Tim-3对肝癌干细胞自我更新能力的调控以及对TGF-β1/smad4通路的影响

Regulation of Tim-3 in the Self-renewal Ability of Hepatocellular Carcinoma Stem Cells and its Effect on TGF-β1/smad4 Pathway

目的:探究T淋巴细胞免疫球蛋白黏蛋白分子-3(Tim-3)在肝癌干细胞自我更新中的调控作用以及对转化生长因子-β1(TGF-β1)/smad4通路的影响。方法:体外培养肝癌细胞Huh7,获取肝癌干细胞,免疫印迹(WB)法检测肝癌干细胞标志物表达,包装si-Tim-3慢病毒载体感染肝癌干细胞分为空白组、si-NC组(添加阴性序列病毒)和si-Tim-3组,CKK-8法检测细胞增殖情况,光镜计数法检测细胞成球率,平板细胞克隆形成实验检测细胞克隆形成情况,划痕实验检测细胞迁移能力,Transwell小室检测细胞侵袭能力,WB法检测细胞中TGF-β1、smad4蛋白表达。结果:与肝癌细胞相比,肝癌干细胞标志物白细胞分化抗原133(CD133)、CD90、上皮细胞黏附分子(Ep CAM)蛋白表达显著升高(P<0.05)。与空白组和si-NC组相比,si-Tim-3组细胞增殖抑制率、迁移抑制率和Smad4蛋白表达显著升高,成球率、克隆形成率、侵袭数量和Tim-3、TGF-β1蛋白表达显著降低(P<0.05)。结论:抑制Tim-3表达后可抑制肝癌干细胞增殖、侵袭、迁移,其机制可能与下调TGF-β1、上调smad4蛋白有关。

Objective: To investigate the regulatory role of Tim-3 in the self-renewal ability of hepatocellular carcinoma stem cells (HCC) and its effect on transforming growth factor-β1 (TGF-β1)/Smad4 pathway. Methods: Hepatocellular carcinoma cells Huh7 were cultured in vitro,and hepatocellular carcinoma stem cells were obtained. The expression of hepatocellular carcinoma stem cell markers was detected by Western blotting (WB),hepatocellular carcinoma stem cells were infected by packaging si-Tim-3 lentiviral vector,and divided into three groups: the blank group,si-NC group (adding negative sequence virus) and si-Tim-3 group. CKK-8 assay was used to detect the cell proliferation,the cell ball forming rate was detected by light microscopy,panel cell clone formation experiment was used to detect the cell clone formation,scratch test was used to detect the cell migration ability,Transwell cells were used to detect the cell invasion,and WB was used to detect the expressions of TGF-β1 and Smad4 protein in cells. Results: Compared with that of hepatocellular carcinoma cells,the expressions of hepatocellular carcinoma stem cell markers CD133,CD90 and Ep CAM were significantly increased (P<0.05). Compared with those in the blank group and si-NC group,the proliferation inhibition rate,the migration inhibition rate and the expression of Smad4 protein were significantly increased,and the globulation rate,the clone formation rate,the invasion number,the expressions of Tim-3 and TGF-beta 1 protein were significantly decreased in si-Tim-3 group (P<0.05).Conclusion: The inhibition of Tim-3 expression can inhibit the proliferation,invasion and migration of hepatocellular carcinoma stem cells,and the mechanism may be related to down-regulation of TGF-β1 and up-regulation of Smad4 protein.