimic NC and inhibitor negative control &#40;NC&#41; were purchased from GenePharma &#40;Shanghai&#44; China&#41;&#46; H9c2 cells were inoculated in 24-well plates and cultured overnight&#46; The above oligonucleotides were mixed with the transfection reagent Lipofectamine 2000 for 10 min at room temperature and then diluted with serum-free DMEM to a final concentration of 50 nM&#46; Cell transfection procedures were performed according to the instructions for Lipofectamine 2000&#46; After transfection for 24 h&#44; cells were subjected to H&#47;R treatment&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Reverse transcription-quantitative polymerase chain reaction</span><p id="par0035" class="elsevierStylePara elsevierViewall">RNA was extracted using TRIzol reagent&#46; The PrimeScript RT kit was used for cDNA synthesis&#44; and the SYBR Green PCR kit was used for the polymerase chain reaction &#40;PCR&#41; reaction&#46; Glyceraldehyde 3-phosphate dehydrogenase &#40;GAPDH&#41; and U6 were used as an internal reference for CASC15 and U6&#44; respectively&#46; The primers used were as follows&#58; CASC15&#58; forward&#58; 5&#8242;-CTTTGTCTGCTCCGGGACT-3&#8242;&#44; reverse&#58; 5&#8242;-TTAAGGGACATTTCCCCCG-3&#8242;&#59; miR-542-3p&#58; forward&#58; 5&#8242;-TCGGGGATCATCATGTCA-3&#8242;&#44; reverse&#58; 5&#8242;-GAGTGGCTCCCAGACCTT-3&#8242;&#59; GAPDH&#58; forward&#58; 5&#8242;-GTGGCTCCCAGACCTTTC-3&#8242;&#44; reverse&#58; 5&#8242;-AGCTTGACAAAGTGGTCG-3&#8242;&#59; U6&#58; forward&#58; 5&#8242;-CTCGCTTCGGCAGCACA-3&#8242;&#44; reverse&#58; 5&#8242;-AACGCTTCACGAATTTGCGT-3&#8242;&#46; Reaction conditions were initial denaturation at 95<span class="elsevierStyleHsp" style=""></span>&#176;C for 10 min&#44; followed by 40 cycles of denaturation at 92<span class="elsevierStyleHsp" style=""></span>&#176;C for 15 s&#44; annealing at 60<span class="elsevierStyleHsp" style=""></span>&#176;C for 1 min and extension at 60<span class="elsevierStyleHsp" style=""></span>&#176;C for 1 min&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Cell viability assay</span><p id="par0040" class="elsevierStylePara elsevierViewall">Cell viability was assessed with the Cell Counting Kit-8 assay &#40;CCK-8&#41;&#46; H9c2 cells were seeded in 96-well plates at a density of 4000 cells&#47;well and cultured overnight&#46; After cell transfection and H&#47;R treatment according to the experimental procedure&#44; cell viability was monitored every 24 h according to the manufacturer&#39;s scheme&#44; whereby CCK-8 solution at a concentration of 10 nM was added to the cells at a preset time point &#40;0 h&#44; 24 h&#44; 48 h&#44; 72 h&#44; 96 h&#41;&#44; and the cells were incubated in the dark at 37<span class="elsevierStyleHsp" style=""></span>&#176;C for 2 h&#44; and then optical density values were measured at 450 nm&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Cell apoptosis assay</span><p id="par0045" class="elsevierStylePara elsevierViewall">Cell apoptosis was detected by flow cytometry&#46; H9c2 cells were seeded in 6-well plates and treated according to the steps of transfection and H&#47;R treatment&#46; They were then harvested and stained with the Annexin V-FITC&#47;PI apoptosis detection kit&#46; After staining&#44; the cell apoptosis rate was immediately determined by flow cytometry&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Assessment of oxidative stress index</span><p id="par0050" class="elsevierStylePara elsevierViewall">Cell supernatants were used to determine superoxide dismutase &#40;SOD&#41;&#44; methylenedioxyamphetamine &#40;MDA&#41;&#44; and glutathione peroxidase &#40;GSH-Px&#41; content&#46; The corresponding kits were purchased from Nanjing Jiancheng Biotechnology Co&#46;&#44; Ltd&#46;&#44; as follows&#58; SOD assay kit &#40;Sigma-Aldrich&#44; cat&#46; CS1000&#41; using the water-soluble tetrazolium method&#59; MDA assay kit &#40;Beyotime&#44; cat&#46; S0131&#41; using the thiobarbituric acid method&#59; and GSH-Px assay kit &#40;Nanjing Jiancheng Bioengineering Institute&#44; cat&#46; A005&#41; using the colorimetric method&#46; Changes in absorbance were detected by spectrophotometry&#46;</p></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Luciferase reporter assay</span><p