SENP5 Promotes Tumorigenesis In Hepatocellular Carcinoma And Regulates DNA Damage Response

OBJECTIVE 1 Biological information of SENP5;2 The expression of SENP5 in hepatocellular carcinoma cells and tissues;3 Effect of SENP5 on the growth of hepatocellular carcinoma;4 Effect of SENP5 on DNA damage response of hepatocellular carcinoma cells;5 The role of SENP5 in regulating DNA damage response.MATERIALS AND METHODS1 In this study,SENP5 was analyzed by bioinformatics methods to obtain the research clues.2 mRNA and representative protein levels of SENP5 were analyzed by Real-time PCR and Western blot in human hepatocellular carcinoma tissues or adjacent normal tissues.Immunohistochemical detection was used to observe the expression of SENP5 in human hepatocellular carcinoma tissues or adjacent normal tissues.The expression level of SENP5 protein in normal hepatocytes and HCC cells was detected by Western blot.Immunofluorescence(IF)was used to detect the subcellular localization of SENP5 in Hep3B cells.3 Cells were transfected with siRNA oligos targeting SENP5 or scramble siRNA(NC)for 48 h before harvest.mRNA level of SENP5 was determined by real-time PCR in HepG2 cells.Protein level of SENP5 was determined by Western blot in HepG2 cells.The cell proliferative potential was determined by BrdU assay in HepG2 cells transfected with siRNA oligos targeting SENP5 or scramble siRNA(NC).The tumor formation activity of HepG2 cells transfected with siRNA oligos targeting SENP5 or scramble siRNA(NC)was determined by plate colony-formation assay.The HepG2 cells were injected subcutaneously to the front legs of the nude mice.The tumor weights were determined at day 40 post tumor cell injection.4 HepG2 cells transfected with siRNA oligos targeting SENP5 or scramble siRNA(NC)were irradiated as cell survival assay(cck8)carried out.HepG2 cells transfected with siRNA oligos targeting SENP5 or scramble siRNA(NC)were treated with or without 10μM Etoposide for 24h and annexin-V+ cells%was determined by flow cytometry.After HepG2 cells transfected with siRNA oligos targeting SENP5 or scramble siRNA(NC)were treated with or without 10 μM Etoposide for 24h,Western blot was used to determine the expression of the proteins involved in cell cycle block and DNA damage repair.5 RNA interference technique and immunoprecipitation(IP)were used to detect the interaction between SENP5 and ATRIP.RESULTS 1 Multiple databases showed that the SENP5 was highly expressed in various tumor tissues,including hepatocellular carcinoma tissues.SENP5 was SUMO’s specific protease.2 We found that SENP5 was over-expressed in HCC sampes in both mRNA and protein levels.It was mainly located in the nucleus.3 The proliferation of HepG2 cells was significantly inhibited after knocked out the SENP5 by BrdU assay and plate colony-formation assay.The tumor weight was lighter in the nude mices injected the HepG2 cells with siRNA oligos targeting SENP5.4 SENP5-depleted HepG2 cells exhibited hypersensitivity to IR and etoposide treatment with defective checkpoint activation including decreased activation of ATR and phosphorylation of ATR targets.5 At the molecular level,we found that SENP5 interacted with ATRIP and promoted ATRIP deSUMOylation to regulate DNA damage response.CONCLUSIONS Our data suggested that SENP5 was required for HCC cell growth and DNA damage response,which might be a promising drug target for HCC.