| Hepatocellular carcinoma(HCC) is one of the most common cancers in China in terms of number of cases,and the invasion and metastasis are the leading cause for the HCC death,so it is of great clinical importance to investigate the mechanism of invasion and metastasis,and the effectual therapeutic way of anti-metastasis and relapse of HCC.Previous study has showed that the HGF/c-Met signal pathway play a crucial role in the invasion and metastasis of HCC,and the more activeness of HGF/c-Met system after hepatectomy due to the regeneration of remnant thepatic tissue,which promotes proliferation of remnant HCC cells.The critical role of HGF/c-Met signaling in HCC has sparked the active interest in searching for the means of the therapy of HCC through targeting it,as a result,many molecules of inhibitor targeting this signaling were come forth during the latest few years,such as the competed and selected inhibitor NK4 and PHA-665752,however,little progression has been achieved due to the cross-talking between the HGF/c-Met signaling pathway and the other signaling pathway,for example the cross-talking between HGF/c-Met and integrin signaling pathway.The latest study had reported the tetraspanin CD151 is involved in the signaling through form a complex with c-Met, but the role and expression of CD151 in HCC,and the detailed mechanism of CD151 in HGF/c-Met signaling pathway remains unknown.In the present study,we investigated the expression of CD151 and c-Met in HCC cell lines with different metastatic potential,and HCC,their adjacent nontumorous and normal liver tissues, and then explored the changes of invasive and metastatic ability in non-metastatic cell line HepG2 through CD151 cDNA transfection and high metastatic cell line HCCLM3 through silencing CD151 in vivo and vitro.We assayed the clinical significance of CD151 and combined expression of CD151 and c-Met in HCC samples using tissue microarrays(TMAs) and quantitative reverse transcription-polymerase chain reaction(qRT-PCR).Finally,the effect of CD151 on the HGF/c-Met signaling pathway was studied. Part one Overexpression of tetraspanin CD151 in hepatocellular carcinomaObjective:To investigate the relationship between the expression of CD151 and the carcinogenesis of HCC.Methods:The expression of CD151 was evaluated in HCC,their adjacent nontumorous and normal liver tissues by qRT-PCR and immunohistochemistry.Result:CD151 mRNA can be detected in all samples of three different type tissues, the CD151 mRNA level was significantly higher in HCC than those in their adjacent nontumorous and normal liver tissues(p＜0.05),the mRNA level in adjacent nontumorous also higher than that in normal liver tissues(p＜0.05).The CD 151 protein was located in the cytoplasm of HCC cell,the level of CD151 protein was consistent with the CD151 mRNA(p＜0.05).Conclusion:The overexpression of CD151 may be related to the carcinogenesis of HCC.Part two Correlation and clinical significance of overexpression of CD151 and/or c-Met in hepatocellular carcinomaObjective:To investigate the correlation and clinical significance of CD151 and/or c-Met with hepatocellular carcinoma.Methods:The qRT-PCR was used to detect the expression of CD151 and c-Met in HCC,the TMA was used to detect the expression of CD151 and c-Met in 520 HCC patients,then the clinical significance of overexpression of CD 151 and/or c-Met was analyzed by SPSS11.5.Result:Overexpresvion CD151 and c-Met accounted of 59.8%(311/520) and 54.4% (283/520) of total patients,respectively.Overexpression CD151 was found to correlate significantly with portal vein tumor thrombus(PVTT),multiple tumor number,high TNM stage and low differentiation(p＜0.05).However,other clinical characteristics,including age,gender,preoperative serum AFP and tumor size were not directly related to the overexpression of CD151.As for the overexpression of c-Met was related significantly to PVTT,large tumor size,multiple tumor number, high TNM stage and low cell differentiation(/7＜0.05).The 3,5,7,year survival rate of CD151~+ was much lower than that of CD151~-(p＜0.01),furthermore,3,5,7 year survival rate of CD151~+/c-Met~+ was much lower than those of CD151~-/c-Met~+ and CD151~-/c-Met~-.Conclusion:The overexpression of CD151 and high expression of CD151/c-Met can be a new marker in predicting the prognosis of HCC,and the tetraspanin CD151 could affect the signaling of HGF/c-Met.Part three Relationship of overexpression CD151 with the invasion and metastasis of hepatocellular carcinomaObjective:To investgate the relationship of CD151 with the invasion and metastasis of HCC cells.Method:the qRT-PCR,western blot and FCM was used to detect the expression of CD151 and c-Met in different HCC cell lines with different different metastatic potential.We then transfected the pcDNA3-CD151 cDNA and pGPU6/GFP/Neo-CD151 plasmid into the non-metastatic cell line HepG2 and the highly metastatic cell line HCCLM3,the expression of CD151 was determined by FCM,western blot in the stable transfection HCC cell lines.The secretion of MMPs was detected by gelatin zymography,and the ability of proliferation and invasion was tested by MTT and transwell in the the stable transfection HCC cell and their parental cell lines,respectively.At the last,metastasis assays in vivo was performed.Result:The secretion of MMP9 was upregulated in CD151 high expression group, while the activity of MMP-2 was not affected by the expression of CD151;the ability of proliferation did not alter between the high and low CD 151 expression groups,the transwell showed that the numbers of invaded cells in HCCLM3,HCCLM3-Mock and HepG2-CD151 were 36.8±14.4,32.3±10.2 and 26±8.8,which were significantly higher than those in HCCLM3-CD151-shRNA and HepG2, HepG2-Mock(20.3±10.7 and 17±7.7,14±6.3).The pulmonary metastasis rates and metastatic tumor clusters per mouse were 100%(5/5) and 220±53,100%(5/5) and 238±55,60%(3/5) and 145±43 in HCCLM3,HCCLM3-Mock cells and HepG2-CD151 cells group respectively,while 40%(2/5) and 114±46,0%(0/5) and 0%(0/5) in HCCLM3-CD151-shRNA cells,HepG2 and HepG2-Mock cells groups, respectively,with a statistical significance(p＜0.05).Conclusion:The CD151 was positive correlation with the ability of invasion and metastasis. Part four Role of CD151 on the HGF/c-Met signaling transduction pathway in Hepatocellular carcinoma cellsObjective:To expatiate the relationship between CD151 and c-Met in HCC cells, and the role of CD151 on HGF/c-Met signal pathway in hepatocellular carcinoma (HCC) cells.Methods:we investigated the expression of CD151 and c-Met in different metastatic potential of HCC cell lines by immunofluorescence and confocal laser scan microscopy,and the presence of CD151/c-Met complex was confirmed in HCCLM3 cells by immunoprecipitation,then the phosphorylation and non-phosphorylation of Akt,FAK and ERK,which are key molecules in HGF/c-Met signal pathway,were tested by western blot after HGF acting on the modified and parental cells.Result:The expression and location of CD151 and c-Met were unanimous,and formed a complex in HCC cells.The phosphorylation of FAK and Akt was higher in high expression of CD151 cells than in low expression cells(p＜0.05),while the phosphorylation of ERK had no difference between the two kinds of HCC cells (p＞0.05).Conelusion:The CD151 affect the HGF/c-Met signal transduction pathway through the signal molecules of FAK and Akt. |
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