id="par0055" class="elsevierStylePara elsevierViewall">Starbase v2&#46;0 was used to analyze RNA&#8211;RNA interactions&#44; and predicted that miR-542-3p had a complementary binding site to CASC15&#46; We hypothesized that these two molecules have a targeted binding relationship&#44; and tested this hypothesis by luciferase reporter gene assay&#46; Briefly&#44; the steps are as follows&#58; wild-type CASC15 &#40;WT-CASC15&#41; and mutant CASC15 &#40;mut-CASC15&#41; were inserted into pmirGLO reporter vectors&#44; and these reporter vectors were co-transfected into H9c2 cells with miR-542-3p mimic or miR-542-3p inhibitor using Lipofectamine 2000&#46; After transfection for 48 h&#44; relative luciferase activity was measured on a dual-luciferase reporter assay system&#46; Renilla was used as an internal reference&#44; and data were expressed as the ratio of Renilla luciferase activity to Firefly luciferase activity&#46;</p></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0115">Statistical analysis</span><p id="par0060" class="elsevierStylePara elsevierViewall">Data are presented as mean<span class="elsevierStyleHsp" style=""></span>&#177;<span class="elsevierStyleHsp" style=""></span>standard deviation&#46; The two-tailed Student&#39;s <span class="elsevierStyleItalic">t</span> test was used to compare the two groups&#44; and multiple groups were compared by one-way analysis of variance&#46; Statistical significance was assumed for p&#60;0&#46;05&#46; Each test was repeated in triplicate&#46;</p></span></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0120">Results</span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0125">Determination of reoxygenation time</span><p id="par0065" class="elsevierStylePara elsevierViewall">Reoxygenation times are presented in <a class="elsevierStyleCrossRef" href="#fig0005">Figure 1</a>&#46; They show that CASC15 expression of cells in the H&#47;R group was significantly higher than in the control group after reoxygenation &#40;except at 0 h&#41; &#40;p&#60;0&#46;05&#41;&#46; In addition&#44; with the time extension&#44; after reoxygenation culture&#44; there were differences between the groups&#46; CASC15 levels in the H&#47;R group at 24 h were significantly higher than at 12 h &#40;p&#60;0&#46;001&#41;&#44; and similarly were significantly higher at 48 h than at 24 h &#40;p&#60;0&#46;01&#41;&#46; Therefore&#44; 24 h was selected as the reoxygenation culture time for this study&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0130">Effect of CASC15 downregulation on cell survival</span><p id="par0070" class="elsevierStylePara elsevierViewall">The intracellular CASC15 expression level was regulated by cell transfection&#46; As shown in <a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>A&#44; CASC15 expression in the H&#47;R model group was significantly increased compared with the control group&#44; however&#44; si-CASC15 transfection significantly downregulated CASC15 levels &#40;p&#60;0&#46;001&#41;&#46; Investigation of cell viability showed that compared with the control group&#44; cell viability in the H&#47;R model group was significantly inhibited&#44; but was significantly promoted after CASC15 was inhibited &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>B&#41;&#46; Concerning cell apoptosis&#44; the proportion of apoptotic cells in the H&#47;R model group was about 20&#37;&#44; which was significantly higher than in the control group&#46; As expected&#44; CASC15 depletion helped alleviate H&#47;R-induced apoptosis &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a>C&#41;&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0135">Effect on oxidative stress of inhibiting CASC15 expression</span><p id="par0075" class="elsevierStylePara elsevierViewall">It is well known that H&#47;R induces oxidative damage&#46; In the assessment of oxidative stress indicators&#44; it was found that MDA levels in the H&#47;R model group increased significantly&#44; while SOD and GSH-Px levels decreased&#44; indicating that H&#47;R treatment caused oxidative damage to H9c2 cells &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Figure 3</a>A&#8211;C&#41;&#46; It is worth noting that after CASC15 expression was