LncRNA H19调控肾小球足细胞焦亡中作用

Role of LncRNA H19 in pyrophosis of glomerular podocyte induced by high glucose

目的探讨lncRNA H19在高糖调控肾小球足细胞焦亡中的作用。方法qRT-PCR方法检测高糖中lncRNA H19本底表达;Western blot检测高糖中肾足细胞结构性相关蛋白Nephrin、Podocin表达;Western blot及免疫荧光方法检测细胞焦亡相关蛋白NLRP3、Caspase-1、IL-1β表达水平;qRT-PCR检测转染Ad lncRNA H19及si lncRNA H19后lncRNA H19的mRNA表达,细胞转染分别设置:(1)对照组(不做处理)、lncRNA H19过表达阴性对照组、lncRNA H19过表达组;(2)对照组(不做处理)、lncRNA H19干扰阴性对照组和lncRNA H19干扰组(si-lncRNA H19-1、si-lncRNA H19-2、si-lncRNA H19-3);Western blot检测转染Ad lncRNA H19及si lncRNA H19后NLRP3、Caspase-1、IL-1β蛋白表达水平,细胞转染分别设置:(1)对照组(不做处理)、高糖组、lncRNA H19过表达对照+高糖组、lncRNA H19过表达+高糖组;(2)对照组(不做处理)、高糖组、lncRNA H19干扰对照+高糖组、lncRNA H19干扰+高糖组。结果(1)与对照组(不做处理)相比,lncRNA H19在高糖诱导肾足细胞损伤过程中呈低表达(P<0.05),而lncRNA H19低表达可调控细胞焦亡相关蛋白NLRP3、Caspase-1、IL-1β表达增高(P<0.05),lncRNA H19表达与细胞焦亡相关蛋白表达两者呈负相关;(2)与对照组(不做处理)相比,在高糖诱导肾足细胞损伤过程中,肾足细胞结构蛋白Nephrin、Podocin表达水平降低(P<0.05);(3)与lncRNA H19过表达对照+高糖组相比,过表达lncRNA H19后,缓解高糖诱导肾足细胞焦亡,细胞焦亡相关蛋白NLRP3、Caspase-1、IL-1β呈低表达(P<0.05);(4)与lncRNA H19干扰对照+高糖组相比,干扰lncRNA H19后,促进高糖诱导足细胞焦亡,而焦亡相关蛋白NLRP3、Caspase-1、IL-1β呈高表达(P<0.05)。结论在高糖诱导足细胞焦亡过程中lncRNA H19具有可调控细胞焦亡相关蛋白表达,为未来治疗糖尿病肾病提供一定的理论依据。

Objective To investigate the role of lncRNA H19 in pyrophosis of glomerular podocyte induced by high glucose(HG).Methods The background expression of lncRNA H19 in HG was detected by qRT PCR;western blot was used to detect the expression of Nephrin and Podocin;detection of NLRP3,Caspase-1 and IL-1βexpression level was performed by using western blot and immunofluorescence.QRT-PCR was used to detect the mRNA expression of lncRNA H19 after transfection of Ad lncRNA H19 and si lncRNA H19.The cell transfection were set as follows:(1)control group(without treatment),negative control group of lncRNA H19 overexpression,lncRNA H19 overexpression group;(2)control group(without treatment),negative control group of lncRNA H19 interference and lncRNA H19 interference group(si lncRNA H19-1,si lncRNA H19-2,si lncRNA H19-3).Detection of NLRP3,Caspase-1 and IL-1βexpression level after transfection with Ad lncRNA H19 and si lncRNA H19 was performed by western blot.The cell transfection were set as follows:(1)control group(without treatment),high glucose group,lncRNA H19 overexpression control+high glucose group,lncRNA H19 overexpression+high glucose group,(2)control group(without treatment),high glucose group,lncRNA H19 interference control+high glucose group,and lncRNA H19 interference+high glucose group.Results(1)Compared with that in the control group(without treatment),the expression of lncRNA H19 was low in the process of renal podocyte injury induced by high glucose(P<0.05),and the low expression of lncRNA H19 led to higher expression of pyrophosis-related proteins NLRP3,Caspase-1,and IL-1β.There was a negative correlation between the expression of lncRNA H19and the expression of pyrophosis-related proteins.(2)Compared with that in the control group(without treatment),the expression level of Nephrin and Podocin in renal podocytes decreased during the process of injury induced by high glucose(P<0.05).(3)Compared with that in the lncRNA H19 overexpression control+high glucose group,overexpression of lncRNA H19 alleviated high glucose induced nephropodocyte pyrophosis,and the expression of pyrophosis-related proteins NLRP3,Caspase-1,and IL-1βwas low(P<0.05).(4)Compared with that in the lncRNA H19 interference control+high glucose group,the interference of lncRNA H19 promoted the high glucoseinduced podocyte pyrophosis,while the pyrophosis-related proteins NLRP3,Caspase-1,and IL-1βwere highly expressed(P<0.05).Conclusion In the process of podocyte pyrophosis induced by high glucose,lncRNA H19can regulate the expression of pyrophosis-related proteins,which provides theoretical basis for the treatment of diabetic nephropathy in the future.