TSSC3通过调控PI3K/Akt-细胞失巢凋亡途径抑制肾小管上皮的纤维化表型

TSSC3 suppresses fibrosis of renal tubular epithelium by regulating PI3K/Akt signaling pathway and anoikis resistance in myofibroblasts

目的研究印记基因STF cDNA 3(TSSC3)对肾脏来源的肌成纤维细胞(myofibroblast,MyoFb)抗失巢凋亡和促纤维化能力的影响及其相关机制。方法使用TGF-β1处理人肾近曲小管上皮细胞系HK-2诱导其成为MyoFb,并采用超低粘附板悬浮培养法以及外源性RGD多肽培养法建立失巢凋亡模型;使用含或不含TGF-β1的细胞培养基处理稳定转染过表达TSSC3慢病毒的HK-2细胞,采用流式细胞术及CCK-8检测TSSC3对MyoFb及HK-2细胞失巢凋亡、增殖的影响;qRT-PCR及Western blot检测PI3K/AKT介导的凋亡途径以及纤维化相关分子mRNA及蛋白表达情况。结果TGF-β1明显增加HK-2中α-SMA的表达、降低Villin的表达(P<0.05),且具有时间依赖性,表明TGF-β1成功诱导HK-2细胞成为MyoFb,且MyoFb的失巢凋亡率明显低于HK-2细胞(P<0.01),表明TGF-β1诱导的MyoFb具有失巢凋亡抵抗的能力。相对于HK-2细胞,MyoFb增殖率明显增加、失巢凋亡率明显降低(P<0.01),PI3K/AKT通路抑制剂LY204002则部分逆转了上述改变。过表达TSSC3明显抑制失巢凋亡模型中MyoFb中p-PI3K、p-AKT的表达,增加cleaved-caspase 3的表达。过表达TSSC3明显抑制MyoFb和HK-2细胞中Ⅰ型胶原、Ⅲ型胶原、纤连蛋白、PAI-1、MMP-2、MMP-9的mRNA及蛋白的表达。结论TSSC3通过下调PI3K/Akt途径抑制肾脏来源MyoFb的失巢凋亡抵抗及促纤维化的能力。

Objective To investigate the effects of tumor suppressing STF cDNA 3(TSSC3)on the anoikis resistance and profibrogenic ability of renal myofibroblasts(MyoFb)and its related mechanisms.Methods Renal tubular epithelial HK-2 cells were stimulated with transforming growth factor beta 1(TGF-β1)to induce the formation of MyoFb.The model of cell anoikis was established by culturing cells in ultra-low adhesion plate suspension or by exogenous RGD-containing peptides culture.In addition,HK-2 cells transfected with TSSC3-overexpressing lentiviral vector were treated with or without TGF-β1,and the effects of TSSC3 on the anoikis and proliferation of cells were observed using flow cytometry and cell counting kit-8(CCK-8)assay.Finally,qRT-PCR and Western blotting were performed to detect the mRNA and protein expression of the PI3 K/AKT pathway and fibrosis related molecules.Results The results demonstrated thatα-SMA expression was significantly increased while that of Villin was decreased in HK-2 cells after stimulation of TGF-β1 in a time-dependent manner(P<0.05),indicating that HK-2 cells were successfully induced to MyoFb,which showed remarkably lower anoikis rate and higher proliferation than HK-2 cells(P<0.01),and had anoikis resistance.However,the above changes were partially reversed by PI3 K/Akt pathway inhibitor LY294002.Further study suggested that overexpression of TSSC3 obviously inhibited the mRNA and protein expression of fibrosis related genes,such as typeⅠand typeⅢcollagen,fibronectin,PAI-1,MMP-2 and MMP-9 in MyoFb and HK-2 cells.TSSC3 overexpression also up-regulated the level of cleaved-caspase 3 and attenuated the expression of p-PI3 K and p-Akt in MyoFb.Conclusion TSSC3 suppresses the anoikis resistance and profibrogenic ability of renal myofibroblasts by down-regulating the PI3 K/Akt pathway.