Research On The Tumor-associated Genes In The Development Of Primary Liver Cancer

Primary liver cancer is the fifth most common malignancy worldwide,majorly affecting those in Asia and of sub-Saharan Africa, among which about half of cases are from China.The long-term prognosis of patients undergoing potentially curative hepatic resection is still poor,with reported 5-year survival rates ranging from 17%to 53%. Despite resection with curative intent,the clinical course is variable and recurrence occurs in a high proportion of cases.The number of death from liver cancer per year are almost equal to that of new cases occurred in the same year.The poor prognosis of liver cancer is majorly due to patients' no response to almost all conventional chemotherapy and over 80%patients diagnosed at advanced stage and lost the opportunity for curative resection.Numorous genes play roles in the multi-step development of liver cancer.At present,numerous genes involved in the development of liver cancer remains unclear.Research on the tumor-associated genes of liver cancer could provide clues for searching the potential biomarkers for early diagnosis and prognosis and screening targets for treatment.In the present study,methods of suppression subtractive hybridization combined cDNA chips were used to identify and screen the differential expression genes in hepatocellular carcinoma.However, samples from the human being only can be obtained from a certain phase, while serial analysis of differential expression genes profiles in the whole process of development of liver cancer could be accomplished through establishment of animal models.Therefore,we established the rat models of liver cancer by DEN and investigated the gene expression profiles of liver tissues from cirrhosis to carcinoma with Affymetrixchip' assisting. Fn14,one upregulated gene persisting from cirrhosis to metastasis in rat models was chosen for further research in liver cancer cells.Part 1 Identification of differential expression of genes in hepatocellular carcinoma by suppression subtractive hybridization combined cDNA microarrayThe genetic background of hepatocellular carcinoma(HCC)has yet to be completely understood.Here,we describe the application of suppression subtractive hybridization(SSH)coupled with cDNA microarray analysis for the isolation and identification of differential expression of genes in HCC.Twenty-six known genes were validated as up-regulated and 19 known genes as down-regulated in HCC.The known genes identified were found to have diverse functions.In addition to the overexpression of AFP,these genes(increased in the presence of HCC) are involved in many processes,such as transcription and protein biosynthesis(HNRPDL,PABPC1,POLR2K,SRP9,SNRPA,and six ribosomal protein genes including RPL8,RPL14,RPL41,RPS5,RPS17, RPS24),the metabolism of lipids and proteins(FADS1,ApoA-Ⅱ,ApoM, FTL),cell proliferation(Syndecan-2,and Annexin A2),and signal transduction(LRRC28 and FMR1).Additionally,a glutathione-binding protein involved in the detoxification of methylglyoxal known as GLO1 and an enzyme which increases the formation of prostaglandin E(2) known as PLA2G10 were up-regulated in HCC.Among the underexpressed genes discovered in HCC,most were responsible for liver-synthesized proteins(fibrinogen,complement species,amyloid, albumin,haptoglobin,hemopexin and orosomucoid).The enzyme implicated in the biotransformation of CYP family members (LOC644587)was decreased.The genes coding enzymes ADH1C, ALDH6A1,ALDOB,Arginase and CES1 were also found.Additionally, we isolated a zinc transporter(Zip14)and a function-unknown gene named ZBTB11(Zinc finger and BTB domain containing 11)which were underexpressed,and seven expression sequence tags deregulated in HCC without signifcant homology reported in the public database.Essentially, by using SSH combined with a cDNA microarray we have identified a number of genes associated with HCC,most of which have not been previously reported.Further characterization of these differentially expressed genes will provide information useful in understanding the genes responsible for the development of HCC.Part 2 Analysis of genomic expressional profiles in the development of liver cancer models of rats induced by DENTo explore the key genes involved in the development and progression of liver cancer at global scale,we established the rat models of liver cancer induced by DEN and investigated the gene expression profiles of liver tissues from cirrhosis to carcinoma with Affymetrixchip' assisting.The pathological changes of the livers in rats are processes including non-special injury,fibrosis&cirrhosis,dysplastic nodules,early cancerous nodules and metastasis.The liver tissues of cirrhosis(12^th week),the dysplasia nodules at the 14^thweek,the cancerous nolules at the 16^thweek and the cancerous nodules from the rats with lung metastasis (20^thweek)were chosen to investigate the differential expression genes(DEGs)by compared with the liver tissues from the normal rats with Affymetrix chip.The differential expression genes obtained by comparing the signals of 4 groups selected with the signals from the liver tissues of the normal rats,respectively.There are 681 upregulated and 687 downregulated genes in the cirrhosis tissue(12^thweek);857 upregulated and 732 downregulated genes in the dysplasia nodules(14^th week);1223 upregulated and 1016 downregulated genes in the cancerous nodules at the 16^thweek;999 upregulated and 906 downregulated genes in the cancer tissue at metastatic stage(20^thweek),respectively.Among all the DEGs,there are 349 upregulated and 345 downregulated genes shared for the 4 stages,among which 246 of 349 upregulated DEGs and 215 of 345 downregulated DEGs are known genes,the remaining are genes inferred or predicted,translocation locus and unknown genes completely.Some differential expression genes were confirmed by the method of real time RT-PCR.The deregulated DEGs play various roles and involve in diverse processes such as metabolism,transport,cell proliferation,apoptosis,cell adhesion,agiogenesis and so on.Some of the DEGs are associated with inflammatory response,immune response and oxidative stress.Upon this,we think that inflammatory response,immune response and oxidative stress provides a niche for liver cells on the changed structure of hepatitis,fibrosis,cirrhosis as well as excellular matrix.The balance between cell proliferation and apoptosis was destroyed due to the deregulation of numerous genes,meanwhile, neovascularization is producive.Therefore,the liver cancer developed and progressed.In the present study,we provide gene expression profiles may play major roles in the development of liver cancer by systemic analysis of profiles of DEGs during the development and progression of liver cancer of rats,which will contribute to the understanding of liver cancer development and offer the opportunity for searching marker of diagnosis and prognosis as well as provide clues for targeting therapy. However,many genes related to liver cancer have not been previously reported.Part 3 The functional assays of Fn14 in liver cancer cellsIn the second part of the thesis,a number of deregulated expression genes were identified in the liver cancer model of rats induced by DEN. Fn14 is one of the up-regulated expression genes occurred persistently from liver cirrhosis to metastastic stage of liver cancer.To explore the roles of Fn14 plays in the development of liver cancer,overexpression of Fn14 in SMMC7721 was established.On the basis of Fn14's overexpression,methods and techniques including MTT, immunoflurence staining and flow cytometry analysis were used to assay the effects of Fn14 on the survival ability and apoptosis of SMMC7721 cells treated by TRAIL.Furthermore,an inhibitor of NF-κB activity was used to determine whether the effects of Fn14 on the survive and apoptosis were dependent on the activation of NF-κB.The results showed that overexpression of Fn14 prompts cellular survive and resists apoptosis induced by TRAIL.Meanwhile,increased expression of Fn14 results in translocation of p65 subunit of NF-κB to the nucleus from cytoplasm.Furthermore,increased survive and resisted apoptosis of cells induced by upregulated expression of Fn14 was dependent on the activation of NF-κB.The results implied that Fn14 may be potential marker of diagnosis and prognosis and serve as a target for therapy of liver cancer.