Establishment Of NCOA5 Conditional Knockout Mouse Model And Study On Effect And Mechanism Of NCOA5T445A In Hepatocellular Carcinoma

Background and purposeHepatocellular carcinoma(HCC)is the fifth common cancer in worldwide,and also the third cause of cancer death.The incidence of HCC has significant gender disparity,with approximately 2-4 times more incidence in male than female.Hepatitis virus remains a major risk factor for HCC,but in developed countries,non-alcoholic fatty liver and diabetes also contributes as important risk factors.Diabetes mellitus(DM)is an independent risk factor for HCC.In addition,DM can significantly increase HCC-related mortality,overall mortality and reduce relapse-free survival,suggesting that DM is a indicator of poor prognosis of HCC.Therefore,DM plays an important role in affecting the development and prognosis of HCC.However,the molecular mechanisms associated with diabetes and HCC remains unclear.Inflammation,aberrant lipid,glucose and energy metabolism,insulin resistance and hyperinsulinemia all contributes to the occurence of diabetes-related liver cancer.Oncogenesis of HCC is a multi-step process and result from accumulation of a plurality of pathway anomalies.The major relevant gene mutations found by far includes TP53,β catenin,AXIN1.Chromosome amplification occurs mainly in 1q,6p,8q,17q,20q,while chromosome deletion occurs mainly in 4q,8p,11q,13q,16q,and 17p.Mechanism underlying epigenetic changes in HCC remains unclear,but studies had shown that inactivation of tumor suppressor like RASSF1,SOCS1,E-caderin HCC and activation of oncogene like MYC are related to HCC.miRNA also plays a role in the development of HCC by regulating oncogene.Above changes result in activation of signaling pathway of cell survival and proliferation like EGFR、RAS、mTOR、HGF、c-MET、Wnt and thus leads to HCC.NCOA5(also called CIA)is an AF2-independent coactivator with both transcriptional activation and repression domain.It can regulate transcription of ER.Genomic analysis showed that NCOA5 is a susceptible gene for type 2 diabetes(T2D),locating in T2D relevant regions chromosome 20q12-13.1.Professor HuaXiao found that haploinsufficient NCOA5 cause HCC in male mice with elevating IL-6 expression,early impaired glucose tolerance,progressive steatohepatitis and liver dysplastic precede to the onset of tumor.We thus measured NCOA5 mRNA expression in human HCC tumor and adjacent tissues using quantitative PCR,and also analyzed relationship between NCOA5 and clinicopathological parameters in HCC;Sequencing analysis revealed a novel splice form of NCOA5 named sNCOA5,we then explored correlation of sNCOA5 expression and clinicopathological parameters in the aforementioned specimens.NCOA5 haploid knockout mouse in previous experiment is not capable of producing homozygous knockout pups,and male fertility defects were found.In order to further study the cellular and molecular mechanism underlying,we constructed a NCOA5 conditional knockout plasmid,transfected ES cell by electroporation and then microinjected into blastocyst to obtain chimeric mice,facilitating research on physiological mechanism of different mouse model with time or spatial-specific NCOA5 knockout.Moreover,we found that a single nucleotide change of NCOA5 in Caucasian HCC population,named NCOA5T445A.We constructed tet on inducible system consisting of NCOA5 plasmids and retroviruses,and established NCOA5/NCOA5T445A overexpression HCC cell lines that can be regulated by doxycyclin,then conducted analysis of the biological behavior of NCOA5T445,providing clues of their impact on oncogenesis and development of HCC.Method1.Quantitative PCR analysis measuring expression of NCOA5 and sNCOA5 mRNA in HCC tumor and adjacent tissueQuantitative PCR analysis was applied to analyze expression levels of NCOA5 mRNA in 30 HCC tumor and adjacent tissues as well as 3 non-HCC liver tissue.Sequencing was carried out in HCC samples and indicated a splice form of NCOA5 named sNCOA5.Information about patient’s age,sex,differentiation grade,tumor size,portal vein invasion,hepatitis viruses and quantitation,AFP,history of diabetes and other characteristics were collected and analyzed.2.Design and construct NCOA5 conditional knockout plasmid and chimeraDesign NCOA5 conditional knockout plasmid using online databases and software.Molecular biology methods like PCR,restriction enzyme digestion,electrophoresis,and ligation were used to construct plox Frt Ncoa5 containing upstream and downstream homologous recombination arm,neo fragment flanked with two FRT sites and floxed NCOA5 exons 3.Linearized plasmid was transfected into embryonic stem cells and underwent neomycin screening for positive clones before microinjecting into blastocysts to obtain chimeric mice.3.Functional analysis of NCOA5T445A on the biological behavior of HCC cell linesPREV-TRE-NCOA5,PREV-TRE-NCOA5T445 plasmid were designed and constructed.Retrovirus were packaged by transfecting Phoenix cells with plasmids mentioned above.Inducible PLC/PRF/5 cells and Hep3B cells were established through infection with PREV tet on plasmid and screening with G418.Inducible efficiency with doxycycline was measured by luciferase assay after transiently transfecting with PREV-TRE-luc and induced with doxycycline.PREV-TRE-CON,PREV-TRE-NCOA5 and PREV-TRE-NCOA5T445A retrovirus were used to infect HepG2 tet on and PLC/PRF/5 tet on cells.After selection with hygromycin,two groups of tetracycline or doxycycline regulated cell lines were obtained.In vitro proliferation of HepG2 and PLC/PRF/5 cells CON/NCOA5/NCOA5T445A cells