| ? Objective ? 1.The expression of(extracellular matrix protein 1,ECM1),(epithelial-mesenchymal transitions,EMT)-related markers(Vimentin and E-cadherin)in(hepatocellular carcinoma,HCC)and normal liver tissues were detected by immunohistochemical staining method,to assess the relationship between the expression of ECM1 and clinicopathologic factors,and the survival of HCC patients after surgery.To investigate the correlations between the expressions of ECM1 with Vimentin and E-cadherin in HCC.Explore the role may play of ECM1 in invasion and metastasis process in HCC.2.Established the HCC Bel-7402 cells which can stable transfected by ECM1,make a experimental basis for the study of the role in HCC progression of ECM1.3.Study the changes of Bel-7402 liver cancer in cell phenotype,invasion and metastasis after stable transfected by ECM1,and to investigate the relationship between epithelial-mesenchymal transformation.4.Explore the possible molecular mechanisms that ECM1 promote HCC Bel-7402 cells occur epithelial-mesenchymal transition.? Methods ? 1.77 patients with pathologically confirmed HCC made surgical liver resection surgery in Anhui Provincial Hospital Affiliated Anhui Medical University between January 2008 and January 2012 were randomly selected.The paraffin blocks archived specimens for 3?m thick paraffin sections.They were done ECM1,Vimentin and E-cadherin immunohistochemical staining to analyze the relationships among the expressions and clinicopathological parameters in HCC.2.The ECM1 gene sequence was provided by Gen Bank database.The human hepatoma cell Bel-7402 was transfected with vector p EGFP-N2-ECM1 by liposome-mediated transfection,which can gained the stable ECM1/Bel-7402 cell model with high expression of ECM1 protein.3.RT-PCR,Western blot analysis were used to detected the expressions of EMT marker(Vimentin,E-cadherin).MTT assay method was used to detect the adherent ability,and Transwell test method was used to examine the ability of invasion of transfected cells.The cell morphology changes of hepatoma cell line ECM1/Bel-7402 were observed and EMT-related marker m RNA and protein levels were detected before and after transfection.So as to explore whether ECM1 could promote the invasion and metastasis of liver cancer cells by regulating epithelial-mesenchymal transition.4.In order to clear the molecular mechanism of ECM1 regulating epithelial-mesenchymal transformation in HCC,SB431542 was used to blocke TGF-?-Smad pathway,the protein expression levels changes of Vimentin and E-cadherin was detected,and changes in tumor cell morphology,invasion and migration abilities also be detected.To clarity the role of TGF-?-Smad pathway in the process of epithelial-mesenchymal transformation in liver cancer cells Bel-7402.? Results ? 1.In 77 HCC tissue specimens,there were 57 cases with different expression levels of ECM1,the positive rate(57/77)was significantly higher than that in normal liver tissues(4/17),the difference was statistically significant.In HCC tissues,the expression of Vimentin positive rate of 61.0%,E-cadherin expression positive rate of 46.8%,53.2% rate of loss of expression.Statistical analysis showed,ECM1 positive expression rate of HCC tissues correlated to vascular invasion and TNM classification,the positive rate of Vimentin and E-cadherin expression loss rate of liver cancer with clinical stage was significantly correlated,and in hepatocellular carcinoma the ECM1 positive rate was positively correlated with the expression of Vimentin,no significant relationship between E-cadherin.2.Construction ECM1 eukaryotic expression vector p EGFP-N2-ECM1,by liposome-mediated transfection of human hepatoma cell Bel-7402,750?g / ml G418 to select positive clones cells after three weeks to expand culture,RT-PCR and immunocytochemistry staining techniques to identify screening expand,which can gain the stable ECM1/Bel-7402 cell model with high expression of ECM1 protein.3.Morphological observation showed ECM1 transfected Bel-7402 cell line make the occurrence of EMT consistent morphological changes,cell growth by the group,round shape to discrete growth,fusiform morphology transformation;compared with the control groups,the experimental group Vimentin expression was upregulated and E-cadherin expression was down-regulated.Vitro migration test(scratch test)and invasive tests(Transwell chamber assay)results show that enhanced expression of transfected genes ECM1 in Bel-7402 cells,the ability of migration and invasion of tumor cells significantly increased than the control group.4.After treated by SB431542 in ECM1 / Bel-7402 cells,the p-Smad expression level was significantly lower than the control group,indicating that SB431542 blocked ECM1 of TGF-?-Smad pathway activation and Vimentin expression level were significantly decreased,epithelial marker E-cadherin expression levels were significantly increased,these results suggest ECM1/Bel-7402 cells induced EMT process,and the TGF-?1 may the key signaling pathways.To further assess the impact of SB431542 blockade on tumor cell invasion,using Transwell experiment found that after SB431542 treated,ECM1/ Bel-7402 cells migration significantly reduced compared with the control group,suggesting that SB431542 may via blocked TGF-?-Smad after the signal transduction pathway to inhibits the invasion of tumor cells.It is further illustrated that by blocking TGF-?-Smad pathway could block the signaling pathway of ECM1 in tumor cells to reverse the transformed phenotype of substance between the epithelium,hindering the abilities of invasion and metastasis in tumor.?Conclusions?1.ECM1 positive rate in liver tissues was significantly higher than normal liver tissue,and related to vascular invasion and TNM classification of liver cancer,it could be as an independent predictor in HCC patients of metastasis and recurrence after surgery;the positive expression rate of interstitial markers Vimentin was positively correlated with the expression of ECM1 in HCC.2.Hepatoma cell line Bel-7402 after stably transfected expression ECM1 genes,tumor cells in morphology and related molecules expression level occurred with EMT consistent phenotypic changes,and the abilities of migration and invasion in tumor cells are significantly enhanced.3.ECM1 possible induced liver cancer cells Bel-7402 occur EMT via TGF-?-Smad transduction pathway,TGF-?1 as the key link in the signal,maybe as a new effective target which can interfere invasion and metastasis in HCC. |
PDF Full Text Request