| Purpose: Connexin(Cx)and its composed gap junction(GJ)with expression and functional abnormalities is closely related to the occurrence and development of tumor.Generally speaking,Cx/GJ is not only one of the tumor-inhibiting factor,but also a potential target for cancer therapy.But the expression and function of Cx/GJ in hepatocellular carcinoma are still unclear.There is considierable controversy on the finding of different research..In our study,we studied thoroughly the expression of Cx32,Cx43 and Cx26 in human hepatocellular carcinoma,which were mainly expressed in human liver cells..we futher investigated the relationship between the expression of the above-mentioned Cx and Clinical progression of hepatocellular carcinoma.Our study aimed to investigate the pattern of expression and distribution of Cx/GJ in different development stages of HCC.To illuminate the effect and mechanism of Cx/GJ in the occurrence and development of HCC.To provide critical evidence for Cx/GJ as a new molecular target for the prevention and treatment of HCC.Methods 1.The fresh tissue and paraffin embedded tissue samples from 97 patients with hepatocellular carcinoma(HCC)were collected,including HCC tissues,adjacent liver tissue and remote normal liver tissue.All cases had complete clinical information and follow-up data.2.Cx32,Cx43,Cx26 protein expression and localization was detected via immunohistochemical staining in 96 cases of paraffin embedding primary hepatocellular carcinoma specimens;3 RT-q PCR and Western Blot used to detect Cx32,Cx43,Cx26 mRNA and protein expression level on fresh specimens of primary hepatocellular carcinoma,including HCC tissues,adjacent live tissue and remote normal liver tissue respectively;4.Chi-Square test and Pearson contigency coefficeent were used to determine relationship between clinicopathological features and Cx32 expression.(such as TNM stage,tumordifferentiation,tumor size et al);5.Kaplan-Meier method,log-ank test and Multivariable Cox regression model were applied to analysis the relationship between abrrent expression of Cx32 with HCC prognosis.6.Detection of EGFR protein and its signal pathway downstream mTOR,ERk and Stat3 protein expression in hepatocellular carcinoma tissue by western blot and immunohistochemistry method,Analysis of the correlation between the expression of EGFR and Cx32.Results: 1.The expression of Cx32,Cx43 and Cx26 in HCC tissues,adjacent live tissue and remote normal liver tissue were detected by immunohistochemistry staining.Cx32 protein expression in HCC is also higher than adjacent live tissue and remote normal liver tissue,mainly located in the cytoplasm and accumulated in perinuclear of HCC cells.But Cx32 protein expression in adjacent live tissue and remote remote normal liver tissue mainly located in cell membrane.Cx26 protein expression mainly located in the cytoplasm and cell membrane;There was no significant difference between HCC tissue,adjacent live tissue and remote normal liver tissue.Cx43 protein expression in HCC tissue is also lower or even negative than adjacent live tissue and remote normal liver tissue;but Cx43 protein expression in adjacent live tissue and remote remote normal liver tissue mainly located in cell membrane.2.The expression levels of Cx protein and mRNA were detected by western blot and RT-qPCR in 96 cases of fresh HCC tissues,adjacent liver tissues and normal liver tissues.(1)the protein expression level of Cx32 in HCC tissue,adjacent live tissue and remote normal liver tissue were 4.6014 ± 0.2388,1.1433 ± 0.1006 and 1.0583 ±0.0693,respectively.Compared with adjacent live tissue and remote normal liver tissue,the protein expression of Cx32 in HCC tissue was significantly higher(P<0.05).the expression level of Cx32 mRNA in HCC tissue,adjacent live tissue and remote remote normal liver tissue were respectively 0.8356±0.1255,0.7754±0.0931 and 0.7779±0.1573,the expression of Cx32 mRNA was no statistical significance among HCC tissue?adjacent live tissue and remote normal liver tissue(P>0.05).(2).The protein expression of Cx43 in HCC tissue,adjacent live tissue and remote normal liver tissue were 0.6303±0.0714,1.3152±0.1456 and 1.5458±0.1792,respectively.Compared with adjacent live tissue and remote normal liver tissue,the protein expression of Cx43 in HCC tissue was significantly depressed(P<0.05).the expression level of Cx43 mRNA in HCC tissue,adjacent live tissue and remote normal liver tissue were respectively1.0772±0.1216,1.1693±0.1317 and 1.0887±0.1260,the expression of Cx43 mRNA was no statistical significance among HCC tissue?adjacent live tissue and remote normal liver tissue(P>0.05).(3)The protein expression of Cx26 in HCC tissue,adjacent livetissue and remote remote normal liver tissue were 1.5203±0.1126,1.4200±0.1212 and1.4200±0.1212,respectively.The protein expression of Cx26 no significant difference among HCC tissue,adjacent live tissue and remote normal liver tissue.the expression level of of Cx26 mRNA in HCC tissue,adjacent live tissue and remote normal liver tissue were respectively 0.6325±0.0873,0.6983±0.0731 and 0.6884±0.0584,the expression of Cx26 mRNA was no statistical significance among HCC tissue?adjacent live tissue and remote normal liver tissue(P>0.05).3.Clinical significance of Cx32 aberrant expression in primary HCC.Cx32 expression is closely related to the clinical-pathologic features of patients with primary HCC: pathological differentiation(P<0.001,r=0.488),the clinical TNM stage(P<0.001,r=0.373).4.The patients with high expression of Cx32 of hepatocellular carcinoma predicts poor prognosis,overall survival time and recurrence free survival time shorter than those patients with low Cx32 expression.Multiple variables Cox regression analysis showed that ‘Cx32 expression'and ‘pathological differentiation' in patients with hepatocellular carcinoma are independent prognostic factors for postoperative overall survival.5.By western blot method,the protein expression of Cx32 was correlated positively with the protein expressions of EGFR and its signal transduction pathway downstream chiefly protein of mTOR.Conclusion: 1.The expression of Cx32 protein in HCC tissue was increased,which was mainly distributed in the cytoplasm,and the distribution of the membrane decreased.Cx43 protein expression in HCC is decreased which was mainly distributed in cell membrane.Cx26 protein expression in HCC is no obvious changes.The gap junction(GJ)decrease or disappear in HCC tissue.2.The expressions of Cx32 was correlated positively with pathological differentiation,clinical TNM stage of HCC.3)Cx32cytoplasmic aberrant expression indicated the poor prognosis of patients with HCC.cytoplasmic expression of Cx32 could be as one of the independent predictors in postoperative HCC.4)The aberrant expression of Cx32 in hepatocellular carcinoma,which increases and accumulates in cytoplasm,might activate EFGR signaling pathway by increasing the expression of EGFR,and then play a role of promoting the proliferation and anti apoptosis of cancer cells. |
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