Role Of Pannexin1 Channel On The Migration And Invasion Of Testicular Cells And Possible Mechanism

Objective: 1.To assess the pannexin1 protein expression in testicular cell.2.To clarify the effect of pannexin1 on invasion and migration of testicular cancer cells and its possible mechanism.Methods: 1.Western blotting was used to detect the expression of pannexin1 protein in TM3 cells and I-10 cells.2.Carbenoxolone(CBX)and probenecid(PBN)were used to inhibit pannexin1 channel in testicular cancer I-10 cells,the function of pannexin1 channel was detected by real-time fluorescence transfer and extracellular ATP assay.The invasion and migration ability of testicular cancer I-10 cells were detected by transwell assay and wound-healing assay.The expression of invasion and migration related-proteins were detected by western blotting.3.Molecular biological silencing(sh RNA)and overexpression(m Panx-1)of pannexin1,real-time fluorescence transfer and extracellular ATP assay were used to detect pannexin1 channel function,transwell assay and wound-healing assay were used to detect the invasion and migration ability of testicular cancer I-10 cells,the invasion and migration related proteins were detected by western blotting.4.U0126(p-ERK inhibitor)was used to inhibit p-ERK in stably transfected cell lines(sh RNA and m Panx-1),transwell assay and wound-healing assay were used to detect the invasion and migration ability of gene silencing and overexpression stable cell lines and western blotting was used to detect invasion and migration-related protein expression.Results: 1.Compared with Leydig cells TM3,the expression of pannexin1 protein in testicular cancer I-10 cells increased significantly.Western blotting showed that the expression of pannexin1 in testicular cancer cells I-10 was significantly higher than that in Leydig cells TM3(P < 0.05).2.Pannexin1 channel functions decreased by CBX and PBN.Compared with control group,Real-time fluorescence transfer experiments showed that the fluorescence transduction ability of cells was significantly decreased after inhibition of pannexin1 channel by CBX and PBN(P < 0.05).The ATP detection kit found that CBX and PBN inhibited pannexin1 channel compared with Control group(P < 0.05).The extracellular ATP decreased significantly(P < 0.05).3.Pannexin1 channel inhibited by CBX and PBN,testicular cancer cell I-10 invasion and migration ability decreased.Compared with the control group,transwell and wound-healing assay showed that the CBX and PBN groups had reduced migration ability(P < 0.05);the invasive ability was reduced.4.Silencing Panx-1 can specifically inhibit the invasion and migration of testicular cancer.Real-time fluorescence transfer experiments showed that pannexin1 channel function was weakened in testicular cancer I-10 cells after silencing of Panx-1(P < 0.05);ATP detection reagents showed that extracellular ATP levels were decreased(P < 0.05);transwell and wound-healing assays revealed silencing of Panx-1,testicular cancer I-10 cell invasion and migration ability decreased.;western blotting revealed expression of MMP-9,vimentin and increased expression of E-cadherin(P < 0.05).5.Overexpression of Panx-1 enhances the invasion and migration of testicular cancer cells I-10.Real-time fluorescence transfer experiments showed that pannexin1 channel function became stronger in testicular cancer I-10 cells after overexpression of Panx-1(P < 0.05);ATP detection reagents showed enhanced extracellular ATP levels(P < 0.05);transwell and wound-healing assays found silencing of Panx-1,enhanced invasion and migration ability of testicular cancer cells I-10(P < 0.05);western blotting revealed that the expression of expression of MMP-9,vimentin and increased expression of E-cadherin(P < 0.05).6.In the overexpressed Panx-1 cell line,m Panx-1 enhanced the invasion and migration ability of testicular cancer cells,and U0126(p-ERK inhibitor)significantly reduced the migration and invasion ability of m Panx-1treament cells.Transwell and wound-healing assay showed that U0126+m Panx-1 significantly inhibited invasion and migration of testicular cancer cells in the overexpressed Panx-1 gene cell line compared with NC group and m Panx-1 group(P < 0.05);western blotting found that U0126 +m Panx-1 significantly inhibited the expression of MMP-9,vimentin and increased expression of E-cadherin(P < 0.05).Conclusion: 1.The expression of Pannexin1 channel proteins are increased in testicular cancer I-10 cells.2.Pannexin1 channel promotes the invasion and migration of testicular cancer I-10 cells via enchance expression of p-ERK1/2 protein.