miR-204-3p靶向CYP2E1介导脂多糖诱导的肾小管上皮细胞损伤

目的探讨miR-204-3p靶向细胞色素P450家族2亚家族E成员1(CYP2E1)介导脂多糖(LPS)诱导的肾小管上皮细胞炎症反应及细胞凋亡的分子机制。方法体外培养人肾小管上皮细胞HK-2,qRT-PCR与Western blot分别检测miR-204-3p、CYP2E1的表达量;实验分组:Con组、LPS组、LPS+miR-NC组、LPS+miR-204-3p组、LPS+si-NC组、LPS+si-CYP2E1组、LPS+miR-204-3p+pcDNA组、LPS+miR-204-3p+pcDNA-CYP2E1组;ELISA检测IL-6、TNF-α的水平;流式细胞术检测细胞凋亡率;双荧光素酶报告实验验证miR-204-3p与CYP2E1的靶向结合关系;Western blot检测Bcl-2、Bax的表达量。结果与Con组比较,LPS组细胞中miR-204-3p的表达水平降低,CYP2E1的表达量升高,IL-6、TNF-α的水平升高,凋亡率和Bax蛋白水平升高,Bcl-2蛋白水平降低,差异有统计学意义(P<0.05);与LPS+miR-NC组比较,LPS+miR-204-3p组IL-6、TNF-α水平降低,凋亡率和Bax蛋白水平降低,Bcl-2蛋白水平升高,差异有统计学意义(P<0.05);与LPS+si-NC组比较,LPS+si-CYP2E1组IL-6、TNF-α的水平降低,凋亡率和Bax蛋白水平降低,Bcl-2蛋白水平升高,差异有统计学意义(P<0.05);双荧光素酶报告实验证实miR-204-3p能够靶向结合CYP2E1;与LPS+miR-204-3p+pcDNA组比较,LPS+miR-204-3p+pcDNA-CYP2E1组IL-6、TNF-α的水平升高,凋亡率和Bax蛋白水平升高,Bcl-2蛋白水平降低,差异有统计学意义(P<0.05)。结论 miR-204-3p过表达可靶向调控CYP2E1的表达从而抑制LPS诱导的肾小管上皮细胞炎症反应及细胞凋亡。

miR-204-3p targets CYP2E1 to mediate lipopolysaccharide-induced renal tubular epithelial cell damage

Objective To investigate the molecular mechanism of miR-204-3 p targeting CYP2 E1 expression mediating LPS-induced renal tubular epithelial cell inflammation and apoptosis. Methods Human renal tubular epithelial cells HK-2 were cultured in vitro,and the expressions of mi R-204-3 p and CYP2 E1 were detected by q RT-PCR and Western blot. Experimental groupings:Con group,LPS group,LPS+mi R-NC group,LPS+mi R-204-3 p group,LPS+si-NC group,LPS+si-CYP2 E1 group,LPS+mi R-204-3 p+pc DNA group,LPS+mi R-204-3 p+pc DNA-CYP2 E1 group. The levels of IL-6 and TNF-α were detected by ELISA.Flow cytometry was used to detect the apoptosis rate. The dual luciferase reporting experiment verified the targeted binding relationship between mi R-204-3 p and CYP2 E1. Western blot was used to detect the expression of Bcl-2 and Bax. Results In the LPS group,the expression level of mi R-204-3 p was decreased,the expression level of CYP2 E1 was increased,the levels of IL-6 and TNF-α were increased,the rate of apoptosis and the level of Bax protein increased,and the level of Bcl-2 protein was decreased,compared with the Con group,the difference was statistically significant(P<0.05). The levels of IL-6 and TNF-α in the LPS+mi R-204-3 p group were decreased,the apoptosis rate and the level of Bax protein were decreased,and the level of Bcl-2 protein was increased,compared with the LPS+mi R-NC group,the difference was statistically significant(P<0.05).The levels of IL-6 and TNF-α in the LPS+si-CYP2 E1 group were decreased,the apoptosis rate and the level of Bax protein were decreased,and the level of Bcl-2 protein was increased,compared with the LPS+si-NC group,there were differences Statistically significant(P<0.05). The dual luciferase report experiment confirmed that mi R-204-3 p could target CYP2 E1. In the LPS+mi R-204-3 p+pc DNA-CYP2 E1 group,the levels of IL-6 and TNF-α were increased,the apoptosis rate and the level of Bax protein were increased,and the level of Bcl-2 protein was decreased,compared with the LPS+mi R Compared with-204-3 p+pc DNA group,the difference was statistically significant(P<0.05). Conclusion Overexpression of mi R-204-3 p can target the regulation of CYP2 E1 expression and inhibit LPS-induced renal tubular epithelial cell inflammation and apoptosis.