HDAC3 Inhibits Hepatocarcinogenesis Via Suppressing A-EJ DNA Repair Pathway

Background:The incidence and mortality of liver cancer in China account for about 50%of the global liver cancer,seriously threatening the life and health of the people.Hepatocellular carcinoma（HCC）is the main form of liver cancer,with the concealed incidence and limited treatment,in addition to liver transplantation,surgical resection,radiotherapy and chemotherapy,only a few targeted therapeutic drugs,such as Sorafenib and Regorafenib can be selected,with unsatisfactory curative effect.Therefore,to explore the molecular pathogenesis of HCC and find new therapeutic targets will provide a more perfect theoretical basis and a broader prevention and treatment strategy for HCC.Genomic instability is an important feature to promote the survival,proliferation and proliferation of cancer cells,which makes the tumor accelerate the acquisition of genetic diversity.In most non-hereditary cancers,genomic instability is mainly caused by DNA damage.DNA double strand break（DSB）which cause the loss of a large amount of genetic material,are the most dangerous type of DNA damage.It can be repaired by non-homologous end joining（NHEJ）pathway,alternative end joining（a-EJ）pathway,single-strand annealing（SSA）pathway or homologous recombinant（HR）pathway.If not repaired in time and correctly,it is easy to lead to genomic instability and gene mutation.Epigenetic regulation,especially histone modification,is an important part of DNA damage repair.Due to the reversible changes of epigenetics,acetylation at different sites of the N-terminal tail of core histone is dynamically regulated by histone acetyltransferase（HAT）and histone deacetylase（HDAC）.HDAC3 is an important member of class I HDAC family,which is necessary for effective replication and damage regulation of DNA.Previous studies have found that DNA damage and spontaneous formation of HCC can be observed in mice with liver specific deletion of HDAC3,which leads us to speculate that HDAC3 may lead to abnormal repair process after DNA injury.The research of HDAC3 in the field of HCC is very limited.In order to explore the molecular mechanism of HDAC3 regulating repair pathway in the process of DSB repair,and to elucidate the important role of HDAC3 in maintaining genomic stability in the occurrence and development of HCC,we intend to construct DSB model in HDAC3 silenced/overexpressed hepatocellular carcinoma cells,and combined with the genotypes of hepatocyte specific knockout mice(HDAC3^LKO).This study will reveal the new mechanism of HDAC3 regulating the occurrence and development of HCC and the important role of HDAC3 in maintaining genomic stability,and provide a new theoretical basis and strategy for clinical targeted treatment of HCC.Methods:1.Bioinformatics analysis of the differential expression of HDAC3 in HCC tissues and normal liver tissues in TCGA database was performed;the relationship between HDAC3 expression and prognosis of patients with HCC was analyzed;the correlation between HDAC3 expression level and genomic stability was evaluated.2.The effects of HDAC3 on the proliferation,metastasis and DNA damage and repair of hepatocellular carcinoma:the overexpression and silencing HDAC3 model of human hepatoma cells was established,and the effect of HDAC3 on proliferation was observed by CCK8 and clone formation assay.The effect of HDAC3 on migration ability was observed by Transwell,and the effect of HDAC3 on DNA damage and repair pathway was detected by Western blot and immunofluorescence.The HDAC3^LKOKO mice were identified and the survival curve and tumorigenesis of HDAC3^LKO mice were observed.3.HDAC3 regulates POLQ-mediatedα-EJ pathway to repair DNA damage in HCC:DSB model of DNA damage induced by CPT was constructed,and the effects of silencing/overexpression of HDAC3 on DNA damage and DSB repair protein induced by CPT in human hepatoma cells were observed.The model of HCC in mice was established by DEN induction,and the formation and survival curve of liver cancer induced by DEN in HDAC3^LKO mice were observed.Bioinformatics was used to analyze the correlation between HDAC3 and POLQ,and the relationship between POLQ expression and prognosis of patients with HCC.Effects of Poly（ADP-ribose）Polymerase 1[Poly（ADP-ribose）polymerase 1,PARP1]inhibitor blocking POLQ on genome stability and apoptosis were detected by Western blot and Flow Cytometry.Results:1.Bioinformatics analysis showed that the survival time of HCC patients with high expression of HDAC3（pathological stageⅡ+Ⅲ）was better than that of patients with low expression（P<0.05）.The mutation ratios of LZTR1,RB1,KRAS,IL6ST,EEF1A1,GPATCH4,CREB3L3,AHCTF1 and APOB in HDAC3 high expression group were lower than those in HDAC3 low expression group.The disease-free/progression-free survival time of the patients without mutation of the above mutated genes was significantly prolonged（P<0.05）.HDAC3 may affect the prognosis of patients with HCC by regulating the mutation rate of genes.2.Spontaneous tumorigenesis was observed in hepatocyte specific knockout mice(HDAC3^LKO),and its survival curve suggested that the survival rate of HDAC3^LKO mice was significantly lower than that of WT mice.By regulating the expression of HDAC3 in human hepatoma cells,we found that silencing HDAC3 promoted the proliferation and migration of hepatoma cells,and the existence of DNA damage was observed.at the same time,the expression of PARP1 and POLQ,which representα-EJ repair pathway,was enhanced.3.An in vitro and in vivo experimental model of DNA damage was established by chemical methods of DEN induction and CPT intervention to observe the effect of HDAC3 on DNA damage and repair.HDAC3^LKOKO mice could be tumorigenic at the size of 3 months after DEN induction,which was earlier than that of WT mice induced by DEN.At the same time,the survival rate of mice in group(HDAC3^LKO+DEN)was lower than that in other groups.Similarly,in the cell model of DNA damage,silencing HDAC3 increased the expression of PARP1 and POLQ and activated theα-EJ repair pathway.Blocking POLQ with PARP1 inhibitor Olaparib enhanced the sensitivity to CPT and promoted apoptosis.Conclusion:The current results of this study reveal that silencing HDAC3 leads to up-regulation of POLQ expression,and activatesα-EJ repair pathway and changesα-EJ from"backup pathway"to"essential pathway".This change in the choice of repair methods has greatly contributed to genomic instability.The study of HDAC3 maintaining genomic stability in the field of HCC may explain the bottleneck of HDAC inhibitors in tumor treatment.Meanwhile,as a PARP1 inhibitor that blocks POLQ,it has gone beyond the research field of"synthetic lethal"drugs.at present,it plays a new and important role in DNA repair and other fields.its unknown surprise awaits us to find out further.