suppressed&#44; oxidative damage was significantly reduced&#44; mainly manifested by decreased MDA and increased SOD and GSH-Px &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Figure 3</a>A&#8211;C&#41;&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0140">Validation of the targeting relationship between miR-542-3p and CASC15</span><p id="par0080" class="elsevierStylePara elsevierViewall">Bioinformatics predicted that CASC15 and miR-542-3p may have a targeting relationship&#44; and the complementary binding sites of the two molecules are shown in <a class="elsevierStyleCrossRef" href="#fig0020">Figure 4</a>A&#46; The luciferase reporter gene was used to verify this relationship&#44; and the results showed that for the WT-CASC15 group&#44; transfection of miR-542-3p mimic or inhibitor respectively weakened or enhanced luciferase activity&#44; while no similar results were obtained in the mut-CASC15 group &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Figure 4</a>B&#41; &#40;p&#60;0&#46;001&#41;&#46; Further verification results were as follows&#58; in H&#47;R model cells&#44; miR-542-3p expression decreased&#44; and at the same time&#44; inhibition of CASC15 helped to improve the expression of miR-542-3p&#44; which further underlines the relationship between CASC15 and miR-542-3p &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0020">Figure 4</a>C&#41;&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0145">Effect of miR-542-3p on cell survival</span><p id="par0085" class="elsevierStylePara elsevierViewall">As shown in <a class="elsevierStyleCrossRef" href="#fig0025">Figure 5</a>A&#44; miR-542-3p expression&#44; which increased due to the restricted expression of CASC15&#44; was significantly downregulated after transfection with miR-542-3p inhibitor &#40;p&#60;0&#46;001&#41;&#46; The cell viability assay showed that inhibition of miR-542-3p reversed the increase in cell viability resulting from silencing CASC15 &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Figure 5</a>B&#41;&#46; Similarly&#44; assessment of cell apoptosis showed that the loss of miR-542-3p promoted cell apoptosis compared to the inhibition of apoptosis induced by the attenuation of CASC15 &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0025">Figure 5</a>C&#41;&#46;</p><elsevierMultimedia ident="fig0025"></elsevierMultimedia></span><span id="sec0090" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0150">Effect of miR-542-3p on oxidative stress in H9c2 cells</span><p id="par0090" class="elsevierStylePara elsevierViewall">As mentioned above&#44; H&#47;R-induced oxidative stress damage was alleviated by the inhibition of CASC15&#46; However&#44; oxidative stress damage was found to be worsened when miR-542-3p in the cells was knocked down&#46; It was observed that after miR-542-3p was inhibited&#44; MDA levels increased and SOD and GSH-Px levels decreased &#40;p&#60;0&#46;001&#41; &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Figure 6</a>A&#8211;C&#41;&#46;</p><elsevierMultimedia ident="fig0030"></elsevierMultimedia></span></span><span id="sec0095" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0155">Discussion</span><p id="par0095" class="elsevierStylePara elsevierViewall">Abnormal lncRNA expression following I&#47;R after AMI may be involved in the occurrence and development of myocardial injury&#46; However&#44; the regulatory mechanism of some lncRNAs in I&#47;R injury remains unclear&#46; The oncogenic effects of CASC15 have been demonstrated by its upregulation in liver cancer and promotion of hepatic carcinoma&#46;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">13</span></a> Recent reports indicate that CASC15 is upregulated in acute ischemic stroke&#44; and can function as a sponge molecule of miR-338-3p&#44; promoting inflammatory damage to nerve cells&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">14</span></a> The results of our study showed that CASC15 expression in cardiomyocytes treated with H&#47;R was significantly increased compared with normal cultured cardiomyocytes&#44; suggesting that the abnormal increase in CASC15 was closely related to the occurrence of H&#47;R injury&#46; Further experiments showed that CASC15 inhibition significantly