| Hepatocellular carcinoma (HCC) remains the most frequent cancer in the world. Since promoter hypermethylation of tumor suppressor genes TSGs is a frequent event in HCC and may play an important role in hepatocarcinogenesis. Thus, identification of novel TSGs will be of great importance in the understanding of the mechanisms of HCC.RAS signaling pathway is frequently activated in human tumors. The Ras-Association Domain Family (RASSF) is a group of proteins that contain Ras association domains that can potentially bind Ras oncoproteins and mediate Ras-regulated functions. Recently a new member of the RASSF family was identified that is termed N-terminal RASSF gene RASSFIO. However, the functional characteristics of the new member in human cancers are largely unclear. Here, we examined the biological functions and related molecular mechanisms of RASSF10in hepatocellular carcinoma (HCC).In this study, we examined the expression of RASSF10in HCC and figured out the relationships between the promoter methylation and the inactivation of RASSFIO. In addition, we determined whether ectopic expression of RASSF10had tumor suppressing effect in HCC in in vivo and in vitro. We also testified the potential downstream targets of RASSF10by adhesion gene-expression assay and qPCR validation. The regulation of MMP2via FAK signaling pathways by RASSF10was further investigated.Material and Methods1. The relationship between promoter hypermethylation and the expression of RASSF10in HCC.①The expression of RASSF10mRNA in20paired HCCs include primary HCCs and adjacent normal tissues were determined by qPCR, and RASSF10expression in a panel of HCC cell lines (n=8) and in normal liver tissue were examined by RT-PCR.②The mRNA expression of RASSF10before and after de-methylated treatment with5-aza-2’-deoxycytidine (5-Aza-DC) were examined by RT-PCR.③The methylation status of RASSF10in HCC cell lines were determined by methylation specific PCR (MSP) and Bisulfite Genomic Sequencing (BGS).2. The functional role of RASSF10in HCC.①Cell proliferation by ectopic expression of RASSF10was confirmed by Colony formation assay and MTS assay.②Cell apoptosis and cell-cycle were assessed by flow cytometry. Further, Apoptosis and cell cycle related proteins were analysised by western blot.③Xenografted HCC model was used to validate the tumor suppressing effect by ectopic expression of RASSF10in vivo.④Cell migration and invasion were investigated in the24-transwell cell migration and collagen-based cell invasion system.3. Screening and validation of the downstream targets related with the adhesion-related signaling pathway of RASSF10in HCC. ①The Adhesion Gene-expression Array and qPCR validation analysis were performed in QGY7703cells transfected with RASSF10or empty vector.②Ectopic expression of MMP2in QGY7703/RASSF10, then cell invasion assay was observed.③Western blot analysis validated the relationship between the expression of MMP2and FAKs (FAK, pFAKY397, pFAKY925) in QGY7703cells transfected with RASSF10or empty vector.Results1. RASSF10is downregulated mainly through promoter hypermethylation①The mean mRNA expression level of RASSF10was significantly lower in primary HCCs as compared to their adjacent normal tissues (P=0.003,n=20). RASSF10is expressed in normal human liver tissue, but silenced or down-regulated in62.5%(5/8) of HCC cell lines.②The expression of RASSF10mRNA was dramatically re-activated after demethylated treatment with5-Aza-DC in HCC cell lines.③By MSP and BGS, full or partial methylation was observed in HCC cell lines (BEL7404, QGY7701,QGY7703, HepG2and Hep3B), whereas methylation was not detected in the cell lines with RASSF10expression(SK-Hepl, PLC5, Huh7).2. RASSF10significantly inhibited proliferation, impaired migration and invasion of HCC cells(DEctopic expression of RASSF10in HepG2and QGY7703cell lines caused a significant decrease in cell viability by MTS (p<0.01). We observed that the number of surviving colonies formed on the plates was significantly reduced in RASSF10transfected cells when compared to the control vector transfectants (p<0.01).②We observed that RASSF10-transfected cells (HepG2and QGY7703) showed high G1phase populations in comparison to the empty vector transfectants.But there is no difference in cell apoptosis. Ectopic expression of RASSF10in QGY7703upregulated p21and P27expressions and downregulated cyclinDl and CKD2expression.③Ectopic expression of RASSF10suppresses tumor growth in a mouse xenograft model.④Ectopic expression of RASSF10(HepG2and QGY7703) significantly impaired cell migration (p<0.01) and invasion (p<0.05).3. RASSF10modulates multiple downstream genes related with cell adhesion especially MMP2via FAK signaling pathways①Our results showed that ADAMTS1, ADAMTS13, ITGA6, PECAM1and TIMP2were up-regulated, while ITGB2, MMP2and TGFBI were down-regulated by overexpression of RASSF10in QGY7703cells.②Ectopic expression of MMP2in QGY7703/RASSF10caused a significant increase in cell invasion property (p<0.01).③To obtain more clues, we examined the role of Focal Adhesion Kinase (FAK), which is involved in the production of MMPs. Western blot fond that overexpression of RASSF10can decrease the total and phosphorylation (Y397, Y925) level of FAK.Conclusions1. DNA methylation may be associated with the transcriptional silencing of RASSF10in HCC cell lines.2. Ectopic expression of RASSF10significantly inhibited proliferation through induction of Cell Cycle Accumulation in G1Phase. In particular, re-expression of RASSF10suppresses tumor growth in a mouse xenograft model. Meanwhile RASSF10impaired migration and invasion of HCC cells. RASSF10functions as a candidate tumor suppressor in hepatocellular carcinoma (HCC). 3. The suppression of cell invasion by RASSF10may be mediated through MMP2via FAK signaling pathways. |
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