下调miR-153-3p可促进Nrf2表达从而减轻H2O2诱导的H9C2细胞损伤

Down-regulation of miR-153-3p attenuates H2O2-induced H9C2 cell injury by promoting Nrf2 expression

目的:研究敲减微小RNA-153-3p(miR-153-3p)的表达在H2O2引起的H9C2细胞氧化损伤中的作用以及具体机制。方法:建立H2O2诱导的H9C2细胞氧化应激损伤模型,分别通过MTT实验和RT-qPCR检测细胞活力和miR-153-3p表达的变化。通过RNA干扰技术敲减miR-153-3p的表达,观察其对氧化应激状态下H9C2细胞氧化损伤的影响,并通过Western blot和双萤光素酶报告基因实验确定miR-153-3p的作用靶点。结果:H9C2细胞活力随H2O2浓度升高而逐渐降低(P<0.05),miR-153-3p的表达则逐渐增加(P<0.05)。敲减miR-153-3p的表达可提高H9C2细胞在氧化应激状态下的细胞活力,减少细胞凋亡,减少丙二醛(MDA)含量并升高超氧化物岐化酶(SOD)活性(P<0.01)。Western blot实验可见核因子E2相关因子2(Nrf2)的表达和抗氧化反应元件(ARE)活性随H2O2浓度升高而升高(P<0.05);TargetScan分析和双萤光素酶报告基因实验结果显示Nrf2是miR-153-3p的潜在靶基因之一。Western blot实验进一步显示miR-153-3p过表达可抑制Nrf2的表达(P<0.01),抑制miR-153-3p则可促进Nrf2表达(P<0.01)。同时敲减H9C2细胞的Nrf2和miR-153-3p表达可显著降低H9C2细胞在H2O2环境下的细胞活力,促进细胞凋亡,增加MDA含量并降低SOD活性(P<0.01)。结论:抑制miR-153-3p表达可上调Nrf2/ARE通路从而减轻H2O2引起的H9C2细胞损伤。

AIM:To study the effect of microRNA-153-3 p(miR-153-3 p)knock-down on oxidative injury of H9 C2 cells induced by H2O2 and its specific mechanism.METHODS:The oxidative stress injury of H9 C2 cell model was induced by H2O2,and then the cell viability and the expression of miR-153-3 p were detected by MTT assay and RT-qPCR,respectively.The effects of miR-153-3 p knock-down on the H9 C2 cell injury under oxidative stress were studied by RNA interference technology.The targets of miR-153-3 p were identified by Western blot and dual-luciferase reporter assay.RESULTS:MTT assay showed that the viability of H9 C2 cells was decreased with the increase in H2O2 concentration(P<0.05).The results of RT-qPCR showed that the expression of miR-153-3 p was increased with the increase in H2O2 concentration(P<0.05).Knock-down of miR-153-3 p increased the viability of H9 C2 cells under oxidative stress,decreased the cell apoptosis and the content of malondialdehyde(MDA),and increased the activity of superoxide dismutase(SOD).The expression of nuclear factor E2-related factor 2(Nrf2)and antioxidant response element(ARE)activity were increased with the increase in H2O2 concentration(P<0.01).TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3 p.The results of Western blot further showed that over-expression of miR-153-3 p inhibited the expression of Nrf2(P<0.01),while down-regulation of miR-153-3 p increased the expression of Nrf2(P<0.01).Dual interference with Nrf2 and miR-153-3 p significantly reduced H9 C2 cell viability,promoted the apoptosis,increased MDA content,and decreased SOD activity in the presence of H2O2(P<0.01).CONCLUSION:Inhibition of miR-153-3 p expression attenuates the injury of H9 C2 cells induced by H2O2 through up-regulating Nrf2/ARE signaling pathway.