Silencing ATAD2 Gene Enhances Sensitivity Of Human Hepatocellular Carcinoma Cells To 5-Fluorouracil In Vitro Study

Objective: Hepatocellular carcinoma(HCC)is a common clinical malignant tumor with high malignant degree and low cure rate,ranking fifth in the incidence of malignant tumors in the world and third in fatality rate.The current treatment of liver cancer,including a variety of means,such as surgery,radiotherapy,chemotherapy,molecular targeted therapy,hepatic artery chemoembolization and TCM.Chemotherapy is still an important means of treatment in advanced liver cancer.Among them,5-fluorouracil(5-FU)is the main drug for advanced liver cancer chemotherapy,however,the clinical efficacy is still not optimistic.The main reason for the failure of treatment is the development of acquired drug resistance in liver cancer.Therefore,in-depth study of 5-FU in liver cancer cell resistance mechanism has important clinical significance.In recent years,with the development of molecular biology theory and gene recombination technology,there are many researches on drug resistance genes of hepatocellular carcinoma.Chemotherapy combined with gene therapy has shown broad application prospects.Related studies have reported ATAD2 and 5-FU chemotherapy is closely related to the effect of ATAD2 is a tumor closely related proto-oncogene,located on chromosome 8q24.13 its expression product ATAD2,also known as ANCCA,in poorly differentiated tissue or malignant tumor high expression in tissues,but low or no expression in differentiated mature tissues.This study knocked out the ATAD2 by using crispr/cas9 knockout technology to observe the change of sensitivity of hepatoma cells to 5-FU and to explore its mechanism.Research methods:In this study,Huh7 cells and HCCLM3 cells isolated from hepatocellular carcinoma were used as research objects.The ATAD2 knockout was performed by using crispr/cas9 knockout technology to establish the cell lines of Huh7-sh-ATAD2 and HCCLM3-sh-ATAD2.The ATAD2 expression in Huh7-sh-ATAD2 cells and HCCLM3-sh-ATAD2 cells was detected by Western blot.The CCK-8 assay was used to detect the differences in the proliferation of Huh7-sh-ATAD2,HCCLM3-sh-ATAD2 and negative control cells at different concentrations of 5-FU for 48 h and 72 h,and then draw the corresponding curves.Cellcycle and apoptosis were detected by flow cytometry after 5-FU treatment.The expression of GLi1 in 5-FU-treated and untreated cancer cells was detected by Western blot.Result:1.Western blot analysis showed that: ATAD2 knockdown HepG2 cells and HCCLM3 cells,ATAD2 is not expressed,ATAD2 silencing stable cell line was successfully established.2.CCK-8 proliferation experiments show that:1)The inhibitory rate of 5-FU on Huh7-sh-ATAD2 cells,HCCLM3-sh-ATAD2 cells,Huh-NC cells and HCCLM3-NC cells increased gradually with the increase of drug concentration and the prolongation of action time(p<0.05);2)Treated with 5-FU,the proliferation inhibition rate of Huh7-sh-ATAD2 cells was significantly higher than that of Huh-NC,HCCLM3-sh-ATAD2 cells and HCCLM3-NC under the same action concentration and action time(p<0.05);3.Annexin V was used to detect apoptosis.The results showed that the apoptotic rates of HCCLM3 and Huh-7 cells were significantly higher than those of the control group after adding 5-FU(p<0.05).Silencing ATAD2 can enhance the cytotoxic effect of5-FU on HCCLM3 and Huh-7 cells;4.PI test,adding 5-FU,the interference group compared with the control group,HCCLM3,Huh-7 cells easily blocked in the S phase;5.Western blot analysis showed that Gli1 expression in knockout group was significantly lower than that in control group after 5-FU treatment.We have the preliminary evidence of ATAD2 knockout,5-FU increased susceptibility may be regu1.Knockdown of ATAD2 increased the sensitivity of helated by the Hedgehog signaling pathway.Conclusion: patoma cells Huh7 and HCCLM3 to 5-FU;2.Knock-out ATAD2,hepatocellular carcinoma cells increased sensitivity to 5-FU mechanism may be mediated by the Hedgehog signaling pathway.