Expression Of Extracellular Matrix Protein1in Hepatocellular Carcinoma Cells And The Effect Of It On Biological Characteristics Of Hepatic Carcinoma Cell Line SMMC-7721and Human Umbilical Vein Endothelial Cells

【Objective】Evaluate extracellular matrix protein1(ECM1) expression in four HCCcell lines with various metastatic potentials and one normal liver cell line by reversetranscriptase polymerase chain reaction (RT-PCR), Western blotting, andImmunocytochemistry to determine the relationship between the expression level andHCC cell lines metastatic potential. In addition, we have constructed ECM1EukaryoticExpression Vector pEGFP-N2-ECM1and transfected it into SMMC-7721cells withlipofectamine, to study the biological effect of ECM1on human hepatic carcinoma cellline SMMC-7721and human umbilical vein endothelial cells (HUVEC).【Methods】 RT-PCR、Western blotting and Immunocytochemistry was used toevaluate ECM1expression in four HCC cell lines with various metastatic potentials andone normal liver cell line, then the specific recombinant plasmids pEGFP-N2-ECM1was designed according to the ECM1sequence in GenBank. The sequence analysis wasperformed. The vector was transfected with lipofectamine into SMMC-7721cells, andthe cells harboring ECM1genes were selected by G418pressure. The SMMC-7721cells expressed pEGFP-N2-ECM1was determined by GFP fluorescence, RT-PCR andImmunocytochemistry. Then we have investigated the biological characteristics ofECM1transfected SMMC-7721. The effect of ECM1on the transfected SMMC-7721adhesive abilities to matrigel was detected by MTT assay. The effect of ECM1oninvasion and motility of the transfected SMMC-7721were investigated by reconstitutedmatrigel invasion and polycarbonate filters migration experiments. The effect of ECM1on proliferation of the transfected SMMC-7721was detected by MTT assay. In addition we also have detected the effect of ECM1on the proliferation of HUVEC by MTTassay and on the capability of forming capillaries of HUVEC by matrigel assay.【Results】we found that ECM1expression was leveled with increasing metastaticpotential of cell lines. Among the four cell lines, the expression level in HCC-LM3,which with highest metastatic potential, was significantly higher than that with lower;the expression level in SMMC-7721with lowest metastatic potential was lowest. Theexpression level in normal liver cell line L-02was similar with that in SMMC-7721.The recombinant eukaryotic expression vector pEGFP-N2-ECM1was obtained and itwas confirmed that ECM1cDNA was inserted into the eukaryotic expression vectorcorrectly using PCR, digestion identification and sequencing．It was showed that ECM1gene had been transfected into SMMC-7721cells using lipofectamine and the resultwas detected by GFP fluorescence, RT-PCR and immunocytochemistry. The shape andbiological characteristics of ECM1transfected SMMC-7721cells were not significantlydifferent with those untreated and mock transfected SMMC-7721cells．Proliferationand the ability of forming capillaries of HUVEC in ECM1transfected group was higherthan those untreated and mock transfected group．【Conclusions】ECM1is expressed the hepatocellular carcinoma cells and correlatedwith the metastasis properties of them， ECM1can significantly promote theproliferation and forming capillaries of HUVEC．but it has no effect on SMMC-7721invitro．...