miR-381-3p靶向调控Sox9对高糖诱导的肾小管上皮细胞凋亡的影响

    Effect of miR-381-3p on high glucose-induced renal tubular epithelial cell apoptosis by targeting and regulating Sox9

    • 摘要:
      目的 探讨微小RNA-381-3p(miR-381-3p)靶向性别决定区Y框蛋白9(Sox9)对高糖(HG)诱导肾小管上皮细胞(HK-2)凋亡的影响。
      方法 体外培养肾小管上皮细胞系HK-2细胞, 将HK-2细胞分为对照组、HG组、HG+miR-NC组(转染miR-NC)、HG+miR-381-3p mimics组(转染miR-381-3p mimics)、HG+si-NC组(转染si-NC)、HG+si-Sox9组(转染si-Sox9)、HG+miR-381-3p mimics+pcDNA组(miR-381-3p mimics与pcDNA共转染)和HG+miR-381-3p mimics+pcDNA-Sox9组(miR-381-3p mimics与pcDNA-Sox9共转染), 除对照组外, 其余组均用30 mmol/L葡萄糖处理。实时荧光定量PCR(RT-qPCR)法检测HK-2细胞miR-381-3p及Sox9 mRNA表达;流式细胞仪检测细胞凋亡情况;免疫印迹法检测各组HK-2细胞Sox9、B淋巴细胞瘤-2相关蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)蛋白表达;双荧光素酶报告基因实验验证miR-381-3p与Sox9的靶向关系。
      结果 与对照组比较, HG组细胞凋亡率、Bax mRNA及蛋白表达水平升高, miR-381-3p、Bcl-2 mRNA及蛋白表达水平降低, 差异均有统计学意义(P < 0.05)。经targetscan数据库预测显示, miR-381-3p与Sox9 mRNA 3′UTR区有结合位点。过表达miR-381-3p或抑制Sox9表达, 细胞凋亡率、Bax、Sox9 mRNA及蛋白表达水平降低, Bcl-2 mRNA及蛋白表达水平升高, 差异均有统计学意义(P < 0.05);过表达Sox9可逆转过表达miR-381-3p对HG诱导的HK-2细胞凋亡的抑制作用(P < 0.05)。
      结论 miR-381-3p在HG诱导的肾小管上皮细胞中低表达, 过表达miR-381-3p可通过靶向抑制Sox9表达, 从而减少了HG诱导的肾小管上皮细胞凋亡。

       

      Abstract:
      Objective To investigate the effect of microRNA-381-3p (miR-381-3p) targeting SRY related HMG box-9 (Sox9) on high glucose (HG)-induced renal tubular epithelial HK-2 cells apoptosis.
      Methods The renal tubular epithelial HK-2 cells were cultured in vitro, and divided into control group, HG group, HG+miR-NC group (transfected with miR-NC), HG+miR-381-3p mimics group (transfected with miR-381-3p mimics), HG+si-NC group (transfected with si-NC), HG+si-Sox9 group (transfected with si-Sox9), HG+miR-381-3p mimics+pcDNA group (co-transfected with miR-381-3p mimics and pcDNA) and HG+miR-381-3p mimics+pcDNA-Sox9 group (co-transfected with miR-381-3p mimics and pcDNA-Sox9).Except the control group, the cells in other groups were treated with 30 mmol/L glucose.The expression of miR-381-3p and Sox9 at mRNA level in HK-2 cells was detected by real-time fluorescent quantitative PCR, the apoptosis was detected by flow cytometry, the protein expressions of Sox9, B lymphoma-2 related protein (Bax) and B lymphoma-2 (Bcl-2) in HK-2 cells were detected by Western blotting, and the targeting relationship between miR-381-3p and Sox9 was verified by the dual luciferase reporter gene experiment.
      Results Compared with the control group, the apoptosis rate, the mRNA and protein expression levels of Bax in HG group were increased, and the expression of miR-381-3p and Bcl-2 at mRNA and protein levels were decreased, which had significant statistical difference (P < 0.05).The targetscan database predicted that miR-381-3p had a binding site with the 3′UTR region of Sox9 mRNA.When overexpression of miR-381-3p or inhibition of Sox9 expression, the apoptosis rate, the mRNA and protein expression levels of Bax and Sox9 were significantly reduced (P < 0.05), and the mRNA and protein expression level of Bcl-2 was significantly increased (P < 0.05).Overexpression of miR-381-3p or inhibition of Sox9 expression decreased the apoptosis rate, mRNA and protein expression levels of Bax and Sox9, and increased the mRNA and protein expression levels of Bcl-2, the difference was statistically significant (P < 0.05).
      Conclusions MiR-381-3p is low-expressed in HG-induced renal tubular epithelial cells, overexpression of miR-381-3p can reduce HG-induced renal tubular epithelial cell apoptosis by targeted inhibition of Sox9 expression.

       

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