Experimental Study Of Basigin/CD147 In Glioblastoma Progression And Its Antisense RNA In Inhibiting Tumor Cell Invasion And Angiogenesis

Gliomas are the most common of brain neoplasms and exhibit a high degree of diffuse local invasion of normal nervous tissue. High invasion is the principle reason to the high mortality of Gliomas. Tumor cells often infiltrate beyond any obviously defined tumor margin and contribute to the high incidence of recurrence. In the invasive and metastatic process of malignant tumor, an important step is the degradation of extracellular matrix (ECM).Molecules existing in ECM and receptors or ligands existing on the surfaces of tumor cells play critical roles.CD147, also named extracelluar matrix metalloproteinase inducer (EMMPRIN). EMMPRIN expressed by cultured tumor cells stimulates fibroblasts to produce very high levels of collagenase activity, which likely facilitates tumor metastasis. Previous studies demonstrated that EMMPRIN concentrated on the surfaces of most tumor cells, promoted invasion of tumor cells by stimulating stromal cells to produce elevated levels of several matrix metalloproteinase (MMPs) which play very important roles in several aspects of tumor progression, including growth, invasion, metastasis, and angiogenesis. Moreover, supporting its key role in the processes of tumorigenesis and metastasis, EMMPRIN was reported as one of the most constantly up-regulated mRNA in metastatic cells. Our interest in gliomas let us to consider the role of CD147 in glioma invasion, metastasis, and angiogenesis.Here, we investigated expression of Basigin/CD147 in normal brain and gliomas of varying malignancy by using tissue array and tissue obtained from surgical removal. We constructed a vector of antisense RNA of CD147 and investigated its inhibitory effects on invasion and angiogenesis of glioblastoma cells in vitro. We also study the effects of antisense RNA of CD147 to tumor formation in nude mice and the therapy effect to tumor-bearing nude mice.Part 1: Expression of Basigin/CD147 in normal brain and gliomas.We investigated expression of Basigin/CD147 in normal brain and gliomas of varying malignancy by immunohistochemistry using tissue array and tissue obtained from surgical removal. Immunolocalization revealed quite different distribution in non-neoplastic brain and glioma: In non-neoplastic portions of the brain, EMMPRIN immunostaining was demonstrated only in vascular endothelial cells, whereas in glioma the tumor cells were stained positively, the positive immunohistochemical staining of gliomas was 74.3%. In astrocytomas, varying percentages of the tumor cells were positive, level of immunohistochemical staining of higher grade astrocytomas(grade III, IV) was higher than that of lower grade astrocytomas(grade I,II). The RT-PCR study indicated that level of Basigin/CD147 mRNA expression was higher in gliomas(0.274±0.087) than in non-neoplastic brain(0.081±0.043). Thus, the Basigin/CD147 mRNA level was up-regulated significantly in gliomas compared with non-neoplastic tissue(P<0.05 by unpaired t-test). Part 2: Construction of the antisense RNA expression vector of Basigin/CD147. Transfection of the antisense RNA vector to U251 and selection of the positive clone.Full length cDNA fragment coding for basigin/CD147 was cloned in the antisense orientation into the XbaI and XhoI site of the eukaryotic expression vector PCI-neo. The antisense vector of basigin, named as PCI-147.Empty vector PCI-neo and antisense vector PCI-147 were transfected into human glioblastoma cell line U251 via cation liposome Lipfectinamine2000 (Gibco) according to the manufacturer's description and the two kinds of transfected cells were named as U251/neo and U251/147 respectively. Stable antisense transfectants were generated and characterized. Basigin/CD147 has been shown to play significant roles in ECM degradation. Transfection of U251 cells with the antisense vector strongly inhibited the expression of CD147 as compared to U251, U251/neo.Part 3: Study on the inhibitory effects of antisense RNA on proliferation of U251 in vitro.Gelatin zymography was used to analyze the effect of antisense RNA on MMPs secretion. The results showed that the secretions of MMP-9 and MMP-2 in the antisense transfectants were inhibited compared to parental- and empty vector-transfected cells in Gelatin zymography assay. Since CD147 has implicated in the invasiveness of tumor cells, we evaluated the migration of parental and stable transfectants. In scrape-wound-migration assay, the mobility of stable antisense transfectants was decreased as compared to parental- and empty vector-transfected cells. Matrigel-Boyden chamber method was used to study the inhibitory ability of antisense RNA on tumor cells invasion and mobility through the reconstituted basement membrane. The results showed that the number of stable transfectants infiltrated cells of U251/147 were less than those of U251/neo and intact U251. More over, ELISA was used to detect the VEGF (Vascular endothelial growth factor) production in the supernatant of selected cells. Downregulation of CD147 by antisense transfection in U251 cells resulted in a reduction of VEGF secreted into media by antisense tumor cells compared to parental- and empty vector-transfected cells.