Cx32调控肝癌细胞上皮间质转化的机制

Connexin32 regulates epithelial-mesenchymal transition by PI3K/Akt signaling pathway in hepatocellular carcinoma cells

目的探讨缝隙连接蛋白32(Cx32)在肝癌细胞上皮间质转化(epithelial-mesenchymal transition,EMT)中的作用及其机制。方法在Hep G2细胞(对阿霉素敏感)中干扰Cx32的表达,在Hep G2/doxorubicin(DOX)细胞(对阿霉素耐药)中过表达Cx32,Western blot测定Cx32、p-Akt的表达水平。20μmol/L的LY294002(PI3K/Akt信号通路的抑制剂)作用于Hep G2/DOX细胞24 h,Western blot检测p-Akt、E-cadherin、vimentin的表达水平,Transwell实验检测细胞的侵袭、迁移能力。在干扰Cx32的Hep G2细胞中,用20μmol/L的LY294002作用于细胞24 h,Western blot检测E-cadherin、vimentin的表达水平,Transwell实验检测细胞的侵袭、迁移能力。结果 Western blot结果显示,在Hep G2细胞中干扰Cx32的表达后,p-Akt的表达升高(P<0.05);在Hep G2/DOX细胞中过表达Cx32后,p-Akt的表达降低(P<0.05)。20μmol/L的LY294002作用于Hep G2/DOX细胞24 h后,p-Akt、vimentin的表达降低,E-cadherin的表达升高(P<0.05),且细胞的侵袭、迁移能力降低(P<0.05)。在Hep G2细胞中干扰Cx32的表达后,细胞中E-cadherin的表达降低,vimentin的表达升高(P<0.05),且细胞的侵袭、迁移能力显著增加(P<0.05);而在干扰Cx32的Hep G2细胞中,用20μmol/L的LY294002作用于细胞24 h后,细胞中E-cadherin、vimentin的表达均没有显著变化(P>0.05),细胞的侵袭、迁移能力也无显著改变(P>0.05)。结论 Cx32可调控肝癌细胞中PI3K/Akt信号通路的活性,LY294002可逆转Hep G2/DOX细胞的EMT,Cx32可能是通过PI3K/Akt信号通路调控肝癌细胞EMT。

Objective To investigate the mechanisms of Connexin32(Cx32)affecting epithelial-mesenchy mal transition(EMT)in hepatocellular carcinoma cells.Methods Cx32 was interfered in HepG2 cells[sensitive to doxorubicin(DOX)]and Cx32 was overexpressed in HepG2/DOX cells(resistant to DOX).Western blot was used to detect the expression levels of Cx32 and p-Akt.The 20μmol/L LY294002(the inhibitor of PI3K/Akt signaling pathway)was given HepG2/DOX cells for 24 h,and then Western blot was used to detect the expression levels of p-Akt,E-cadherin and vimentin,and Transwell assays were used to assess the abilities of invasion and migration.The 20μmol/L LY294002 was given HepG2 cells for 24 h,in which Cx32 was interfered,and then Western blot was used to assess the expression levels of E-cadherin and vimentin,and Transwell assays were used to assess the abilities of invasion and migration.Results Western blot results showed that the expression of p-Akt was enhanced after interference of Cx32 in HepG2 cells(P<0.05),while the expression of p-Akt was decreased after overexpression of Cx32 in HepG2/DOX cells(P<0.05).After HepG2/DOX cells were treated with 20μmol/L LY294002 for 24 h,the expression of p-Akt and vimentin was decreased(P<0.05),E-cadherin was enhanced(P<0.05),and the invasive and migratory abilities of cells were decreased(P<0.05).After interference of Cx32 in HepG2 cells,the expression of E-cadherin was decreased(P<0.05),the expression of vimentin was enhanced(P<0.05),and the invasive and migratory abilities of cells were enhanced(P<0.05).After HepG2 cells were treated with 20μmol/L LY294002 for 24 h,in which Cx32 was interfered,the expression of E-cadherin and vimentin showed no obvious change(P>0.05),and the invasive and migratory abilities also had no change(P>0.05).Conclusion Cx32 can regulate the activity of PI3K/Akt signaling pathway in hepatocellular carcinoma cells,and LY294002 can reverse EMT in HepG2/DOX cells.Cx32 may mediate EMT by affecting PI3K/Akt signaling pathway in hepatocellular carcinoma cells.