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长链非编码RNA组蛋白去甲基化酶1同系物D反义RNA1靶向微小RNA-421对过氧化氢诱导的心肌细胞氧化损伤的影响

Effect of lncRNA JHDM1D-AS1 targeting microRNA-421 on hydrogen peroxide induced oxidative damage of cardiomyocytes

    摘要
  • 摘要:
    目的 探讨长链非编码RNA组蛋白去甲基化酶1同系物D反义RNA1(lncRNA JHDM1D-AS1)靶向微小RNA-421(miR-421)对过氧化氢(H2O2)诱导的心肌细胞(H9C2细胞)氧化损伤的影响。
    方法 将H9C2细胞分为对照(Con)组、H2O2组、H2O2+pcDNA组、H2O2+pcDNA-JHDM1D-AS1组、H2O2+anti-miR-NC组、H2O2+anti-miR-421组、H2O2+pcDNA- JHDM1D-AS1+miR-NC组、H2O2+pcDNA-JHDM1D-AS1+miR-421组。采用实时荧光定量PCR(RT-qPCR)检测JHDM1D-AS1和miR-421表达。采用比色法测定H9C2细胞中超氧化物歧化酶(SOD)活性、丙二醛(MDA)水平以及培养液中乳酸脱氢酶(LDH)水平。采用流式细胞术评估H9C2细胞凋亡率。JHDM1D-AS1和miR-421的靶向关系通过双荧光素酶报告基因法验证。
    结果 与Con组比较, H2O2组H9C2细胞JHDM1D-AS1水平、SOD活性降低, MDA水平、LDH水平、细胞凋亡率、miR-421水平升高，差异均有统计学意义(P < 0.05)。与H2O2+pcDNA组比较, H2O2+pcDNA-JHDM1D-AS1组H9C2细胞SOD活性升高, miR-421表达水平、MDA水平、LDH水平、细胞凋亡率降低，差异有统计学意义(P < 0.05)。与H2O2+anti-miR-NC组比较, H2O2+anti-miR-421组H9C2细胞SOD活性升高, MDA水平、LDH水平、细胞凋亡率降低，差异有统计学意义(P < 0.05)。miR-421是JHDM1D-AS1的靶基因。与H2O2+pcDNA-JHDM1D-AS1+miR-NC组比较, H2O2+pcDNA-JHDM1D-AS1+miR-421组H9C2细胞SOD活性降低, MDA水平、LDH水平、细胞凋亡率升高，差异有统计学意义(P < 0.05)。
    结论 lncRNA JHDM1D-AS1通过靶向下调miR-421表达抑制细胞凋亡和氧化应激，减轻H2O2诱导的心肌细胞氧化损伤。

     

    Abstract:
    Objective To investigate the effect of long non-coding RNA Jumonji C domain containing histone demethylase 1 homolog D antisense RNA1 (lncRNA JHDM1D-AS1) targeting microRNA-421 (miR-421) on hydrogen peroxide (H2O2) induced oxidative damage in cardiomyocytes (H9C2 cells).
    Methods H9C2 cells were divided into control (Con) group, H2O2 group, H2O2+pcDNA group, H2O2+pcDNA-JHDM1D-AS1 group, H2O2+anti-miR-NC group, H2O2+anti-miR-421 group, H2O2+pcDNA-JHDM1D-AS1+miR-NC group, and H2O2+pcDNA-JHDM1D-AS1+miR-421 group. The expressions of JHDM1D-AS1 and miR-421 were detected by real-time fluorescent quantitative PCR (RT-qPCR). The superoxide dismutase (SOD) activity, malondialdehyde (MDA) level, and lactate dehydrogenase (LDH) level in the culture medium of H9C2 cells were measured by colorimetry. Flow cytometry was used to evaluate the apoptosis rate of H9C2 cells. The targeting relationship between JHDM1D-AS1 and miR-421 was verified by dual luciferase reporter gene assay.
    Results Compared with the Con group, the H2O2 group showed decreased levels of JHDM1D-AS1 and SOD activity, and increased levels of MDA, LDH, apoptosis rate, and miR-421 in H9C2 cells, with significant between-group differences (P < 0.05). Compared with the H2O2+pcDNA group, the H2O2+pcDNA-JHDM1D-AS1 group exhibited increased SOD activity and decreased miR-421 expression level, MDA level, LDH level, and apoptosis rate in H9C2 cells, with significant between-group differences (P < 0.05). Compared with the H2O2+anti-miR-NC group, the H2O2+anti-miR-421 group showed increased SOD activity and decreased MDA level, LDH level, and apoptosis rate in H9C2 cells, with significant between-group differences (P < 0.05). MiR-421 was identified as a target gene of JHDM1D-AS1. Compared with the H2O2+pcDNA-JHDM1D-AS1+miR-NC group, the H2O2+pcDNA-JHDM1D-AS1+miR-421 group exhibited decreased SOD activity and increased MDA level, LDH level, and apoptosis rate in H9C2 cells, with significant between-group differences (P < 0.05).
    Conclusion LncRNA JHDM1D-AS1 inhibits apoptosis and oxidative stress by targeting and downregulating miR-421 expression, thereby alleviating H2O2-induced oxidative damage in cardiomyocytes.

     

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