过表达LncRNA H19对糖尿病肾病足细胞生物学行为的影响及相关作用机制

Effect of overexpressed LncRNA H19 on the biological behavior of podocytes of patients with diabetic nephropathy and its related mechanism

目的探讨过表达LncRNA H19对糖尿病肾病足细胞生物学行为的影响及相关作用机制。方法选取小鼠足细胞系MPC5,培养后分为正常组、高糖组、沉默组、过表达组,并对4组细胞分别进行干预。MTT法检测细胞增殖能力,TUNEL法检测细胞凋亡能力,Transwell小室实验检测细胞侵袭能力,细胞划痕实验检测细胞迁移能力,RT-PCR检测LncRNA H19、E-cadherin、vimentin表达,Western-blot法检测细胞凋亡相关蛋白PI3K、Akt、mTOR及细胞自噬相关蛋白Beclin-1、PINK1、Parkin相对表达量。结果24 h、48 h、72 h时,与正常组比较,其他三组细胞增殖率较低,凋亡率较高;与高糖组、沉默组细胞比较,过表达组细胞增殖率较高、凋亡率较低(P<0.05)。与正常组细胞比较,其他三组细胞侵袭指数、迁移指数较高;与高糖组、沉默组细胞比较,过表达组细胞侵袭指数、迁移指数较低(P<0.05)。与正常组比较,其他三组细胞E-cadherin表达较高,LncRNA H19、vimentin表达较低,PI3K、Akt、mTOR相对表达量较低,Beclin-1、PINK1、Parkin相对表达量较高;与高糖组、沉默组细胞比较,过表达组细胞E-cadherin表达较低,LncRNA H19、vimentin表达较高,PI3K、Akt、mTOR相对表达量较高,Beclin-1、PINK1、Parkin相对表达量较低(P<0.05)。结论过表达LncRNA H19表达能够抑制糖尿病肾病足细胞凋亡,促进糖尿病肾病足细胞增殖、侵袭、迁移,其作用机制可能为过表达LncRNA H19能够靶向调控细胞凋亡相关蛋白及细胞自噬相关蛋白表达,从而对糖尿病肾病足细胞起到保护作用。

Objective To investigate the effect of overexpressed lncRNA H19 on the biological behavior of podocytes in patients with diabetic nephropathy and its related mechanism. Methods Mouse podocyte cell line MPC5 was selected.After the incubation,they were divided into the normal group,high glucose group,silencing group and overexpression group,and the four groups of cells were intervened respectively. MTT assay was used to detect cell proliferation.TUNEL assay was used to detect cell apoptosis. Transwell chamber assay was used to detect cell invasion. The cell wound scratch assay was used to detect cell migration. RT-PCR was used to detect the expressions of lncRNA H19,Ecadherin and vimentin. Western blot was used to detect the relative expressions of apoptosis-related proteins PI3K,Akt,mTOR and autophagy-related proteins Beclin-1, PINK1, Parkin. Results At 24 h,48 h and 72 h,compared with the normal group, the other three groups showed higher rate of the cell proliferation and lower rate of apoptosis. Compared with the high glucose group and silencing group,the proliferation rate of cells in overexpression group was higher and the apoptosis rate was lower(P<0.05). Compared with the normal group,the other three groups had higher invasion index and migration index in cells. Compared with the high glucose group and silencing group,the overexpression group exhibited lower invasion index and migration index in cells(P<0.05). Compared with the normal group, the expression of E-cadherin in the other three groups was higher, the expression of lncRNA H19 and vimentin was lower,the relative expression of PI3K,Akt and mTOR was lower,and the relative expression of Beclin-1,PINK1 and Parkin was higher.Compared with the high glucose group and silencing group, the expression of E-cadherin in overexpression group was lower, the expression of LncRNA H19 and vimentin was higher,the relative expression of PI3K,Akt and mTOR was higher,and the relative expression of Beclin-1,PINK1 and Parkin was lower(P<0.05). Conclusion Overexpressed lncRNA H19 can inhibit the apoptosis of podocytes and promote the proliferation, invasion and migration of podocytes in diabetic nephropathy. The mechanism of action may be that the overexpression of lncRNA H19 can target the regulation of the expressions of apoptosis-related proteins and autophagy-related proteins,thus playing a protective role in podocytes in diabetic nephropathy.