circ_ZNF609/miR-423-5p轴对高糖诱导肾小管上皮细胞HK2凋亡和氧化应激的机制研究

Experimental study of the effects of circ_ZNF609/miR-423-5p axis on the apoptosis and oxidative stress of renal tubular epithelial cells HK2 induced by high glucose

目的探讨circ_ZNF609对高糖诱导的肾小管上皮细胞凋亡和氧化应激的影响及可能机制。方法体外培养肾小管上皮细胞HK2,用含25 mmol/L葡萄糖的培养液培养24 h后,RT-qPCR法检测细胞中circ_ZNF609和miR-423-5p表达。分别转染circ_ZNF609小干扰RNA、miR-423-5p模拟物或共转染circ_ZNF609小干扰RNA和miR-423-5p抑制剂至HK2细胞,然后用含25 mmol/L葡萄糖的培养液培养24 h,CCK-8法检测细胞增殖活性,流式细胞术检测细胞凋亡,蛋白印迹法检测细胞中Cleaved-caspase3蛋白表达,试剂盒检测细胞中SOD活性及MDA含量。双荧光素酶报告基因实验验证circ_ZNF609和miR-423-5p调控关系。结果HK2细胞经高糖处理后,细胞中circ_ZNF609表达量增加(P<0.05),miR-423-5p表达量减少(P<0.05)。与高糖处理的HK2细胞比较,高糖处理的下调circ_ZNF609或上调miR-423-5p的HK2细胞增殖活性和细胞中SOD活性升高(P<0.05),细胞凋亡率、细胞中Cleaved-caspase3蛋白表达和MDA含量降低(P<0.05)。circ_ZNF609靶向结合miR-423-5p,且下调circ_ZNF609的HK2细胞中miR-423-5p的表达量增加。下调miR-423-5p逆转下调circ_ZNF609对高糖诱导的HK2细胞凋亡和氧化应激的影响。结论下调circ_ZNF609可能通过靶向上调miR-423-5p抑制高糖诱导的肾小管上皮细胞HK2凋亡和氧化应激。

Objective To investigate the effects of circ_ZNF609/miR-423-5p axis on the apoptosis and oxidative stress of renal tubular epithelial cells HK2 induced by high glucose,and to explore its possible mechanism.Methods The renal tubular epithelial cells HK2 were cultured in vitro and treated by the culture medium containing 25 mmol/L glucose for 24h,and then the expression levels of circ_ZNF609 and miR-423-5p in the cells were detected by RT-qPCR.After circ_ZNF609 small interfering RNA,miR-423-5p mimic or circ_ZNF609 small interfering RNA and miR-423-5p inhibitor were transfected into HK2 cells,which were cultured for 24h in culture medium containing 25mmol/L glucose.The CCK-8 method was used to detect cell proliferation activity;flow cytometry was used to detect cell apoptosis;Western Blot was used to detect the expression levels of Cleaved-caspase3 protein in cells,and the kits were used to detect the activity of SOD and the content of MDA in cells.Moreover the dual luciferase reporter gene experiment was used to verify the regulatory correlation between circ_ZNF609 and miR-423-5p.Results After HK2 cells were treated by high glucose,the expression levels of circ_ZNF609 in the cells were significantly increased(P<0.05),however,the expression levels of miR-423-5p were significantly decreased(P<0.05).Compared with HK2 cells treated by high glucose,the proliferation activities of high glucose-induced HK2 cells that down-regulated circ_ZNF609 or up-regulated miR-423-5p and the activity of SOD were significantly increased(P<0.05),however,the apoptosis rate,and the expression levels of Cleaved-caspase3 protein as well as the content of MDA in cells were significantly decreased(P<0.05).Moreover circ_ZNF609 could target miR-423-5p,and the expression levels of miR-423-5p were increased in HK2 cells that down-regulated circ_ZNF609.in addition the down-regulation of miR-423-5p reversed the effects of down-regulation of circ_ZNF609 on HK2 cells apoptosis and oxidative stress induced by high glucose.Conclusion Down-regulation of circ_ZNF609 may inhibit the apoptosis and oxidative stress of renal tubular epithelial cells HK2 induced by high glucose through targetedly up-regulation of miR-423-5p.