improved the H&#47;R-induced dysfunction of H9c2 cells &#40;including inhibition of cell viability and enhancement of cell apoptosis&#41; and oxidative stress response&#46; In addition&#44; dual-luciferase reporter assay revealed that CASC15 targets miR-542-3p and that CASC15 expression was negatively regulated by miR-542-3p&#44; indicating that CASC15 may promote H&#47;R injury in H9c2 cells by suppressing the expression of miR-542-3p&#46;</p><p id="par0100" class="elsevierStylePara elsevierViewall">I&#47;R injury is a common perioperative complication&#44; especially in the reoxygenation phase&#44; in which oxidative stress and a large increase in reactive oxygen species activate the mitochondrial apoptotic program&#44; triggering the caspase cascade and ultimately leading to cardiomyocyte apoptosis&#44;<a class="elsevierStyleCrossRefs" href="#bib0220"><span class="elsevierStyleSup">15&#44;16</span></a> a key factor in the development of heart failure after I&#47;R injury&#46; It is therefore important to inhibit cardiomyocyte apoptosis for prevention and treatment of the consequences of I&#47;R injury&#46;<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">17</span></a> In the present study&#44; cell viability was significantly inhibited after H&#47;R treatment&#44; and the proportion of apoptotic cells was significantly greater than in the control group&#46; Surprisingly&#44; after CASC15 knockout and H&#47;R treatment&#44; the effect on cell viability and apoptosis was no longer serious&#44; implying that excessive CASC15 plays an important role in H&#47;R injury&#46; Induction of apoptosis by CASC15 has been demonstrated in several studies&#46; For example&#44; Qin et al&#46; showed that CASC15 expression increased in diabetes-induced chronic renal failure&#44; and overexpression of CASC15 promoted podocyte apoptosis by sponging miR-34c&#46;<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">18</span></a> The molecular mechanism by which CASC15 promotes apoptosis of cardiomyocytes in our study needs further investigation&#46;</p><p id="par0105" class="elsevierStylePara elsevierViewall">Under normal physiological conditions&#44; there is a dynamic balance between oxidation and antioxidation in the body&#46; In the process of I&#47;R damage&#44; excessive production of oxidants leads to an imbalance between the oxidant and antioxidant systems&#46;<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">19</span></a> This imbalance results in oxidative stress&#44; which leads not only to inflammation&#44; but also to apoptosis&#44; which is a major factor in aging and disease&#46;<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">20</span></a> Presently&#44; the levels of three substances are commonly used as indices to assess the degree of oxidative stress&#58; MDA&#44; SOD and GSH-Px&#46; MDA is an end-product of lipid peroxidation&#44; and when increased indicates oxidative damage to the cell membrane&#46;<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">21</span></a> SOD activity is closely associated with oxidative damage caused by free radicals&#44; and may indirectly reflect the body&#39;s ability to scavenge free radicals and prevent peroxidation of low-density lipoprotein&#46;<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">22</span></a> GSH-Px catalyzes the formation of glutathione disulfide &#40;GSSG&#41; from reduced monomeric glutathione &#40;GSH&#41;&#44; and its activity can be used as a biochemical index to measure antioxidant levels in cells&#46;<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">23</span></a> In the current study&#44; after H&#47;R treatment&#44; oxidative stress indicators such as MDA increased while SOD and GSH-Px decreased&#44; indicating that H9c2 cells were damaged by oxidation&#46; However&#44; after downregulation of CASC15&#44; MDA concentrations in cells decreased&#44; while SOD and GSH-Px levels increased&#44; indicating that reduction of CASC15 alleviates the oxidative damage in cells caused by H&#47;R and significantly enhances the antioxidant capacity of cells&#46;</p><p id="par0110" class="elsevierStylePara elsevierViewall">miRNAs can inhibit the expression of target genes by recognizing the corresponding