Study On Proliferation And Apoptosis Of Hepatocarcinoma Cells And Effects Of SH2B1 To Akt/mTOR Pathway

Objective: To investigate the inhibitory effect of apatinib and 5-Fu on the proliferation of hepatoma cell lines;SH2B1 is a key enhancer of the proto-oncogene RET.Previous studies have shown that SH2B1 contributes to the malignant progression of tumors,including the digestive system.Such as gastric cancer,respiratory system,such as non-small cell lung cancer.This study is dedicated to exploring new treatments for liver cancer,and exploring new ways to treat liver cancer in the future,providing patients with more lines and reliable treatments,and exploring small and effective treatments for patients with liver cancer.Methods: 1.Culture of liver cell line: normal liver cell line QSG-7701,liver cancer cell line MHCC-97,liver cancer cell line HepG2;2.Apatinib and 5-Fu single drug and combined use for liver cancer cell line MHCC-97 H The IC50 and cell proliferation inhibition rate after 48 hours of CCK8 detection and treatment were calculated.3.The cell proliferation was detected after 48 hours of CCK8 treatment,and the cell proliferation inhibition rate was calculated.CompuSyn software calculated the combined effect of the two drugs;4.Recombination slow The virus was transfected into HepG2 cells to obtain stable transfected cell lines;5.The relative expression of SH2B1 mRNA was detected by qRT-PCR;6.The protein expression was detected by Western blot;7.The cell cycle was detected by flow cytometry;8.CCK-8 The method was used to detect cell proliferation ability;9.Double staining flow cytometry was used to detect apoptosis ability;10.SPSS 17.0 was used to analyze the data.Results: 1.Apatinib single drug and combined 5-Fu drug can inhibit the proliferation of MHCC-97 cells in a dose-dependent manner;2.Chou-Talalay median effect method to calculate the combined effect of the two drugs showed slight synergy between the two drugs 3.Compared with normal hepatocytes,the relative expression of SH2B1 mRNA and SH2B1 protein in hepatocarcinoma cells was significantly increased(P>0.05).4.The expression of SH2B1 in HepG2 cells after lentivirus transfection,blank group and NC shRNA group There was no significant difference in the expression of SH2B1 mRNA and SH2B1 protein(P>0.05).Compared with the blank group and NC shRNA group,the expression of SH2B1 mRNA and SH2B1 protein in SH2B1 shRNA group was significantly decreased(P>0.05).5.Silencing SH2B1 expression The effect of HepG2 cell proliferation,CCK8 test showed that there was no significant difference in OD value between the NC shRNA group and the blank group at each time point(P>0.05).Compared with the NC shRNA group and the blank group,the SH2B1 shRNA group was transfected.After 48 h,the OD value decreased significantly(P>0.05).6.The effect of silencing SH2B1 expression on HepG2 cell cycle;compared with the blank group,the proportion of cells in the NC shRNA group was not significant(P>0.05);Group compared to blank group,SH2B1 The proportion of cells in G0/G1 phase of shRNA group decreased,the change of S phase was not significant,and the proportion of cells in G2/M phase increased significantly(P<0.05).7.The effect of silencing SH2B1 expression on apoptosis of HepG2 cells blank group and NC shRNA group There was no significant difference in apoptosis rate between the two groups(P>0.05).Compared with the blank group and the NC shRNA group,the apoptosis rate of SH2B1 shRNA group was significantly increased(P<0.05).8.Silencing SH2B1 expression on HepG2 cells There was no significant difference in the levels of Bax,Bcl-2 and Caspase 3 between the blank group and the NC shRNA group(P>0.05).Compared with the blank group and the NCshRNA group,the SH2B1 shRNA group was compared with the blank group and the NCshRNA group.The levels of Bax and Caspase 3 protein increased significantly(P<0.05),and the content of Bcl-2 protein decreased(P<0.05).The effect of silencing SH2B1 expression on the Akt/mTOR pathway was not significantly different between the blank group and the NCshRNA group(P>0.05).Compared with the blank group and the NCshRNA group,the SH2B1 shRNA group was compared.The levels of p-AKT and mTOR protein were significantly decreased(P<0.05).Conclusion: 1.Apatinib and 5-Fu were used in the MHCC-97 hepatoma cell line,and the single-drug and combination drugs inhibited the proliferation of MHCC-97 hepatoma cell line in a concentration-dependent manner.2.The Chou-Talalay median effect method calculates the combined effect of the two drugs to show that there is a synergistic effect between the two drugs.3.SH2B1 is highly expressed in hepatocellular carcinoma cell line HepG2,and silencing SH2B1 expression may inhibit proliferation and promote apoptosis by inhibiting Akt/mTOR signaling activation.