Effect Of CD147 Overexpression On Murine Hepatocacinoma Hepa1-6 Cells

Background and ObjectiveInvasion and metastasis are the most important biological character of malignanttumor and factor of malignant tumor causing death. The study of tumor metastasis mechanism is important significance for the early diagnosis and therapy of tumor metastasis. The modes of tumor metastasis have lymphatic metastasis, hematogenous metastasis and implantation metastasis. But the mechanism of metastasis has not yet been thoroughly explored. Several previous studies have demonstrated that tumor metastasis is not only related with organism microenvironment, but also associated with tumor cells property. The glycosylation of cell surface glycoprotein play significant roles in regulating tumor metastasis.CD147 (EMMPRIN) is a highly glycosylated immunoglobulin superfamily transmembrane protein, which is enriched on the surface of many malignant tumor cells and a membrane spanning molecule. CD147 is composed of two extracellular Ig domains, a single transmembrane domain, and a cytoplasmic domain. The first Ig domain of CD147 is required for counter receptor binding activity, which is involved in MMP induction and oligomerization. The second Ig domain of CD147 is known to associate with Caveolin-1(Cav-1). As a result of heterogeneous N-glycosylation, CD147 exists in both a highly glycosylated form, HG-CD147 (~40-66KD) and lowly glycosylated form, LG-CD147 (~33KD). But the relationship of CD147 glycosylation and tumor metastasis is not clear. CD147-mediated MMPs induction could be a common mechanism in physiological and pathological situation, such as tissue remodeling, development, reproduction, tumor, arthritis and ulceration. On the other hand, CD147 is also highly expressed on the cell surface of various tumors. It has been confirmed that the expression of CD147 is high in many malignant tumor. Later studies have shown that CD147 level was detected in numerous malignant tumors, such as lung cancer, hepatoma, neurogliocytoma, esophageal cancer, laryngeal carcinoma, ovarian cancer and breast cancer. As an adhesion molecule of the tumor cell surface, CD147 can mediate the interaction of tumor and matrix, induce stromal fibroblast sand tumor cells themselves to produce MMPs, promote tumor angiogenesis, and promote tumor metastasis. Potential regulators that influence CD147 level and its MMP inducing activity include glycosylation,growth factors, hormones and membrane shedding.The main focus of this article was to clone CD147 cDNA, construct CD147 gene eukaryotic expression vector, stably overexpress CD147 in Hepa1-6 cells and observe some related biological changes and the changes of glycoform of CD147. Our experiment could provide a basis for the research of relationship of CD147 glycosylation and the metastatic capability of Hepa1-6, and supply a new target of drug design for anti- tumor.Methods1. Total RNA was extracted from mouse lung tissue, then the murine CD147 gene was amplified by RT-PCR and cloned into the clone vector named PMD18-T simple vector. The recombinant vector was transformed into E.coli. DH5a, the positive clones were selected and plasmid DNA was identified by PCR and restriction enzyme analysis. Then, CD147 gene was subcloned into the pcDNA3.1 expression vector, the positive clones were selected and plasmid DNA was identified by restriction enzyme analysis and sequencing.2. The recombinant vector was transfected into Hepa1-6 cells by lipofectamine, the positive cell clone was selected by G418. PCR and Western blot were used to detect the expression of CD147.3. The changes of glycoform of transfection positive cells were analyzed by Western blot. The biological behavior change was observed by ECM invasion assay. Results1. The cDNA of CD147 was successfully cloned from the total RNA of mouse lung tissue. Restrictive enzyme analysis and DNA sequencing showed that the murine CD147 cDNA of 850bp was successfully inserted into pcDNA3.1 express vector which was assigned as pcDNA3.1/CD147.2. Western blot and RT-PCR analysis results showed that CD147 was overexpressed stably in Hepa1-6 cells by transfecting pcDNA3.1/CD147.3. In Hepa1-6 cells of CD147 overexpression, both levels of HG-CD147 and LG-CD147 were increased, and the ability of invasion was enhanced significantly.ConclusionsOverexpression of CD147 in Hepa1-6 cells didn't affect the CD147 glycosylation, but enhanced the invasion ability of Hepa1-6 cells.