target site in the 3&#8217;-UTR region of the target gene and binding to it through complementary base pairing&#46;<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">24</span></a> Studies have shown that miRNAs can influence the process of I&#47;R injury by inhibiting oxidative damage and apoptosis of cardiomyocytes&#46; As a tumor inhibitor&#44; miR-542-3p mRNA expression is abnormally downregulated in many human tumors and other diseases&#44; including colon cancer&#44;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">25</span></a> prostate cancer&#44;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">26</span></a> gastric cancer&#44;<a class="elsevierStyleCrossRef" href="#bib0280"><span class="elsevierStyleSup">27</span></a> and ischemic stroke&#46;<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">28</span></a> A previous study showed that miR-542-3p was significantly reduced in mice with middle cerebral artery occlusion&#44; and overexpression of miR-542-3p mitigated apoptosis and activation of inflammatory responses caused by oxygen and glucose deprivation&#46;<a class="elsevierStyleCrossRef" href="#bib0290"><span class="elsevierStyleSup">29</span></a> Another study confirmed that overexpression of miR-542-3p has a protective effect on H&#47;R-induced cardiomyocytes and reduces cell apoptosis&#46;<span class="elsevierStyleSup">30</span> After a literature search&#44; we selected miR-542-3p&#44; which is clearly associated with cardiovascular disease&#44; as the target of CASC15 in this study&#46; Our results showed that the expression of miR-542-3p in cells was reduced after H&#47;R treatment&#44; suggesting that the loss of miR-542-3p may be involved in I&#47;R injury&#46; Furthermore&#44; luciferase reporter assay confirmed that CASC15 targeted miR-542-3p&#44; and that the latter can negatively regulate the expression of CASC15&#46; In this study&#44; H9c2 cells were co-transfected with si-CASC15 and miR-542-3p inhibitor&#44; and then treated with H&#47;R for 24 h&#46; The results showed that CASC15 inhibition promoted H&#47;R-induced cell viability&#44; inhibited cell apoptosis and attenuated oxidative damage&#46; However&#44; the reduction of miR-542-3p significantly inhibited cardiomyocyte viability and promoted oxidative damage&#44; which suggests that CASC15 overexpression may promote H&#47;R-induced myocardial injury by inhibiting the expression of miR-542-3p&#46;</p></span><span id="sec0100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0160">Conclusion</span><p id="par0115" class="elsevierStylePara elsevierViewall">In conclusion&#44; CASC15 expression increased in H&#47;R-induced H9c2 cells&#46; Overexpression of CASC15 aggravated oxidative stress injury in H&#47;R-induced cells&#44; inhibited cardiomyocyte viability&#44; and promoted cardiomyocyte apoptosis&#46; CASC15 can target miR-542-3p&#46; miR-542-3p expression decreased in the H&#47;R group&#44; which aggravated H&#47;R-induced oxidative stress&#44; inhibited cell viability and promoted apoptosis&#46; Therefore&#44; based on the above results&#44; we preliminarily speculate that CASC15 may promote H&#47;R-induced myocardial injury by inhibiting the expression of miR-542-3p in H&#47;R-induced H9c2 cells&#46;</p></span><span id="sec0105" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0165">Funding</span><p id="par0120" class="elsevierStylePara elsevierViewall">No funding was received for conducting this study&#46;</p></span><span id="sec0110" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0170">Conflicts of interest</span><p id="par0125" class="elsevierStylePara elsevierViewall">The authors have no conflicts of interest to declare&#46;</p></span></span>"
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            0 => array:2 [
              "identificador" => "sec0020"
              "titulo" => "Cell culture and treatment"
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            1 => array:2 [
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              "titulo" => "Cell transfection"
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              "titulo" => "Reverse transcription-quantitative polymerase chain reaction"
