HZ-6d Targeted HER C5 To Regulate P53 ISGylation In Human Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is a primary malignant tumor of the liver,arising from hepatocytes.It is regarded as the third leading cause of all cancer-related mortality in the world.In spite of advances in the diagnostic techniques and therapeutic strategies of HCC,such as liver transplant,radiation,chemotherapy,or drug combination,the prognosis for HCC patients remains depression.Now,evidence has emerged that the development of HCC is multiple steps,multiple phases procession,which involved in inactivation of tumor suppressor genes and/or activation of multiple proto-oncogenes.Among these altered genes,p53 is a significant one.With the in-depth study of p53 signaling pathway,tumor therapy based on p53 has been paid more and more attention.Emerging evidence have identified interferon-stimulated gene 15(ISG15)can covalently modified p53 at 2 sites in the N and C terminus,and in turn degraded by the 20 S proteasome.During this process,HERC5,an IFN-induced HECT-type E3 ligase,mediated p53 ISGylation.However,very little or no information is available on the role of HERC5 in HCC.In this study,the expressions of ISG15 and HERC5 in HCC tissues and cells were observed.Besides,we screened a 7,11-disubstituted quinazoline derivative HZ-6d that could bind to the HERC5 G-rich sequence in vitro.Finally,the regulatory mechanism of HZ-6d was explored in HCC.As follows:(1)The expression of HERC5 and ISG15 in HCCTissue samples were collected from The First Affiliated Hospital of Anhui Medical University and the Human HepG2 and SMMC-7721 cells and L-02 cell line were cultured.The expression of HERC5 and ISG15 was detected by Immunohistochemistry,Western blot and qPCR.Results have revealed that HERC5 and ISG15 were over-expressed in both HCC tissue samples and cell lines.(2)HERC5 promotes cancerous proliferation and involves in cell apoptosis,which associated with p53-mediated pathway in HCCHepG2 and SMMC-7721 cells were transfected by HERC5 small interfering RNA(siRNA)and pEGFP-HERC5 plasmid.MTT and Colony forming assay were performed to test the effect of HERC5 on cell proliferation.We next evaluated the effect of HERC5 using flow cytometry.The protein levels of p53,p21 and Bax/ bcl-2 were tested by Western blot.The results showed that cell proliferation and colony formation were suppressed by silencing of HERC5 in both HepG2 and SMMC-7721 cells.Meanwhile,reduced expression of HERC5 significantly increased cell apoptosis.More importantly,silencing of HERC5 remarkably increased the protein level of p53,p21 and Bax/Bcl-2.In contrast,up-regulation of HERC5 promoted cell proliferation colony formation and led to lower expression of p53,p21 and Bax/Bcl-2 in HepG2 and SMMC-7721 cells.(3)The combination of HERC5 and HZ-6dThe stability of the interaction between HZ-6d and HERC5 gene G-quadruplex was elucidated using molecular docking,and CD spectroscopy assay.These results revealed that compound HZ-6d could bind and stabilize the HERC5 G-quadruplex structure and inhibition of the G-rich sequence expression.(4)To detect the anti-tumor activity of HZ-6d in vivo and vitro.We evaluated antitumor effects of HZ-6d as demonstrated in vivo within nude mice bearing SMMC-7721 xenografts.The expression of HERC5 and ISG15 was detected by Immunohistochemistry and Western blot.The data together demonstrated that HZ-6d treatment effectively inhibited tumor growth in vivo and blocked the expression of HERC5 and ISG15.To investigate whether HZ-6d treatment suppress cell growth on HCC cells,MTT assay,Colony forming assay,Wound healing assay and flow cytometry were used in HepG2 and SMMC-7721 cells treated with various concentrations of HZ-6d for 24 h,respectively.Western blot and qPCR were used to detect the expression of HERC5,ISG15 and p53.our data indicated that HZ-6d treatment can effectively inhibit tumor growth of HCC cells,reduce the expression of HERC5 and ISG15 and activate p53.(5)To further explore the underlying mechanism of HZ-6d.Aim to investigate the related mechanism of HZ-6d in HCC,siRNA specific for human HERC5 was used to knockdown the HERC5 expression.Then,we treated the cells with HZ-6d and analyzed the expression of p53 pathway proteins by Western blot.Our results revealed that down-regulation of HERC5 could reactivate p53 pathway and the trend is the same as the treatment of HZ-6d.Consistently,down-regulation of HERC5 in combination with HZ-6d treatment induced p53 pathway proteins expression to more degree compared with HZ-6d alone or siRNA treatment alone.In conclusion,our studies determined that HERC5 may play an important role in HCC.HZ-6d possessed high effectiveness in the inhibition of HCC growth was due to block the expression of HERC5.Accordingly,the decreased of HERC5 induced p53 protein accumulation through inhibited ISGylation mediated p53 degradation pathway.