| HCC (hepatocellular carcinoma) is one of the malignant tumors worldwide, which is tremendously harmful to human health and survival. HCC is the most common disease of liver cancers, frequently seen in East Asia, Southeast Asia and Africa. However, the pathogenesis of HCC has not been understood and remains unclear. During the development period of tumors, especially HCC, chromosome deletion, amplification and gene mutation have been reported to be accompanied with the tumor progression. In our previous studies, single nucleotide polymorphisms 6.0 array (SNP 6.0) was adopted to analyze HCC patients’genome, and 1,241 copy number alterations (CNA) were noted, including many novel copy number alteration regions, like deletion region 14q31.1-32.13 and amplicon 19p13.2. We focused on characterization and analysis of genes located in these two regions. Firstly, we integrative analyzed copy number alteration and expression profile results, six genes were derived in 14q31.1-32.13 region. We observed that SERP1NA5 and SERPINA10 was downregulated in large scale HCC patient sample sets by q-PCR, and by Pearson’s correlation analysis, downregulation of SERP1NA5 and SERPINA10 expression was partially correlated with gene dosage decrease. In addition, SERPINA5 downregulation was negatively related to tumor stage and intrahepatic metastasis. In vivo and in vitro experiments verified that SERPINA5 inhibited metastasis of HCC cells. Secreted and purified SERPINA5 protein also inhibited migration of HCC cells. Co-IP combined with LC-MS/MS and GST pull down demonstrated that SERPINA5 was directly associated with Fibronectin protein, disrupted fibronectin-integrin β1 pathway to suppress HCC cells migration potential. Secondly, we found SMARCA4 gene in 19p13.2 amplicon via integrative analysis of copy number alteration and expression profile results. Q-PCR examination in large scale of HCC tissue sample set indicated that SMARCA4 was an upregulated gene in HCC. Analysis in TCGA and Oncomine database further demonstrated that SMARCA4 gene was obviously upregulated and positively related to tumor grade. In vivo and in vitro experiments showed that SMARCA4 could notably promote HCC cells proliferation. Furthermore, cell cycle and apoptosis analysis suggested that knockdown of SMARCA4 gene lead to Gl phase arrest and apoptosis. In conclusion, we focused on two novel copy number alteration regions in HCC genome, discovered two new HCC related genes, SERPINA5 and SMARCA4, which provided new evidences for HCC pathogenesis mechanisms.Part I Characterization of cancer related genes in 14q31.1-32.13 copy number deletion regionIn our previous studies,14q31.1-32.13 is identified as a novel frequent copy number loss region in HCC genome. To illustrate the function of this region in HCC progression, we planned to characterize candidate cancer related genes in this region through integrative analysis methods. Firstly, by integrating copy number alteration and expression profile data, we identified six downregulated genes, RIN3, KIAA1622, SERPINA3, SERPINA6, SERPINA5 and SERPINA10, located in this region. Furthermore, we examined the six genes in genomic DNA of 125 pairs of HCC patient samples and mRNA of 130 pairs. Only SERPINA5 and SERPINA10 were identically downregulated in both genomic DNA and mRNA. In addition, correlation analysis showed that DNA copy number loss could cause downregulation of both genes. All together, we integrated copy number alteration and expression profile data, observed that frequent loss of 14q31.1-32.13 resulted in downregulation of SERPINA5 and SERPINA10.Part II Functional study of SERPINA5 in hepatocelluar carcinomaWe focused on biological function and clinical significance of SERPINA5 downregulation in HCC. Firstly, we further analyzed SERPINA5 expression levels in the Oncomine database, demonstrated that SERPINA5 was significantly downregulated in HCC. Analysis of SERPINA5 expression level in HCC tumor tissues and pathologic indexes showed that downregulation of SERPINA5 was negatively correlated with tumor stage and intrahepatic metastasis, suggesting that SERPINA5 may play an important role in HCC malignant progression. Stable knockdown of SERPINA5 via shRNA in HCC cell lines promoted cell migratory abilities. In addition, exogeous overexpression of SERPINA5 by lentivirus was able to obviously inhibit in vitro and in vivo metastasis potential of HCC cells. Considering that SERPINA5 is a secreted protein, we collected the conditioned culturing medium from stable knockdown and overexpression cell lines to treat HCC cells. The secreted SERPINA5 also inhibited HCC cells migration. To exclude influences from other factor in conditioned medium, we treated HCC cells with purified human recombinant SERPINA5. We observed concentration-dependent migration inhibiting phenomena in HCC cells. Notably, SERPINA5 did not affect cell proliferation. In conclusion, SERPINA5 was able to inhibit HCC cell in vivo and in vitro metastasis.Part Ⅲ Mechanism study of SERPINA5 inhibiting HCC cell metastasisOur studies focused on underlying mechanisms of SERPINA5 suppressing HCC cell metastasis potential. Firstly, we used Co-IP combined with LC-MS/MS to identify immunoprecipitated SERPINA5 and HCC cell membrane protein associated complex, and found that SERPINA5 was associated with Fibronectin. Moreover, association of SERPINA5 and Fibronectin were verified by bi-direction Co-IP, and GST pull down further demonstrated that SERPINA5 directly binded to Fibronectin. Addition of fibronectin to cells after SERPINA5 treatment could partially rescue the potential of cell migration. We also found that shRNA-mediated knockdown of fibronectin expression attenuated the increased migration of HCC cells caused by targeted knockdown of SERPINA5. Moreover, the migration potential of HCC cells on chambers coated with fibronectin was suppressed by secreted SERPINA5. We observed that SERPINA5 affected phosphorylation level of molecules in integrin β1 pathway, including integrin β1 itself and downstream factors, like FAK, Src. As fibronectin, inhibition of integrin β1, a classical receptor of fibronectin, reversed increased migration of HCC cells caused by targeted knockdown of SERPINA5. Briefly, SERPINA5 directly associated with Fibronectin, disrupted fibronectin-integrin β1 signaling pathway and inhibited HCC cell metastasis.Part IV Functional study of 19p13.2 amplicon and related SMARCA4 gene in hepatocelluar carcinomaIn our previous studies of copy number alteration on HCC genome, a novel amplicon 19p13.2 was identified. Researches showed that this amplicon could specifically cause SMARCA4 upregulation, suggesting that SMARCA4 was a potential cancer related gene play a vital role in HCC progression. We aimed to study SMARCA4 biological function and mechanisms, provide clues for HCC pathological mechanisms and therapy. Firstly, q-PCR and bioinformatical methods showed that SMARCA4 was significantly upregulated and positively related with tumor grade. Then, in vivo and in vitro experiments found that SMARCA4 was able to promote HCC cell proliferation. Moreover, cell cycle analysis revealed that downregulation of SMARCA4 could cause G1 phase arrest and increase percentage of apoptosis cells. Identically, overexpression of exogenous SMARCA4 resulted in cell proliferation increment. In conclusion, we firstly determined that SMARCA4 is not only an upregulated gene, but also a cell proliferation promoting gene in HCC. |
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