Effects Of Irradiation And Aspirin On TAB182 Gene Silencing In Hepatocellular Carcinoma HepG2 Cells

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors which threaten the health of our country.In China,the incidence rate of male liver cancer is 15,the female is the highest in the world,and the mortality rate is ranked at the top of the list of second.Liver cancer is characterized by aggressive growth,and the early symptoms are not obvious,the disease progresses rapidly,China is a big country of hepatitis B,50.5% of new patients with liver cancer will appear in China.At present our country in the treatment of liver cancer include surgical tumor resection,radiotherapy,chemotherapy,hepatic artery chemoembolization and local ablation therapy,but the effect is unsatisfactory,thus preventing the occurrence of liver cancer and improve the prognosis of liver cancer has become one of the key.With the development of radiotherapy technology,radiotherapy can kill liver cancer cells,but also cause damage to the surrounding tissue.TAB182 also called TNKS1BP1,is through the yeast hybrid experimental research on Tankyrase 1 was discovered for the first time,a protein involved in DNA repair after injury,and can interact with DNA-Pkcs,can also affect the phosphorylation of DNA-Pkcs and further participate in the DNA damage response,In our laboratory,TAB182 can increase the sensitivity of radiation,and has the function of regulating cell cycle,but there is no in-depth study in the liver cancer cells.Aspirin is antipyretic and analgesic and has a long history,is now widely used in cardiovascular disease,aspirin in the prevention of gastric cancer,esophageal cancer has been confirmed in aspirin inhibits the proliferation of hepatocellular carcinoma cells and promote the apoptosis of hepatoma cells has been studied,but the effect of aspirin for silencing of TAB182 in hepatoma cells.There is no relevant study.We can further inhibit cell proliferation,promote apoptosis of hepatoma cells,providing a new target for gene therapy and drug therapy for liver cancer,improve the prognosis of patients,through the aspirin TAB182 silencing HepG2 cells.Objective: In this experiment,we research hepatoma cell body as object and research the effect gamma ray and aspirin on TAB182 silencing in HepG2 cells through the establishment of HepG2 cell model for screening stable transfection.Methods:The changes of cell proliferation and survival rate of HepG2 cells after gamma ray irradiation were studied by CCK-8 and cell plate cloning,and the changes of HepG2 cell cycle were detected by flow cytometry.CCK-8 assay was used to test the effect of aspirin on the proliferation of TAB182 silenced HepG2 cells.Flow cytometry was used to detect the changes of HepG2 cell cycle and apoptosis in TAB182 cells treated with aspirin.Western and blot were used to detect the expression of apoptosis protein in TAB182 cells after exposure to aspirin and HepG2.Results: 1 Inhibiting the expression of TAB182 gene can incrase the radiosentivity of HepG2 cells.1.1 To verify the successful establishment of TAB182 silencing cell lines: compared with HepG2-NC cells,the expression of TAB182 protein in HepG2-sh-TAB182 cells was reduced,indicating that the TAB182 silencing cell line was successfully established.1.2 Knocking down TAB182 could inhibit the proliferation of HepG2 cells after irradiation: In 0,2,4,6 and 8Gy gamma ray irradiation on HepG2-sh-TAB182 cells and HepG2-NC cells,using CCK-8 method to detect cell proliferation ability,2Gy gamma ray irradiation,HepG2-sh-TAB182 cells did not change significantly with the unirradiated HepG2-sh-TAB182 cells compared with the increment rate,HepG2-sh-TAB182 cell and HepG2-sh-TAB182 cell proliferation rate did not differ significantly(P>0.05),in 4,6 and 8Gy gamma ray irradiation,HepG2-sh-TAB182 cell proliferation rate decreased obviously compared with the unirradiated HepG2-sh-TAB182 cells(P<0.05),there are also obvious differences compared with the same dose rate of HepG2-NC cell proliferation(P<0.05),and HepG2-NC in 8Gy cells after gamma irradiation and unirradiated HepG2-NC cells appeared difference(P<0.05),a simple description of the radiation