【摘要】 目的探讨LAMA4调控TGF-β1/SMAD Western blot检测LAMA4在肝癌细胞Huh7和HepG2及正常肝细胞L02中的表达;MMT法和流式细胞技术检测LAMA4和TGF-β1/SMAD信号传导途径对Huh7细胞增殖和凋亡的影响;RT-qPCR和Western blot检测LAMA4对TGF-β1/SMAD信号转导通路和细胞免疫因子TNF-α、IFN-γ、TGF-β1的影响。结果 RT-qPCR和Western blot检测结果显示:LAMA4在肝癌细胞Huh7和HepG2中mRNA表达显著上调;LAMA4在肝癌细胞Huh7和HepG2中蛋白表达显著上调;沉默LAMA4使TGF-β1和SMAD4表达显著上调,SMAD6、IFN-γ和TNF-α的含量显著降低,过表达LAMA4则结果相反;MMT法和流式细胞技术检测结果显示:敲低LAMA4可使Huh7细胞活力下降,细胞凋亡率增加,而过表达LAMA4则结果相反;Ly364947干扰TGF-β1/SMAD信号传导路径后,Huh7细胞的活力增加,细胞凋亡率显著下降,IFN-γ和TNF-α的含量显著升高,TGF-β1显著降低,且Ly364947可部分逆转沉默LAMA4对肝癌细胞Huh7的免疫逃逸因子和凋亡的调控作用。结论 LAMA4可通过调控TGF-β1/SMAD信号传导途径影响肝癌细胞免疫逃逸和凋亡。更多还原

【Abstract】 This study was performed to investigate the mechanism of LAMA4 affecting immune escape and apoptosis of hepatoma cells by regulating TGF-β1/SMAD signaling pathway. RT-qPCR and Western blot were used to detect the expression of LAMA4 in Huh7 cells, HepG2 cells and Human hepatocytes L02; MTT and flow cytometry assays were performed to analyze the effects of LAMA4 and TGF-β1/SMAD signaling pathway on the cell proliferation and apoptosis in Huh7 cells. RT-qPCR and Western blot were performed to analyze the effect of LAMA4 on TGF-β1/SMAD signaling pathway and cellular immune factors IFN-γ, TNF-α and TGF-β1. Data showed that the expression of LAMA4 was significantly upregulated in Huh7 and HepG2 cells. LAMA4 knockdown significantly increased the expression of TGF-β1 and SMAD4, while decreased the expression of SMAD6, IFN-γand TNF-α, which could be reversed by overexpression of LAMA4. MMT and flow cytometry showed that LAMA4 knockdown inhibited proliferation and induced apoptosis in Huh7 cells, while LAMA4 overexpression demonstrated the opposite effect. Ly364947 increased the expression of IFN-γ and TNF-α, decreased the expression of TGF-β1, promoted proliferation and blocked apoptosis by interfering the TGF-β1/SMAD signaling pathway in Huh7 cells. Ly364947 could partially reverse the effect of LAMA4 silence on cell immune escape factors and cell apoptosis in Huh7 cells. In conclusion, LAMA4 is involved in the immune escape and apoptosis of hepatoma carcinoma cell by regulating the TGF-β1/SMAD signaling pathway.更多还原