Connexin32 Affects Epithelial-mesenchymal Transition In Hepatocellular Carcinoma Via PI3K/Akt Signaling Pathway

Objective: 1.To observe the effect of Connexin32 on the epithelial-mesenchymal transition(EMT)of HepG2 and HepG2/DOX cells.2.To investigate the role of PI3K/Akt signaling pathway in Connexin32(Cx32)regulation of EMT in hepatocellular carcinoma cells(HCC).Methods: 1.Immunohistochemistry assay was used to detect the expression levels of Cx32,E-cadherin,Vimentin in HCC tissues and corresponding paracancerous tissues of several HCC patients.2.MTT assay was used to determine the effect of Dox on the proliferation of HepG2 and HepG2/ DOX cells.3.Transwell assay was used to detect the invasion and migration abilities of HepG2 and HepG2/DOX cells.4.Western blot assay was used to determine the expression levels of Cx32,E-cadherin,Vimentin,Akt and p-Akt in HepG2 and HepG2/DOX cells.5.The pcDNA/Cx32 and Cx32-shRNA plasmid was constructed and was respectively transfected into HepG2/DOX and HepG2 cells via Lipofectamine2000 mediation,G418 was used to screen stable cell lines,the expression levels of Cx32,E-cadherin,Vimentin,Akt and p-Akt in HepG2 and HepG2/DOX cells was determined with Western blot assay,the invasion and migration abilities of HepG2 and HepG2/DOX cells was detected by Transwell assay.6.After blocked the PI3K/Akt signaling pathway used by LY294002 in HepG2/DOX cells,the expression levels of E-cadherin,Vimentin,Akt and p-Akt was determined with Western blot assay,the invasion and migration abilities of HepG2/DOX cells was detected by Transwell assay.7.The PI3K/Akt signaling pathway was blocked by LY294002 in HepG2 cells and Cx32-shRNA plasmid was transfected into HepG2 cells via Lipofectamine2000 mediation,the expression levels of E-cadherin,Vimentin was determined with Western blot assay,the invasion and migration abilities of HepG2 cells was detected by Transwell assay.Results: 1.The expression level of EMT marker proteins are related to the expression of Cx32 in HCC The results of immunohistochemistry showed that the expressions of Cx32 and E-cadherin in HCC specimens were significantly lower than those in paracancerous tissues;The expression of Vimentin in HCC specimens was significantly higher than that in paracancerous tissues,Spearman correlation analysis showed that there was a positive correlation between the expression of Cx32 and the expression of E-cadherin in HCC,there was a was negative correlation between the expression of Vimentin and the expression of Cx32 in HCC.2.HepG2/DOX cells acquire drug resistance to DOX MTT assay show that the IC50 value of HepG2 cells was 0.182?g/m L,the IC50 value of HepG2/DOX cells was 2.113?g/m L,HepG2/DOX cells line showed a 11.61-fold higher resistance to DOX than the HepG2 cells line.These results demonstrated that a doxorubicin resistance HepG2 cells lines was established.3.The expression level of Cx32 decreased in HepG2/DOX cells Western blot assay showed that compared with HepG2 cells,the expression level of Cx32 in HepG2/DOX cells was significantly decreased.4.HepG2/DOX cells undergone significantly EMT Western blot assay showed that compared with the HepG2 cells,the expression level of E-cadherin was significantly decreased and the expression level of Vimentin was significantly increased in HepG2/DOX cells;Transwell assay showed that compared with HepG2 cells,the abilities of invasion and metastasis of HepG2/DOX cells was significantly increased.These results indicated that the HepG2/DOX cells undergone significantly EMT.5.Cx32 affects EMT in HCC After slienced Cx32 in HepG2 cells,Western blot assay showed that the expression level of E-cadherin was significantly decreased and the expression level of Vimentin was significantly increased,Transwell assay showed that the abilities of invasion and migration of HepG2 cells was significantly enhanced.After overexpressed Cx32 in HepG2/DOX cells,Western blot assay showed that the expression level of E-cadherin was significantly increased and the expression level of Vimentin was significantly decreased,Transwell assay showed that the abilities of invasion and migration of HepG2/DOX cells was significantly decreased.These results indicated that Cx32 can affect EMT of HCC.6.Cx32 regulates the PI3K/Akt signaling pathway in HCC After slienced Cx32 in HepG2 cells,Western blot assay showed that the expression of p-Akt was significantly increased,indicated that the PI3K/Akt signaling pathway was activated;After overexpressed Cx32 in HepG2/DOX cells,Western blot assay showed that the expression of p-Akt was significantly decreased,indicated that the PI3K/Akt signaling pathway was inhibited;These results demonstrated that Cx32 affect PI3K/Akt signaling pathway in HCC.7.Blocking the PI3K/Akt signaling pathway reversed the occurrence of EMT in HepG2/DOX cells Western blot assay showed that compared with HepG2 cells,the expression of p-Akt in HepG2/DOX cells was significantly increased.After used LY294002 to block PI3K/Akt signaling pathway in HepG2/DOX cells,Western blot assay showed that compared with control group,the expression of E-cadherin was increased,the expression levels of p-Akt and Vimentin was reduced in LY294002 group;Transwell assay showed that compared with control group,the abilities of invasion and migration of HepG2/DOX cells was significantly decreased in LY294002 group.These results demonstrated that the PI3K/Akt signaling pathway was activated after HepG2/DOX cells occurred EMT,blocking the PI3K/Akt signaling pathway can reverse EMT in HepG2/DOX cells.8.LY294002 blocks the effect of shRNA-Cx32 on EMT in HepG2 cells After slienced Cx32 in HepG2 cells,Western blot assay showed that the expression level of E-cadherin was significantly decreased and the expression level of Vimentin was significantly increased,Transwell assay showed that the abilities of invasion and migration of HepG2 cells was significantly enhanced.After used LY294002 to block PI3K/Akt signaling pathway and slienced Cx32 by transfecting Cx32-shRNA in HepG2 cells at the same time,compared with negative control group,Western blot assay showed that the expression levels of E-cadherin and Vimentin did not change obviously in shRNA-Cx32+LY294002 group;Transwell assay showed that compared with negative control group,the abilities of invasion and migration of HepG2 cells did not change obviously in shRNA-Cx32+LY294002 group.These results showed that LY294002 could abolish the occurrence of EMT caused by transfecting Cx32-shRNA into HepG2 cells,suggested that PI3K/Akt signaling pathway mediated Cx32 regulation of EMT in HCC.Conclusion: 1.Cx32 affects the occurrence of EMT in HCC.2.Cx32 affects EMT in HCC via the PI3K/Akt signaling pathway.