Inhibition Of The Hepatocarcinoma Cells? Proliferation By Alpha-1,3-fucosyltransferase-? SiRNA Through Decreasing The Expression Of SLe~X

Objective:To observe the expression of FUT7 and SLe~x in hepatocarcinoma tissues and discuss the correlation with clinical pathological characteristics,to observe the effect of FUT7 siRNA on SLe~x expression and hepatocarcinoma cell proliferation and discuss the mechanisms.Methods:1.Fifty-six primary hepatocarcinoma and paired para-cancer tissues were collected in operation from Jan.2017 to Jan.2018 in China medical university the first affiliated hospital and the first affiliated hospital of Jinzhou medical university.The patient clinical and pathological datas were collected,including age,blood vessels invasion,HbsAg,AFP,Sex,Liver scirosis,TNM grade,tumor diameter and differentiation degree.IHC,qRT-PCR and Western Blot were used to detect the expression of FUT7 and SLe~x at tissue protein and mRNA,to observe the correlation with clinical pathological characteristics.Pearson Rank analyzed the correlation between the FUT7 and SLe~xexpression.2.SLe~xantigen expression was detected by cytometry assay in human hepatocarcinoma cells(QGY7703 and MHCC97)and normal live cells(THLE-2)surface.3.FUT7 in QGY7703,MHCC97 and THLE-2 cell on protein and mRNA level was detected by qRT-PCR and Western Blot.4.The SLe~Xantigen on MHCC97 cells was inhibited by antibodies,MTT assay was used to detect the cell survival.5.Specical siRNA was used to knockdown FUT7 expression in QGY7703 and MHCC97 cells.Transfection efficiecy was detected by qRT-PCR and Western Blot.Post-transfection cell proliferation was detected by MTT assay,cell cycle was detected by cytometry assay,SLe~x antigen was detected by cytometry assay.6.In MHCC97 cells,PLC?inhibitor U73122,PKA inhibitor KT5720,ERK inhibitor ErkI,PI3K inhibitor LY294002 and CDC25 inhibitor CDC25I were used to inhibit PKA,PI3K/AKT and PLC?/ERK signaling pathway,FUT7 expression and cell proliferation were ovserved.Results:1.IHC showed that SLe~x and FUT7 expression in hepatocarcinoma tissues was higher than in para-cancer tissues(P<0.001);Western Blot and qRT-PCR showed that SLe~x and FUT7 protein and mRNA expression in hepatocarcinoma tissues was higher than in para-cancer tissues(P<0.001).2.SLe~x and FUT7 were higher in molti tumor,blood vessel invasion positive,TNM grade(?and?)(P<0.05).3.Pearson rank analysis showed that SLe~xand FUT7 protein expression were positively correlated(r=0.752,95%CI:0.609~0.847,P<0.0001).4.Flow cytometry showed that compared with THLE-2 group,SLe~x expression was higher in MHCC97 and QGY7703 cells(P<0.001).5.QRT-PCR and Western blot showed FUT7 mRNA and protein in MHCC97 and QGY7703 cells were higher than in THLE-2 cells(P<0.001).6.Different monoclone antibody was used to inhibit MHCC97 and THLE-2 cell surface SLe antigen,MTT assay was used to detect prolifeatrion.Compared with PBS group,anti-SLe~Xmonoclone antibody KM93 was the strongest one to inhibit MHCC97 cell proliferation(P<0.05),and not inhibited the THLE-2 proliferation(P>0.05).7.QGY7703 and MHCC97 cells were transfected with siRNAs to down-regulate FUT7 expression,Western blot and qRT-PCR were used to detect the transfection efficiency,FUT7 protein and mRNA was decreased obviously in siRNA1 and siRNA2 in QGY7703 and MHCC97 cells(P<0.001).8.MTT assay showed that in siRNA-1 group and siRNA-2 group,cell survival rate was decreased(P<0.001).Flow cytometery assay showed that in siRNA-1 group and siRNA-2 group,more cells stayed in S phase(P<0.05),SLe~x was decreased(P<0.05).9.To discuss the mechanism of FUT7 on hepatocarcinoma,several signal pathways protein inhibitor were given to observed FUT7 mRNA expression.QRT-PCR showed in MHCC97 cells,compared with control group,PLC?inhibitor,PKA inhibitor,PI3K inhibitor could induce FUT7 mRNA decreasing(P<0.01,P<0.05,P<0.01).MTT assay showed that in THLE-2 cells,PLC?and Erk inhibitors could decrease cell proliferation(P<0.05;P<0.01).In MHCCC97 cells,CDC25,PI3K,PLC?and Erk inhibitor could inhibitor cell prolifetation(P<0.05;P<0.05;P<0.01;P<0.01).PLC?inhibitor showed the most powerful inhibitor effect.Western Blot was used to detect PLC?1 and p-PLC?1,post-siFUT7 transfection,cytoplasm PLC?1 was decreased,cell membrane p-PLC?was decreased.Down-regulating FUT7 expression might inhibit PLC?translocation and phosphylation and inhibit PLC?activating,FUT7 modulated QGY7703 and MHCC97 cell proliferation via PLC?/Erk signal pathway.Conclusion:FUT7 and SLe~X are high expression in hepatocarcinoma tissue,it may be related to tumor proliferation,metastasis and recurrence.FUT siRNA modulates hepatocarcinoma proliferation by modulating SLe ~X expression.