Inhibitory Effect And Mechanism Of The Raf Kinase Inhibitor Protein Expression In Heparanase On HCCLM3Cell

Hepatocellular carcinoma (hepatocellular carcinoma, HCC), is theworld's fifth most common malignancy, accounting for the cancer cause ofdeath of HCC is of the most common malignant most malignant tumor of theeasy recurrence, metastasis and poor prognosishigh mortality characteristics.Its poor prognosis reasons for the high-level fatality and hepatocellularcarcinoma Yi biological characteristics of invasion and metastasis. Due to thecomplexity of tumor invasion and metastasis and its regulation, its exactmechanism is unclear, and still can not, in theory, for clinical treatment toprovide a clear direction for clear malignant tumor invasion and metastasisand its regulation of the molecular mechanism of has become the urgent needof clinical improve cancer treatment in this area has also become one of themajor hot spots in molecular oncology and other disciplines of basic theory.Heparanase (heparanase Hpa) is a glucuronic acid enzyme, tumordevelopment, metastasis and tumor angiogenesis plays an important role. Rafkinase inhibitory protein (Raf kinase inhibitor, protein, of RKIP) is a highlyconserved phosphatidylethanolamine-binding protein (phosphatidylethano-lamine binding protein, PEBP) family, widely exist in various organisms. TheRKIP involved in regulation of multiple signal transduction pathways,including inhibition of Raf-MEK1/2-ERK1/2and NF-κB signal transductionpathways play an important role in the physiological process of cell growth,proliferation, differentiation, and apoptosis. Recent studies have shown thatRKIP and tumor invasion and metastasis, but the role of RKIP expression inhepatoma cells and its mechanism is not clear.In this study, first observed the RKIP gene stably transfected in vitrocultured liver cancer cells, detect RKIP role, Raf-MEK1/2-ERK1/2signaltransduction pathway related proteins and Hpa expression, to understand the RKIP expression in hepatoma cellin the mechanism, followed by liver cancercells Raf-MEK1/2-ERK1/2signal transduction pathway activating agents EGF,the detection Raf-MEK1/2-ERK1/2signal transduction pathway relatedproteins and Hpaexpression, to further clarify the RKIP inhibition of hepatomacell Hpa expression and Raf-MEK1/2-ERK1/2signal transduction pathway.Objective:To HCCLM3liver cancer cells,empty vector transfectedHCCLM3hepatoma cells and stable transfection of RKIP gene HCCLM3liver cancer cells of RKIP in the detection of liver cancer cells Hpa, P-ERKexpression to further clarify the RKIP inhibited the expression of liver cancercells Hpa whether the Raf-MEK1/2-ERK1/2signal transduction pathway.Methods:1Empty vector transfected HCCLM3hepatoma cells andstably transfected into RKIP gene of the HCCLM3liver cancer cells, extractcell protein, the BCA Protein Assay. Western blotting detection of RKIP,P-ERK and Hpa protein expression, the observed expression of the P-ERKHpa whether RKIP expression.2HCCLM3liver cancer cells, RKIP gene HCCLM3hepatoma cells stablytransfected cell extract protein, BCA Protein Assay. Western blotting detectionof RKIP, P-ERK and Hpa protein expression observed expression of RKIPinhibition of hepatoma cell Hpa and Raf-MEK1/2-ERK1/2signal transductionpathway.3HCCLM3liver cancer cells, stably transfected with RKIP geneHCCLM3liver cancer cells by adding EGF to activate the ERK pathway, cellprotein was extracted after15minutes, the BCA Protein Assay. Westernblotting detection of RKIP, P-ERK and Hpa protein expression, furthervalidate RKIP inhibition of the expression of liver cancer cells Hpa andinhibition Raf-MEK1/2-ERK1/2signal transduction pathway activation.Results:1RKIP expression in RKIP transfected the group HCCLM3cells was significantly higher than the empty vector transfected group (P<0.05), while the expression of P-ERK and Hpa was significantly lower thanthe empty vector transfected group (P <0.05); With HCCLM3cellsof RKIPexpression was increased in the P-ERK, of Hpa expression decreased. 2RKIP expression in RKIP transfected the group HCCLM3cells wassignificantly higher than the blank control group (P <0.05), while theexpression of P-ERK and Hpa was significantly lower than the blank controlgroup (P <0.05); With HCCLM3cells of RKIP expressionincreased, theP-ERK, of Hpa expression decreased.3The EGF (+) control Group HCCLM3cells, P-ERK expression wassignificantly higher than that of EGF (+) of RKIP transfected group (P <0.05),EGF (+) control group HCCLM3cells Hpa expression was significantlyhigher than that of EGF (+)RKIP transfer dye group (P <0.05); RKIPexpression as HCCLM3cells increased ERK pathway activation, the P-ERKHpa expression is still down.Conclusions:1Low expression of RKIP in the empty vector transfectedHCCLM3cells, while the P-ERK Hpa highly expressed high RKIP expressionin RKIP transfected HCCLM3cells, while the P-ERK the Hpa low expression,indicating that the RKIP gene transfection HCCLM3cellssuccessful.2In HCCLM3cells RKIP low expression, while the P-ERK Hpa highexpression, while high expression of RKIP in RKIP transfected HCCLM3cells, the P-ERK Hpa low expression of RKIP in HCC cells may be via ERKpathway to inhibit the expression of Hpa.3Control of RKIP the group HCCLM3cells with low expression, theP-ERK Hpa high expression of EGF (+), and in the EGF (+) high expressionof RKIP transfected the group HCCLM3cells of RKIP, and the P-ERK theHpa low expression, indicating thatthe RKIP the expression in hepatoma cellsinhibit the expression of Hpa and inhibition of ERK signaling pathwayactivation.