RNF20 Regulates The Malignant Phenotype And DNA Damage Repair In Hepatocellular Carcinoma

Objective: To investigate the effects of ring finger protein 20(RNF20)deficiency on proliferation,invasion,migration and DNA damage repair in hepatocellular carcinoma cells,and its mechanism was explored.Methods: 1.The RNF20 knockdown hepatocellular carcinoma cells lines were established.(1)Three sets of short hairpin RNA targeting RNF20 gene were designed which are RNF20-sh RNA1?RNF20-sh RNA2 and RNF20-sh RNA3.(2)The lentiviral vector p Lent-U6-GFP-Puro-sh RNF20 were constructed.(3)The RNF20 knockdown SMMC-7721 and Huh7 cell lines were established by packaging lentivirus and infecting hepatocellular carcinoma cells.The expression of green fluorescence protein(GFP)was observed under fluorescence microscope after the screen by puromycin.(4)The RNF20 m RNA were detected by Q-PCR.The level of RNF20 protein was detected by immunofluorescent staining and Western blot.2.The effects of RNF20 deficiency on both of proliferation,invasion and migration in hepatocellular carcinoma cells.(1)The proliferation of hepatocellular carcinoma cells was detected by Brd U incorporation assay,cell counting kit-8 assay and plate clone formation assay.The invasion of hepatocellular carcinoma cells was detected by transwell assay.The migration of hepatocellular carcinoma cells was detected by scratch test.(2)The level of T-Akt and P-Akt protein was detected by Western blot.(3)The RNF20,Wee1,p27 and p53 gene transcription levels were detected by RNAseq.3.The effects of RNF20 deficiency on DNA damage repair in hepatocellular carcinoma cells.(1)The expression of green fluorescence protein was analysed under fluorescence microscope.The level of RNF20,H2 B and H2Bub1 protein was detected by Western blot.(2)The DNA double strand breaks were created by ionizing radiation.The level of ?H2AX?RPA32s33 and Rad51 protein was detected by immunofluorescent staining.(3)The situation of DNA breaks,special structure and sister chromatid exchanges was detected by Sister chromatid exchange assay.Results: 1.The RNF20 knockdown SMMC-7721 and Huh7 cell lines were established.(1)The infecting efficiency of the two groups was more than 85% under the fluorescence microscope.(2)The expressions of RNF20 of defective groups were significantly lower than that in the control groups by Real-time quantitative PCR.(3)The expressions of RNF20,Wee1,p27,p53 gene and RNF20 protein of defective groups were significantly lower than that in the control groups by immunofluorescence staining and Western blotting.2.The effects of RNF20 deficiency on both of proliferation,invasion and migration in hepatocellular carcinoma cells.(1)The capability of proliferation of the RNF20 defective group were higher than those in control group.(2)The capability of invasion of the RNF20 defective group were higher than those in control group.(3)The capability of migration of the RNF20 defective group were higher than those in control group.(4)The expressions of Wee1,p27 and p53 gene in defective groups were significantly lower than that in the control groups.The capability of both proliferation and migration of the RNF20 defective group which treated by Perifosine were higher than those in control group.3.The effects of RNF20 deficiency on DNA damage repair in hepatocellular carcinoma cells.(1)The expressions of H2Bub1 of RNF20 defective cells and H2BK120 R expressing cells were significantly lower than that in the control groups.(2)The disappearance of ?H2AX of RNF20 defective cells and H2BK120 R expressing cells(SMMC-7721)was much more delayed than that of controls.(3)The expressions of RPA32s33 and Rad51 in RNF20 defective cells and H2BK120 R expressing cells were significantly lower than that in the control groups.(4)The number of DNA breaks and special structure of RNF20 defective cells and H2BK120 R expressing cells were significantly more than that in the control groups,with the number of sister chromatid exchanges being lower than that in the control groups.Conclusion: 1.The deficiency of RNF20 may promote the proliferation,invasion and migration of hepatocellular carcinoma cells in vitro.2.The deficiency of RNF20 may enhance the proliferation and migration of hepatocellular carcinoma cells by regulating Akt signaling pathways in vitro.3.The deficiency of RNF20 may contribute to the depletion of H2Bub1 in hepatocellular carcinoma cells.4.The deficiency of RNF20 may resulte in the defect of DNA damage repair in hepatocellular carcinoma cells.5.The deficiency of RNF20 may contribute to malfunction of the homologous recombination repair in hepatocellular carcinoma cells.