miR-16-5p靶向NLRP1抑制缺氧复氧诱导的BRL-3A细胞焦亡

miR-16-5p targets NLRP1 to inhibit BRL-3A cell pyrolysis induced by hypoxia-reoxygenation

目的探讨mi R-16-5p对缺氧复氧诱导的大鼠正常肝细胞系BRL-3A焦亡的作用以及作用机制。方法通过细胞缺氧复氧的方法构建肝缺血再灌注损伤(HIRI)的体外模型,通过流式细胞术检测细胞焦亡率;通过荧光实时定量PCR(RT-qPCR)检测mi R-16-5p的表达水平;运用mi R-16-5p mimic在细胞中过表达mi R-16-5p并研究mi R-16-5p对细胞焦亡的影响;通过蛋白免疫印迹实验(Western blot)检测焦亡相关蛋白caspase1、IL-1β、IL-18的蛋白表达水平;通过生物信息学分析和双荧光素酶报告基因实验预测并鉴定mi R-16-5p与NLRP1的靶向关系;通过过表达和干扰NLRP1,研究NLRP1及mi R-16-5p/NLRP1轴对细胞焦亡的影响。结果与空白对照组相比,模型组焦亡率极显著上升(P<0.01),mi R-16-5p表达量显著降低(P<0.05)。过表达mi R-16-5p极显著抑制细胞焦亡(P<0.01)并抑制焦亡相关蛋白caspase1、IL-1β、IL-18的表达(P<0.01或P<0.05)。双荧光素酶报告基因实验显示NLRP1是mi R-16-5p的靶基因,过表达mi R-16-5p显著降低了NLRP1的m RNA和蛋白水平(P<0.01或P<0.05)。此外,过表达NLRP1逆转了mi R-16-5p对细胞焦亡的抑制作用(P<0.01)和对caspase1、IL-1β、IL-18表达的抑制作用(P<0.01或P<0.05)。结论MiR-16-5p通过靶向NLRP1来抑制caspase1、IL-1β、IL-18的表达,从而改善缺氧复氧引起的BRL-3A细胞焦亡。

Objective To investigate the effect and mechanism of miR-16-5 p on pyrolysis of rat normal liver cell line BRL-3 A induced by hypoxia-reoxygenation.Methods An in vitro model of hepatic ischemia reperfusion injury was established by cellular hypoxia-reoxygenation and the pyrolysis rate was measured by flow cytometry.RT-qPCR was used to measure the expression level of miR-16-5 p.Overexpressed miR-16-5 p in cells by miR-16-5 p mimics was used to examine the effect of miR-16-5 p on pyrolysis.Western blot was used to measure protein expression levels of pyrolysis-related proteins caspase 1,IL-1β,and IL-18.The relationship between miR-16-5 p and NLRP1 was predicted through bioinformatics analysis and dual luciferase reporter gene assays.Interference and overexpression of NLRP1 were performed to investigate the effect of NLRP1 and the miR-16-5 p/NLRP1 axis on pyrolysis.Results Compared with the blank control group,the pyrolysis rate of the model group was increased significantly(P<0.01)and expression of miR-16-5 p was decreased significantly(P<0.05).Overexpression of miR-16-5 p significantly decreased the pyrolysis rate(P<0.01)and inhibited the expression of caspase 1,IL-1β,and IL-18(P<0.01 or P<0.05).The dual luciferase reporter gene assay showed that NLRP1 was a target gene of miR-16-5 p.Overexpression of miR-16-5 p significantly reduced the m RNA and protein levels of NLRP1(P<0.01 or P<0.05).Additionally,overexpression of NLRP1 reversed the inhibitory effect of miR-16-5 p on pyrolysis(P<0.01)and the inhibitory effect on the expression of caspase 1,IL-1β,and IL-18(P<0.01 or P<0.05).Conclusions MiR-16-5 p inhibits expression of caspase 1,IL-1β,and IL-18 by targeting NLRP1,thereby improving pyrolysis of BRL-3 A cells caused by hypoxia-reoxygenation.