Establishment Of The Cell Line By Knockdowning TAB182 Gene In HepG2 Cells And The Impact Of Chemotherapy Sensitivity On It

Objective:Primary liver cancer(Primary liver cancer,PLC)is a kind of high malignant degree,low cure rate of clinical common malignant tumor.It ranks the sixth in the incidence of malignant tumors and the third in mortality in the world.There are many ways of the treatment of liver cancer,such as surgery,chemotherapy,radiation therapy,hepatic artery embolism chemotherapy,molecular targeted therapy,Chinese medicine treatment,and so on.Due to liver cancer onset conceals,lack of effective methods of early diagnosis,the diagnosis often has reached late or distant metastasis,missed the best time of the operation,so the surgical treatment of liver cancer is limited.The chemical treatment is still important means for the treatment of middle-late stage liver cancer.Although there have been new chemotherapy drugs and new treatment options,the existence of the chemotherapy drug resistance genes not only limits the chemotherapy drug treatment effect,but also is the important cause of the recurrence and metastasis of liver cancer.In recent years,with the maturing of the theory of molecular biology and genetic recombination technology,studies of drug resistance of liver cancer gene emerge in endlessly.Chemotherapy with gene therapy shows a broad application prospect.TAB182,as an acceptor of poly(ADP-ribosyl)ation by tankyrase 1,was originally identified in a two hybid screen with the Tankyrase 1,also named TNKS1BP1,and localizes to the nucleus in a heterochromatic staining pattern and to the cytoplasm,where it co-stains with the cortical actin network.But the function of TAB182 is largely unknown.In large-scale identification of phosphorylated substrates of DNA-PKcs,which is the key regulator of DNA double strand breaks signal response and repair,our lab early found that TAB182 may participate the regulation of DNA damage response induced by ionizing radiation.Further study found TABl82 gene silencing increased cellular radiosensitivity;TAB182 can adjust the double-stranded DNA rupture activation of homologous end connection signal pathway,and participate in the regulation of cell cycle progression.Radiation,and quite a number of anti-cancer drugs can be directly or indirectly acts on the DNA or DNA metabolic processes,which can lead to DNA damage,caused a series of cellular responses,including the repair of damaged DNA,which as a result,improve cell survival.This is also one of the mechanisms of tumor cells to radiation and chemotherapy resistance.If we can inhibite the DNA damage repair,we can also improve the cellular sensitivity to radiation and chemotherapy.Based on this,I assumed that TAB182 gene involved in DNA damage repair,increase the tumor cell radiation sensitivity,so silented TAB182 whether can improve the effect of tumor chemotherapy drugs which caused DNA damage? Therefore,I used liposome as a carrier,transfected HepG2 liver cancer cells,regulated the expression of TAB182 through shRNA technology,selected TAB182 protein stable silence cell,using Cell Counting Kits-8(CCK 8)method researched the effect of chemotherapy drug sensitivity after TAB182 gene was silenced,in order to further explored screening provide evidence for the targeted gene therapy of liver cancer.Methods:1 The shRNA plasmid was stably transfected into HepG2 cells to inhibit the expression of the TAB182 gene by Lipofectamine2000.Through the cultivation of hygromycinB,we established the cell lines of the low expression of the TAB182 gene(HepG2-sh-TAB182).We also set a negative control(HepG2-NC).2 Detecting HepG2-sh-TAB182 cells and HepG2-NC cells TAB182 protein expression by Western blot.3 The CCK-8 test was used to evaluate the cell proliferation inhibition differences of HepG2-sh-TAB182 and HepG2-NC at different concentratons of cisplatin(1,2,4,8 ?g/mL),metformin(5,10,20,40 mmol/L)in different time(24 h,48 h and 72 h)and draw the corresponding curve.4 Statistical software 17.0 was used to analyse the experimental data.The experiment results indicated with mean±deviation.Two independent samples were analysed with t-test.P<0.05 represent the statistical difference.Results:1 Western Blot showed: Transfection of TAB182 shRNA plasmid HepG2 cells did not express TAB182 protein.We had successfully established the cell lines of the low expression of the TAB182 gene.2 CCK-8 proliferation assay demonstrated that:1)Cisplatin,metformin effected on HepG2-sh-TAB182 and HepG2-NC cells,with the increased of drug concentration and acted time,both groups of HepG2 cell proliferation inhibition rate increased gradually.(P<0.05)2)In the same concentration and time under given cisplatin,metformin,HepG2-sh-TAB182 proliferation inhibition rate increased significantly compared with HepG2-NC cells.(P<0.05)Conclusion:1 The study had successfully established the cell lines of the low expression of the TAB182 gene(HepG2-sh-TAB182 cell lines).2 Inhibitting TAB182 gene expression can promote HepG2 cells drug sensitivity to cisplatin,but its mechanism remains to be further research.3 Mefformin can inhibit the growth of HepG2 cells.Inhibitting TAB182 gene expression can promote HepG2 cells drug sensitivity to metformin,but its mechanism remains to be further research.