Inhibition Of JAK-STAT3 Signal Pathway Of PPeOp Polypeptide From Omphalia Lapidescens In Ganstric Cancer Cells

Objective:To observe the effect of pPeOp polypeptide on cell proliferation,morphology,cell migration in MC-4 and SGC-7901 cells,analys different expression between treamtnet with and without pPeOp polypeptide in human gastric cancer cells MC-4 and SGC-7901 on cell migration,and apoptosis,reaearch relationship between pPeOp polypeptide and JAK-STAT3 signal pathway,and investigate the anticancer molecular mechanism of pPeOp polypeptide,provide a novel therapeutic agent for gastric cancer.Method:MC-4 and SGC-7901 were treated with 0,30,60,90 μg/mL pPeOp polypeptide,and 90 μg/mL PVP was used as the negative control,obtained its inhibition cell proliferation ability by MTS assay.Wound-healing method analysed different effect of the inhibition of cell migration after pPeOp polypeptide treated 24h on MC-4 and SGC-7901 cells.Using flow cytometry to detecte the effect of different concentrations of pPeOp on apoptosis.Western bloting analysed different effect on MMP2,MMP9,Bcl-2,P53,Caspase-3,JAK1,JAK2,STAT3,P-STAT3 protein expression.qRT-PCR analyzed the mRNA level on MMP2,MMP9,Bcl-2,P53,Caspase-3,JAK1,JAK2,STAT3.Using right concentration of agonists and inhibitors solely or combin with pPeOp polypeptide 24h,extracted total protein and RNA,Wsetern blot analyzed STAT3、p-STAT3 protein expression.Result:(1)MTS array showed that pPeOp polypeptide could inhibit the proliferation of MC-4 and SGC-7901 in a dose-and time-dependent.After 24h treated,it had a significant difference.(2)Combine with wound heating,Western blot and qRT-PCR to analyse the effect on cell migration,the result shown that the pPeOp could inhibit the migration ability of MC-4 and SGC-7901.And pPeOp could significantly inhibit the protein and mRNA expression level of MMP2 and MMP9.(3)Combine with flow cytometry,Western blot and qRT-PCR to analyse the effect on cell apoptosis.The result shown that the pPeOp could significantly iuduced cell to apoptosis.Except that,pPeOp could significantly inhibit the protein and mRNA expression level of Bcl-2,and significantly inhance the protein and mRNA expression level of P53 and Caspase-3.(4)Combine with MTS,Western blot and qRT-PCR to screen the optimal condition for IL-6 and NSC-74859.The results shown that 100ng/ml IL-6 significantly increased the expression of STAT3 and p-STAT3 in MC-4 and SGC-7901.50ng/ml NSC-74859 significantly decreased the expression of STAT3 and p-STAT3 in MC-4,100ng/ml NSC-74859 significantly decreased the expression of STAT3 and p-STAT3 in MC-4.(4)Combine with Western blot and qRT-PCR to analyse the effect on JAK-STAT3 signal pathway,the result shown that the STAT3 and p-STAT3 protein level on MC-4 and SGC-7901 cell treated combine with IL-6 and pPeOp was higher than which treated only pPeOp,but lower than.And the STAT3 and p-STAT3 protein level on MC-4 and SGC-7901 cell treated combine with NSC-74859 and pPeOp was lower than Ctrl and which treated only pPeOp.Conclusion:pPeOp polypeptide could significantly inhibit the proliferation of human gastric cancer MC-4 and SGC-7901 cell,down-regulation the expression of migration related factors MMP2 and MMP9,apoptosis related factor Bcl-2,and up-regulation apoptosis related factors P53,Caspase-3.pPeOp polypeptide could restrain JAK-STAT3 signal pathway,down-regulation the expression of JAK1,JAK2,STAT3,p-STAT3,up-regulation SOCS1 and SOCS3.This may imply that pPeOp could enhance SOCS1 and SOCS3 protein expression to inhibit JAKs-STAT3 pathway key nodes level finally promote the cell activity decreases.