The Preliminary Function Study Of A Novel Hepatoma Associated Gene HTA

Background and objectiveHepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and poor prognosis. The nosogenesis of HCC was involved with abnormal expression of multiple gene and unusual activation and inactivation of multiple singal pathways. Traditional therapy of HCC was not satisfactory, and immuotherapy and genetherapy become research hotspots now, theoretic core of which was HCC associated genes. Discovering and investigating new biomarkes for HCC are still important aspect and tough task in cancer research. Researchs focus on HCC associated gene can provide useful tools for diagnosis and melocular immunotherapy and prognostic analysis. The studies on of HCC associated genes can enrich the molecular mechanism and theoretical system of HCC besides providing powerful biomarkers and theray targets, which was meaningful for the cancer research field.HTA was a novel HCC associated gene screened via bioinformatic appoarch by our reaserch group. Former experiment result suggested that HTA was not only a HCC biomarker with high specificity, but also played improtant role in HCC process, so it could be a hopeful tumor marker and therapy target. Because HTA was screened from EST database, we have only primarily detected its expression specific and tumor promotive role but didn’t refer to its gene struction and specific function. Therefore, we planed to obtain the full length sequence of HTA gene and analyze its gene struction, then identify the protein encoded by HTA gene. Investigate the function of HTA gene by overexpression strategy and study the functional mechanism of HTA gene use the advantage of multiple life science technique such as cDNA array, western blot, Y2H and immunofluorescence.Methods(1) The full length cDNA of HTA gene was amplified by RACE PCR, its gene struction was analyzed and its protein characteristic was predicted. Expression of different transcripts of HTA gene in HCC and normal Hepatic cell lines was detected by Northern blot.(2) Prokaryotic expression vector pGEX-4T-3-HTA-His was constructed for expression of GST-HTA-His fusion peotein in E.coli. GST-HTA-His peotein was purified by GST agarose gel and the GST-tag was digested and removed by thrombin then active HTA protein was recovered by His-tag purification magnetic bead. Using HTA protein as antigen, anti-HTA monoclonal antibody was produced by hybridoma technique, which was used in western blot and immunohistochemistry to detect the expression of HTA protein in hepatic cell lines. The tumor suppressive role of anti-HTA monoclonal antibody was also investigated by cell toxicity test.(3) Eukaryotic expression vector pcDNA3.1(+)-HTA was constructed and HTA gene was stably overexpressed in hepatic cell line QSG7701and HepG2. Cell proliferation test, cell cycle analysis, colony formation assay and nude mouse transplantation tumor experiment were performed to verify the influence on hepatic cell line caused by HTA gene overexpression.(4) Using HTA gene knock down HepG2cand control cell line’s total RNA as material, the HTA associated differential expression genes were screened by genome-wide mRNA expression microarray. Then GO analysis and pathway analysis were performed to study the HTA associated genes and pathways. Real time PCR and western blot was operated to identify HTA associated genes. Gelatin zymography, cell adheresion experiment and cell invasion experiments experiment were carried out to further confirm the biological function of HTA gene.(5) Using fusion protein containing HTA and DNA binding domain as a bait, proteins which can interact with HTA were screened from a human normalized cDNA library by yeast two-hybrid (Y2H). Selected HTA interactive proteins were analyzed and predicted by bioinformatic softwares. Intersted interactive protein LRP1was chosen via protein binding site and integrating degree analysis. Specific Y2H and immunofluorescence were proceeded to identify the interaction and co-localization of HTA and LRP1.Results(1)Full length cDNA of HTA gene was1414bp, ORF of which was constituted by3exons and2introns. There was an alternative splicing in HTA gene, the252bp second introns of HTA gene can be spliced, or retained as a part of mRNA. Northern blot showed that the two tanscripts of HTA expressed in HCC cell lines HepG2and QGY-7703but not in normal cell lines L-02and HUVEC. The length of HTA transcript was according to full length amplification.(2)36kD GST-HTA fusion protein was expressed and purified. lOkD HTA protein without GST-Tag was obtained after thrombin digestion. Anti-HTA monoclonal antibody produced had a titer of1:10000, which can be used in western blot and immunocytochemistry to detect the expression of HTA protein. Anti-HTA monoclonal antibody was confirmed to have a proliferation suppressive effect to HCC cell lines rather than normal cell lines.(3) Compared to control cell lines, hepatic cell lines which overexpressed HTA gene got faster proliferation speed, higher ability of colony formation, quicker cell cycle proceed and stronger ability of tumor formation in nude mice. In the nude mice transplantation tumor, the group overexpression of HTA had a higer HTA expression and higher malignancy grade.(4) In HTA knock-out HepG2cell line, a large number of gene expression changed, most of which were tumor associated genes and involved with cell growth, cell proliferation, cell metabolism, etc. Intersting, knock out of HTA has caught synchronous expression of many members of metalloprotease gene families such as ADAM9、 ADAM12、ADAM10、ADAM17、MMP9and MMP2, which was confirned by qPCR and western blot. Gelatin zymography showed that the activity of MMP2and MMP9in cell lines was significantly decreased after knock down of HTA gene. It was also proved that HTA gene played an important role in cell adhesion, invasion and metastasis.(5) Several potential HTA interactive protein was screened by Y2H, Specific Y2H and immunofluorescence verified that HTA could interact with C terminal of LRP1and they were co-located in cell membrane and cytoplasm.Conclusion(1) Cloned the full length cDNA of HTA gene and identified by克Northern blot.(2) Obtained HTA protein with biologiacal activity and produced high titer anti-HTA monoclonal antibody.(3) Overexpressing HTA gene in hepatic cell lines can promote growth, proliferation and in vivo tumor formation ability of Hepatic cell lines.(4) Expression and activity of many members of metalloprotease gene families positively correlated with HTA gene. HTA gene might promote the adhesion, invasion, matastasis ability of hepatic cell via metalloprotease family members.(5) HTA interacts with LRP1and they were co-located in cell membrane and cytoplasm.