Function And Mechanism Of DEPDC1 In Hepatocellular Carcinoma Cell Line HepG2

Object: As a cancer protein containing a DEP domain,DEPDC1 is undetectable in normal tissues except testis.Previous studies have shown that DEPDC1 is overexpressed in bladder cancer,breast cancer,non-small cell lung cancer and liver cancer.DEPDC1 is involved in the tumorigenesis in bladder cancer via regulating the proliferation and apoptosis.The role of DEPDC1 in bladder cancer is relatively clear.It was found that zinc finger protein 224(ZNF224)is an important protein related to the function of DEPDC1.The experiments in vivo and in vitro show that the interfering polypeptides(DEPDC1 611 to 628 amino acids with 11 arginine at its N terminal,abbreviated as 11R-DEP:611-628)block ZNF224 and DEPDC1 to form complex,thus can significantly reduce the cell viability.However,up to now,the specific role and mechanisms of DEPDC1 in hepatocellular carcinoma is not clear.The purpose of this study is to explore the function and mechanism of oncoprotein DEPDC1 in hepatocellular carcinoma,providing experimental evidences for the clinical treatment for hepatocellular carcinoma.Method:(1)Western blot was used to detect the expression of DEPDC1 in hepatocarcinoma cells.(2)MTT assay was used to determine the optimalconcentration of interfering peptide with inhibiting effect on the growth of HepG2 cells.(3)Microscopy was used to observe the morphological change of HepG2 cells treated with interfering peptide at post-treatment time points3 h,6h,12 h and 24 h.(4)In the interfering peptides group and the control group,we detected apoptosis of HepG2 cells by Tunel staining.(5)After the treatment of HepG2 cells in the interfering peptides group and the control group,we detected the expression of casepase-3 and cleavedcasepase-3 in HepG2 cells.(6)Nuclear located NF-κB was detected with fluorescent immunostaining in cells treated with interfering peptides for 1h,6h and 12 h.(7)After the peptide intervention treatment HepG2 cells,using Western blot detection DEPDC1 expression.Results:(1)Western blot results showed that the expression of DEPDC1 in HepG2 cells was significantly higher than in SK-hep1,SMMC7721,BEL7402,QGY7701 cells.(2)MTT assay results showed that interfering peptide inhibits proliferation of HepG2 cell,and the optimal concentration of interfering peptide is 3 u M.(3)Microscopic results showed that the cell density of Hep G2 cells treated with interfering peptides was decreased significantly.(4)Tunel staining results showed that the positive rate of HepG2 cell,the level of cell growth inhibition and cell apoptosis in interfering peptides group were significantly higher than in control groups.(5)Western blot showed that in the interfering peptides group,the expression of casepase-3 was decreased while the expression of cleaved casepase-3 was increased compared with the control group.(6)After the DAPI nuclear fluorescence staining,we found nuclear localized NF-κB in the interfering peptides group was decreased.(7)Using Western blot assay,we found that the expression of DEPDC1 in the interfering peptides group was lower than that in the control group,which suggests that the expression ofDEPDC1 may be regulated by DEPDC1-ZNF224.Conclusion: The expression of DEPDC1 is up-regulated in a variety of hepatocellular carcinoma cell lines.DEPDC1 can inhibit cell apoptosis and promote cell proliferation through NF-kappa B signaling pathway in HepG2,which is beneficial to the development of hepatocellular carcinoma.DEPDC1-ZNF224 may be a potential therapeutic target for hepatocellular carcinoma.Peptides 11R-DEP:611-628 may be a potential therapeutic agent for hepatocellular carcinoma.