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              "titulo" => "Cell viability assay"
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              "titulo" => "Cell apoptosis assay"
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              "titulo" => "Assessment of oxidative stress index"
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              "titulo" => "Luciferase reporter assay"
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          "titulo" => "Results"
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            0 => array:2 [
              "identificador" => "sec0065"
              "titulo" => "Determination of reoxygenation time"
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            1 => array:2 [
              "identificador" => "sec0070"
              "titulo" => "Effect of CASC15 downregulation on cell survival"
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            2 => array:2 [
              "identificador" => "sec0075"
              "titulo" => "Effect on oxidative stress of inhibiting CASC15 expression"
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            3 => array:2 [
              "identificador" => "sec0080"
              "titulo" => "Validation of the targeting relationship between miR-542-3p and CASC15"
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              "identificador" => "sec0085"
              "titulo" => "Effect of miR-542-3p on cell survival"
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              "identificador" => "sec0090"
              "titulo" => "Effect of miR-542-3p on oxidative stress in H9c2 cells"
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    "fechaRecibido" => "2023-02-21"
    "fechaAceptado" => "2023-04-24"
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          "clase" => "keyword"
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          "palabras" => array:5 [
            0 => "Hypoxia&#47;reperfusion"
            1 => "Oxidative stress"
            2 => "CASC15"
            3 => "MiR-542-3p"
            4 => "Myocardial infarction"
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            0 => "Hip&#243;xia&#47;reperfus&#227;o"
            1 => "Tens&#227;o oxidativa"
            2 => "CASC15"
            3 => "MiR-542-3p"
            4 => "Enfarte do mioc&#225;rdio"
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    "resumen" => array:2 [
      "en" => array:3 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0010">Introduction and Objectives</span><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">The increasing incidence of ischemic heart disease is a serious threat to human health&#46; Increased CASC15&#44; a long non-coding RNA&#44; has been shown to adversely affect cardiac muscle&#46; The objective of this paper was to explore the effect of CASC15 on a cell model of myocardial infarction and its possible mechanism&#46;</p></span> <span id="abst0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Methods</span><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">H9c2 cells were selected to establish the myocardial infarction model through hypoxia&#47;reoxygenation &#40;H&#47;R&#41; treatment&#46; The expression of CASC15 was attenuated by cell transfection in vitro&#46; The level of CASC15 was detected by RT-qPCR&#46; Cell viability was detected by CCK-8 assay&#44; and cell apoptosis was assessed by flow cytometry&#46; The content of MDA and the activity of SOD and GSH-Px were measured by ELISA&#46; Luciferase reporter gene assay was used to determine the relationship between CASC15 and miRNA&#46;</p></span> <span id="abst0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Results</span><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">CASC15 expression was increased in H&#47;R-treated H9c2 cells&#46; Overexpression of CASC15 adversely affected cell viability and promoted H&#47;R-induced oxidative stress&#46; Inhibition of CASC15 promoted cell viability and suppressed cell apoptosis and oxidative stress damage&#46; Additionally&#44; luciferase reporter gene assay confirmed the targeting relationship between CASC15 and miR-542-3p&#44; and attenuating CASC15 expression enhanced the level of miR-542-3p&#46; Reduction of miR-542-3p weakened the viability of the H&#47;R cell model&#44; increased apoptosis&#44; and enhanced oxidative stress damage&#46;</p></span> <span id="abst0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Conclusion</span><p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">This study suggests that overexpression of CASC15 may inhibit the viability of H9c2 cells&#44; promote apoptosis and induce oxidative stress through targeted regulation of miR-542-3p expression&#46;</p></span>"
