lncRNA CASC9靶向miR-513a-5p调控高糖诱导的肾小管上皮细胞损伤的机制研究

Mechanism of lncRNA CASC9 targeting miR-513a-5p to regulate renal tubular epithelial cell damage induced by high glucose

目的:探讨长链非编码RNA(lncRNA)CASC9对高糖(HG)诱导的人肾小管上皮细胞损伤的影响和可能机制。方法:体外培养人肾小管上皮细胞HK-2,用25 mmol/L葡萄糖干预24 h,RT-qPCR法检测细胞中CASC9和miR-513a-5p表达。分别转染CASC9过表达载体、miR-513a-5p抑制剂或共转染过表达载体与miR-513a-5p模拟物至HK-2细胞,然后用25 mmol/L葡萄糖干预24 h,ELISA检测细胞培养上清中IL-6和TNF-α表达,流式细胞术检测细胞凋亡,Western blot检测细胞中cleavedcaspase3和cleaved-caspase9蛋白表达。双荧光素酶报告基因实验验证CASC9和miR-513a-5p的调控关系。结果:HK-2细胞经高糖处理后,细胞中CASC9的表达量减少(P<0.05),而miR-513a-5p的表达量增加(P<0.05)。上调CASC9或下调miR-513a-5p的HK-2细胞经高糖处理后,细胞培养上清中IL-6和TNF-α表达量减少(P<0.05),细胞凋亡率和细胞中cleaved-caspase3和cleaved-caspase9蛋白表达量降低(P<0.05)。CASC9可靶向结合miR-513a-5p,且上调CASC9抑制HK-2细胞中miR-513a-5p表达。上调miR-513a-5p逆转上调CASC9对高糖诱导的HK-2细胞培养上清中IL-6和TNF-α表达量及细胞凋亡率、cleaved-caspase3和cleaved-caspase9蛋白表达的影响。结论:上调CASC9可能通过靶向下调miR-513a-5p阻碍高糖诱导的人肾小管上皮细胞HK-2分泌炎症因子及细胞凋亡。

Objective:To investigate the effect and possible mechanism of long non-coding RNA(lncRNA)CASC9 on human renal tubular epithelial cell injury induced by high glucose(HG).Methods:Human renal tubular epithelial cells HK-2 were cultured in vitro and treated with 25 mmol/L glucose for 24 h,and then the expressions of CASC9 and miR-513a-5p in the cells were detected by RT-qPCR.After HK-2 cells were transfected with CASC9 overexpression vector or miR-513a-5p inhibitor,or co-transfected with overexpression vector and miR-513a-5p mimic,they were treated with 25 mmol/L glucose for 24 h,and then ELISA was used to detect the expressions of IL-6 and TNF-αin the cell culture supernatant.Flow cytometry was used to detect cell apoptosis,and the Western blot method was used to detect the protein expressions of cleaved-caspase3 and cleaved-caspase9 in the cells.The dual luciferase reporter gene experiment verified the regulatory relationship between CASC9 and miR-513a-5p.Results:After HK-2 cells were treated with high glucose,the expression of CASC9 in the cells was decreased(P<0.05),but the expression of miR-513a-5p was increased(P<0.05).After high glucose treatment of HK-2 cells that up-regulated CASC9 or down-regulated miR-513a-5p,the expressions of IL-6 and TNF-αin the cell culture supernatant were decreased(P<0.05),and apoptosis and the protein expression of cleaved-caspase3 and cleaved-caspase9 were decreased(P<0.05).CASC9 could target miR-513a-5p,and up-regulating CASC9 inhibited the expression of miR-513a-5p in HK-2 cells.Up-regulation of miR-513a-5p reversed the effect of up-regulation of CASC9 on the expressions of IL-6 and TNF-αin the supernatant of HK-2 cells induced by high glucose,the apoptosis rate,and the protein expressions of cleaved-caspase3 and cleaved-caspase9.Conclusion:Up-regulation of CASC9 may block the secretion of inflammatory factors and apoptosis of human renal tubular epithelial cells HK-2 induced by high glucose by down-regulation of miR-513a-5p.