were measured by MTT method;Westernblot and flow cytometry were used to analyze affects on cell cycle with NCOA5 or NCOA5T445A overexpression.4.Statistical analysisSPSS 13.0 was used for statistical analysis.Differences between HCC tumor and adjacent tissues were compared with independent t-test.Independent t test was used to analyze correlation between NCOA5,sNCOA5 mRNA and age,gender,portal vein thrombosis,tumor size,histological grade and HBsAg.Spearman analysis was used to analyze correlation between NCOA5,sNCOA5 mRNA and quantitative HBV,AFP,ALB,ALT,AST,DBIL,TBIL,IBIL and PTINR.Western blot,MTT assay were compared with factorial analysis.Cell cycle data were compared with ONE WAY ANOVA and LSD method between two groups with homogeneity of variance.P<0.05 was considered statistically significant.Result1.Quantitative PCR analysis of NCOA5 and sNCOA5 mRNA levels in human hepatocellular carcinoma tumor and adjacent tissuesIn 30 HCC patients,NCOA5 mRNA expression were significantly reduced in about 40%HCC tumor tissues comparing to adjacent tissues(P<0.05).63%adjacent tissues showed for over 50%decreased(P<0.05)expression of NCOA5 mRNA comparing to three normal liver tissues.Sequencing result of 9 pairs of HCC tumor and adjacent tissues revealed a novel alternative splicing of NCOA5 lacking transcriptional activation domain named sNCOA5.sNCOA5 is composed of 406 amino acids,with extra 23 amino acids from intron 7 following exon 7,causing extension and frameshift for early appearance of STOP codon and premature termination of protein translation.In 30 HCC patients,about 43%had significantly increased sNCOA5 mRNA expression(P<0.05)than adjacent tissues.Western Blot indicated elevation of sNCOA5 protein levels in 2 of 4 HCC tumor samples than adjacent tissues.Correlation analysis of 29 cases showed that NCOA5 or sNCOA5 mRNA expression levels had no significant correlation with age,tumor size,histological grade,portal vein invasion,AFP,ALB,ALT,AST,DBIL,TBIL,IBIL,PT-INR,HBV Ag(P>0.05),while sNCOA5 displayed a positive correlation with HBV viral quantification(spearman coefficient value=0.592,P = 0.043).2.Construction of plox Frt Ncoa5 plasmid and conditional knockout mice2.0Kb 5 ’end of the homologous recombination arm,3.55Kb 3’ end of the homologous recombination arm as well as 1.25Kb exon 3 and its adjacent intronic fragment(Exon3)were amplified with PCR from SVJ129 mouse cDNA library.PCR2.1 up,PCR2.1 down,PCR2.1 Ex3,PBS E3,PCR Cla-E3-loxp,ploxP E3,ploxP ED,ploxP FRT-NEO ED and plox Frt Ncoa5 were constructed sequentially.ES cell electroporation and ES cell clone expansion were carried out before PCR screening and microinjection into C57/BL6 mice.31 Chimera mice were obtained.3.Impact of NCOA5T445A on the biological behaviors of HCC cell linesPREV-TRE-CON,PREV-TRE-NCOA5,PREV-TRE-NCOA5T445A retroviral vectors were constructed,stable Hep 3B and PLC/PRF/5 tet on cell lines which can be regulated by doxycycline were established.Retrovius was packaged and applied to establish HepG2 and PLC/PRF/5 tet on NCOA5/NCOA5T445A cell lines.Western blot showed that NCOA5 and NCOA5T445A expression are regulated by doxycycline in HepG2 tet on and PLC/PRF/5 tet on groups.MTT assay indicated that proliferation of HepG2 tet on con/NCOA5/NCOA5T445A cells are significantly different before or after induction by doxycycline(F = 2.956,P = 0.049;F = 67.662,P = 0.000),PLC/PRF5 also showed similar trends before or after the induction(F = 10.794,P = 0.000;F =81.829,P = 0.000).Flow cytometry showed that leaky overexpression of NCOA5/NCOA5T445A can increase G2M phase in HepG2 cells(F = 522.991,P = 0.000).When induced by doxycycline and produced a large amount of NCOA5 or NCOA5T445A,HepG2 tet on cells also had similar changes(F = 87.773,P = 0.000).Western blot suggested that CyclinBl expressed lower in NCOA5 and NCOA5T445A overexpressing cells than the control group,while NCOA5 group had lower expression than the NCOA5T445A group.Above results demonstrate that overexpression NCOA5 and NCOA5T445A can significantly inhibit cell growth of HCC cell lines(P<0.001),and NCOA5T445A suppression function is impaired(P<0.001).Conclusion1.NCOA5 decreased expression in hepatocellular carcinoma tissues suggesting NCOA5 as an be important tumor suppressor in human hepatocellular carcinoma;2.Expression of inactivated NCOA5 splicing form sNCOA5 increased in hepatocellular carcinoma and is associated with HBV quantification,indicates it could possibly be a potent tumor marker for monitoring and detection of liver cancer;3.Obtained chimeric mice with construction of NCOA5 conditional knockout plasmid,which may be facilitates establishment of experimental model studying time and spatial-specific NCOA5 expression and underlying physiological mechanism.4.NCOA5 is an important tumor suppressor in the development of liver cancer through G2M arrest and cell growth inhibition.One single amino acid change in NCOA5 can attenuate the inhibitory affects.Novelty1.Confirmed that decreased NCOA5 and increased sNCOA5 expression are involved in the development of liver cancer,providing theoretical basis for exploring individualized treatment for liver cancer;2.Established a chimeric mice that facilitates production of tissue or cell,time-specific NCOA5 knockout mice or homozygous NCOA5 knockout mice.3.Found a new amino change in NCOA5 that is related to liver cancer and attenuate inhibitory function of wildtype NCOA5.Established two strains capable of overexpressing NCOA5 or NCOA5T445A with retroviral vectors,providing models to study machinery of NCOA5 or NCOA5T445A function.