        "secciones" => array:4 [
          0 => array:2 [
            "identificador" => "abst0005"
            "titulo" => "Introduction and Objectives"
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          1 => array:2 [
            "identificador" => "abst0010"
            "titulo" => "Methods"
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          2 => array:2 [
            "identificador" => "abst0015"
            "titulo" => "Results"
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        "titulo" => "Resumo"
        "resumen" => "<span id="abst0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Introdu&#231;&#227;o e objetivos</span><p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">O aumento da doen&#231;a isqu&#233;mica card&#237;aca representa uma s&#233;ria amea&#231;a &#224; sa&#250;de humana&#46; Um aumento no CASC15&#44; um RNA n&#227;o codificante longo&#44; est&#225; associado &#224; les&#227;o do m&#250;sculo card&#237;aco&#46; O objetivo deste trabalho foi explorar o mecanismo celular atrav&#233;s do qual a CASC15 contribui para o enfarte do mioc&#225;rdio&#46;</p></span> <span id="abst0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">M&#233;todos</span><p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">C&#233;lulas H9c2 expostas a hip&#243;xia&#47;reoxigena&#231;&#227;o &#40;H&#47;R&#41; foram selecionadas como modelo para estudar os mecanismos envolvidos no enfarto do mioc&#225;rdio&#46; A express&#227;o de CASC15 foi inibida pela transfec&#231;&#227;o com siRNA&#46; Os n&#237;veis de CASC15 foram avaliados por RT-qPCR e a viabilidade celular e apoptose determinadas pelo ensaio CCK-8 e citometria de fluxo&#44; respetivamente&#46; Os teores de MDA&#44; SOD e GSH-Px foram medidos por ELISA&#46; O ensaio do gene rep&#243;rter da luciferase foi utilizado para verificar a rela&#231;&#227;o entre CASC15 e os n&#237;veis do miRNA miR-542-3p&#46;</p></span> <span id="abst0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Resultados</span><p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">C&#233;lulas H9c2 sujeitas a H&#47;R apresentam n&#237;veis aumentados de CASC15&#46; A sobre-express&#227;o de CASC15 afetou negativamente a fun&#231;&#227;o celular e promoveu a les&#227;o induzida pelo <span class="elsevierStyleItalic">stress</span> associado H&#47;R&#46; Por outro lado&#44; a inibi&#231;&#227;o do CASC15 promoveu viabilidade celular e suprimiu apoptose celular e os danos oxidativos&#46; Al&#233;m disso&#44; o ensaio do gene rep&#243;rter da luciferase confirmou a rela&#231;&#227;o alvo entre CASC15 e miR-542-3p&#44; com uma diminui&#231;&#227;o da express&#227;o de CASC15 a resultar num aumento do n&#237;vel de miR-542-3p&#46; Uma diminui&#231;&#227;o do miR-542-3p comprometeu a viabilidade do modelo de c&#233;lulas H&#47;R e aumentou a apoptose e o dano oxidativo&#46;</p></span> <span id="abst0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Conclus&#227;o</span><p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">Este estudo sugere que um aumento da express&#227;o de CASC15 durante a H&#47;R pode contribuir para diminuir a viabilidade dos cardiomi&#243;citos&#44; promover apoptose e induzir <span class="elsevierStyleItalic">stress</span> oxidativo&#44; atrav&#233;s da regula&#231;&#227;o da express&#227;o de miR-542-3p&#46;</p></span>"
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          "en" => "<p id="spar0045" class="elsevierStyleSimplePara elsevierViewall">Expression level of CASC15 under different reoxygenation times&#46; CASC15 levels increased with prolonged reoxygenation&#46; &#42;&#42;p&#60;0&#46;01&#44; &#42;&#42;&#42;p&#60;0&#46;001 vs&#46; control group&#59; <span class="elsevierStyleSup">&#35;</span>p&#60;0&#46;05&#44; <span class="elsevierStyleSup">&#35;&#35;</span>p&#60;0&#46;01&#44; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; H&#47;R group of the previous group&#46; H&#47;R&#58; hypoxia&#47;reoxygenation&#59; lncRNA&#58; long non-coding RNA&#46;</p>"