is not very good to reduce the proliferation rate of HepG2 cells,knocking down TAB182 gene Can reduce the proliferation rate of HepG2 cells after irradiation.1.3 Knocking down TAB182 gene could inhibit the survival rate of HepG2 cells after irradiation: In 2Gy gamma ray irradiation,HepG2-sh-TAB182 cells compared with the unirradiated HepG2-sh-TAB182 cells had obvious difference(P<0.01),HepG2-NC cells and HepG2-NC cells in non irradiated cells,cell survival rate did not decrease(P>0.05),two groups also have obvious difference(P<0.01).In 4,8Gy gamma ray irradiation,compared with their non irradiated HepG2-sh-TAB182 cells and HepG2-NC cells,the survival rate obviously decreased(P<0.01),between the same dose of both survival differences still exist(P<0.05),experiments show that after irradiation the silence of TAB182 gene in the HepG2 cell survival rate decreased,and the within 4Gy under gamma ray irradiation increased sensitivity.1.4 Knocking down TAB182 gene could prolong the G2 / M cycle arrest after irradiation of HepG2 cells: After irradiation of HepG2-sh-TAB182 cells and HepG2-NC cells are G2 / M phase arrest,but in 24 hours after irradiation,HepG2-sh-TAB182 cell cycle arrest in G2 / M phase of the cycle still exists,and the G2 / HepG2-NC cell cycle arrest in M phase has been lifted,that knocking down TAB182 can make the extension of HepG2 cells cell cycle arrest after gamma irradiation,inhibition of cell division,and may lead to cell apoptosis.2 Inhibiting the expression of TAB182 gene can improve the effect of aspirin on HepG2 cells 2.1 Knocking down TAB182 gene can increase the inhibitory effect of aspirin on proliferation of HepG2 cells: HepG2-sh-TAB182 cells and HepG2-NC cells,with the increasing of concentrations of aspirin on cell proliferation,the inhibition rate also increased gradually,at the same concentration,the cells proliferation inhibition rate of HepG2-sh-TAB182 cells is higher than HepG2-NC(P<0.05),indicating that aspirin can inhibit the proliferation of HepG2 cells,and knocking down TAB182 gene can further increase the proliferation of aspirin on the inhibition of HepG2 cells.2.2 Knocking down TAB182 gene can prolong the cycle arrest of G2 / M after irradiation of HepG2 cells: HepG2-sh-TAB182 cells and HepG2-NC cells were treated with 8mmo/l aspirin,we found HepG2-sh-TAB182 cells had G2 / M cycle arrest(P<0.01)in the 4h,and HepG2-NC cells do not have G2 / M phase arrest in 4h,in 12 and 24 h,both had G2 / M cycle arrest(knock down group P=0.00000001<0.01,group P=0.046<0.05),in 36 h,HepG2-sh-TAB182 cell cycle arrest still existed,HepG2-NC cell cycle arrest released,Knocking down TAB182 can prolog G2 / M phase cycle,inhibit cell division.2.3 Knocking down TAB182 could increase the apoptosis of HepG2 cells induced by aspirin: HepG2-sh-TAB182 cells and HepG2-NC cells was treaded with 0,1,8,16mmol/l aspirin for12 h,fter being treaded with 1mmol/l aspirin,HepG2-sh-TAB182 appears apoptosis,with the increase of concentrations of aspirin,cell apoptosis rate also increased,after being treaded with 8,16mmol/l aspirin,HepG2-NCjust appears apoptosis,at the same concentration,the apoptosis rate of HepG2-sh-TAB182 was significantly higher than that of HepG2-NC cells,that aspirin can promote HepG2 cell apoptosis and TAB182 gene silencing can promote the apoptosis of HepG2 cells.2.4 Irradiation and aspirin can promote the expression of HepG2 cell apoptosis protein after TAB182 gene silencing: After treating with the 4Gy gamma ray irradiation and 16mmo/L aspirin to the HepG2-sh-TAB182 cells and HepG2-NC cells,we found that cleaved-Caspase-3 and cleaved-Caspase-9 increased significantly,expression of cleaved-Caspase-3 and cleaved-Caspase-9 in HepG2-sh-TAB182 cells was higher than HepG2-NC cells,indicating TAB182 silencing can promote the expression of apoptosis protein.Conclusions:1 To verify the successful establishment of TAB182 silenced cell lines 2 After silencing TAB182,it can inhibit the cell proliferation rate,survival rate,inhibit cell division and induce cell apoptosis.It was found that TAB182 may be a sensitive gene in the treatment of liver cancer,which may provide a new target for the treatment of liver cancer.