Down-regulated Connexin32 Promotes Tumor Invasion And Migration Through Inducing Snail-mediated EMT In Hepatocellular Carcinoma

Objective:The aim of this study was to investigate the expression of connexin32(Cx32)during hepatocarcinogenesis,and further to explore its effect on tumor invasion and migration and possible mechanisms in hepatocellular carcinoma(HCC).The findings will be helpful in seeking new potential anti-metastasis biological targets and providing new strategies and ideas for the treatment of HCC.Methods:The expression and localization of Cx32 were detected by immunohistochemistry in 20 cases of normal liver tissues and 76 cases of HCC.Possible association between Cx32 expression and clinicopathologic parameters of HCC was also analyzed.In virto,human normal hepatic LO2 cell line,and HCC cell lines SMMC-7721,Hep G2 and Huh7 were used.The expression and localization of Cx32 were measured by western blot and immunofluorescence assay,respectively.The invasive capabilities of different cell lines were detected by transwell assay.Based on the findings regarding Cx32 expression change and its relationship to HCC invasive ability,we performed loss-and gain-of-function assays in vitro to modulate Cx32 expression and investigated the changes in abilities of HCC migration and invasion,and the epithelial-mesenchymal transition(EMT)-related cell morphologic and molecular markers' changes were also determined in this process.Finally,the correlation between Cx32 and EMT markes was verified in HCC tissues.Results:1.Expression of Cx32 in HCC and its clinical significance Compared with normal liver tissue,the expression of Cx32 in HCC tissue is down-regulated,and the positively expressed protein shows significant ectopic expression from the cell membrane to the cytoplasm.We further found that the expression of Cx32 was not correlated with age,gender,tumor size,TNM stage,background of liver disease,or vascular embolus(all P > 0.05),but negatively correlated with histological grade and lymph node metastasis(all P<0.05).Moreover,in comparison to the normal hepatic cell line LO2,Cx32 expression in the three HCC cell lines was remarkedly decreased.Among the three HCC cell lines,SMMC7721 cell line was identified relatively expressing higher Cx32,while Hep G2 cell line was most deficient in Cx32 expression.Immunofluorescence assay further demonstrated that Cx32 was expressed in membrane and cytoplasm of LO2 cells,while Cx32 mainly located in the cytoplasm in SMMC-7721 cells.Thus,we confirmed that Cx32 was down-regulated as well as ectopically expressed during hepatocarcinogenesis both histologically and cytologically.2.Cx32 negatively regulates HCC migration and invasion in vitro LO2 cells was most abundant in Cx32 expression but did not show significant invasive ability.SMMC-7721 cells had a higher Cx32 expression than that of the other two HCC cell lines Hep G2 and Huh7,but the invasive potential was significantly lower.To investigate whether Cx32 inhibits malignant phenotype of HCC cells,we first established SMMC-7721 cells with silencing of the Cx32 expression by si RNA.On the contrary,Hep G2 cells were transfected with Cx32 c DNA to up-regulate Cx32 expression.After the successful establishment of cell models,we found that the invasive ability of SMMC-7721 cells was significantly enhanced by Cx32 down-regulation,and the invasive ability of Hep G2 cells was significantly decreased by Cx32 up-regulation as evidenced by transwell assay.Similar results were also shown in wound healing assay.These results indicated that HCC migration and invasion in vitro could be negatively regulated by Cx32.3.Cx32 affects EMT and MET process in HCC cells Compared with the negative control group,Cx32 down-regulation in SMMC-7721 cells led to apparent changes in the morphology compatible with EMT,which included elongated,spindle-shaped morphology,pseudopodia formation,and increased cell scattering.In contrast,Hep G2 cells over-expressing Cx32 became more rounded and showed no/decreased filamentous or lamellipodia,and increased intercellular connectivity.With the down-regulation of Cx32,a decreased expression of E-cadherin,and an increased expression of Vimentin were detected in the SMMC-7721 cells.For the Hep G2 cells transfected with Cx32 c DNA,the EMT markers showed opposite changes.Immunofluorescence assay further confirmed the changes of expression of E-cadherin and Vimentin by Cx32.These results suggest that down-regulation of Cx32 in HCC accelerated cell migration and invasion through induced EMT.4.Cx32 negatively regulates snail expression and associates with EMT markers in HCC tissues Cx32 down-regulation in the SMMC-7721 cells led to a significant increase of Snail expression,but not of Slug and Twist-1.Meanwhile,over-expression of Cx32 in Hep G2 cells resulted in a significant decrease of Snail with no alteration in Slug or Twist-1 expression.Immunofluorescence assay further demonstrated that Cx32 negatively regulated Snail expression in both HCC cell lines.In addition,we found that Snail was deficient in normal hepatic LO2 cell line which was abundant in Cx32 expression,and was highly expressed in HCC cell lines(SMMC-7721 cells and Hep G2 cells)that expressed down-regulated Cx32.We further performed IHC of EMT markers' expression in 34 of 76 HCC clinical samples.A significant reduction or loss of E-cadherin expression was detected in 18 cases.Snail stained both cytoplasm and nucleus and was recorded positively in the nucleus in 21 cases.For?-catenin staining,41.18%(14/34)of HCC cases was positive for nuclear accumulation with cytoplasmic staining,while all the other samples showed membrane localization.When further analyzed in comparison with expression ofEMT markers,Cx32 expression showed a strong correlation with expression of the loss or reduction of E-cadherin(r=0.528,P=0.001),nuclear Snail(r=–0.448,P=0.008),and nuclear accumulation of ?-catenin(r=-0.457,P=0.007)5.Snail is vital for the biological effect of Cx32 exerting in HCC To further investigate whether Snail mediates the Cx32-induced EMT,Snail was knocked down in SMMC-7721 cells using si RNA and over-expressed in Hep G2 cells using c DNA.Knockdown of Snail resulted in reversal of the Cx32 inhibition-induced EMT-associated phenotype changes including EMT-like morphology,down-regulation of E-cadherin,up-regulation of vimentin,and an enhanced ability of cell invasion in SMMC-7721 cells.Similarly,over-expression of Snail can counteract the biological effects of up-regulation of Cx32 in Hep G2 cells.These data indicated that Cx32 regulated EMT-associated invasion in HCC cells by affecting expression of Snail.Conclusion:Cx32 was down-regulated as well as ectopically expressed during hepatocarcinogenesis,and the down-regulated Cx32 promotes tumor invasion and metastasis through inducing snail-mediated EMT in hepatocellular carcinoma.