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          "en" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">Validation of cell transfection&#44; viability&#44; and apoptosis&#46; &#40;A&#41; Transfection of si-CASC15 effectively inhibited CASC15 expression in the H&#47;R group&#59; &#40;B&#41; inhibition of CASC15 promoted cell viability in the H&#47;R group&#59; &#40;C&#41; loss of CASC15 alleviated cell apoptosis in the H&#47;R group&#46; &#42;&#42;&#42;p&#60;0&#46;001 vs&#46; control group&#59; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#46; H&#47;R&#58; hypoxia&#47;reoxygenation&#59; lncRNA&#58; long non-coding RNA&#59; OD&#58; optical density&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#46;</p>"
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          "en" => "<p id="spar0055" class="elsevierStyleSimplePara elsevierViewall">Measurement of oxidative stress index&#46; Silencing CASC15 alleviated H&#47;R-induced oxidative stress injury&#44; as shown by decreased production of &#40;A&#41; MDA and increased activity of &#40;B&#41; SOD and &#40;C&#41; GSH-Px&#46; &#42;&#42;&#42;p&#60;0&#46;001 vs&#46; control group&#59; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#46; GSH-Px&#58; glutathione peroxidase&#59; H&#47;R&#58; hypoxia&#47;reoxygenation&#59; MDA&#58; methylenedioxyamphetamine&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#59; SOD&#58; superoxide dismutase&#46;</p>"
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          "en" => "<p id="spar0060" class="elsevierStyleSimplePara elsevierViewall">Verification of the relationship between CASC15 and miR-542-3p&#46; &#40;A&#41; Binding site of CASC15 and miR-542-3p&#59; &#40;B&#41; luciferase reporter gene assay&#59; &#40;C&#41; miR-542-3p expression increased after inhibition of CASC15 in the H&#47;R group&#46; &#42;&#42;&#42;p&#60;0&#46;001 vs&#46; control group&#59; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#46; H&#47;R&#58; hypoxia&#47;reoxygenation&#59; mut-CASC15&#58; mutant CASC15&#59; NC&#58; negative control&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#59; WT-CASC15&#58; wild-type CASC15&#46;</p>"
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          "en" => "<p id="spar0065" class="elsevierStyleSimplePara elsevierViewall">Effect of miR-542-3p on cell viability and apoptosis&#46; &#40;A&#41; Transfection with miR-542-3p inhibitor counteracted the enhanced effect of CASC15 knockdown on miR-542-3p expression&#59; &#40;B&#41; downregulation of miR-542-3p reduced cell viability enhancement caused by CASC15 silencing&#59; &#40;C&#41; loss of miR-542-3p increased the number of apoptotic cells in the si-CASC15 group&#46; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#59; <span class="elsevierStyleSup">&#38;&#38;&#38;</span>p&#60;0&#46;001 vs&#46; Si-CASC15 group&#46; NC&#58; negative control&#59; OD&#58; optical density&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#46;</p>"
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          "en" => "<p id="spar0070" class="elsevierStyleSimplePara elsevierViewall">Effect of miR-542-3p on oxidative stress injury&#46; Downregulation of CASC15 expression had an inhibitory effect on oxidative stress injury in the H&#47;R group&#44; and this inhibitory effect was abolished by the deletion of miR-542-3p&#46; With the absence of miR-542-3p&#44; &#40;A&#41; MDA concentration and &#40;B&#41; SOD and &#40;C&#41; GSH-Px contents increased&#46; <span class="elsevierStyleSup">&#35;&#35;&#35;</span>p&#60;0&#46;001 vs&#46; Si-NC group&#59; <span class="elsevierStyleSup">&#38;&#38;&#38;</span>p&#60;0&#46;001 vs&#46; Si-CASC15 group&#46; GSH-Px&#58; glutathione peroxidase&#59; MDA&#58; methylenedioxyamphetamine&#59; si-CASC15&#58; small interfering RNA against CASC15&#59; si-NC&#58; small interfering RNA as negative control&#59; SOD&#58; superoxide dismutase&#46;</p>"
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