| IntroductionGliomas are the most common primary malignant central nervous system, accounting for 44% of intracranial tumors. The tumor showed invasive growth, so surgery is difficult to accurately determine the tumor tissue and normal brain tissue boundaries. In so doing, unnecessary expansion of the region due to surgery greatly increase patient disability, risk of death; the other hand, even in advanced surgical microscope, tumor tissue will be assisted under the total removal of the eye, but there are still about 104 to 105 with strong proliferation of residual tumor cells, which is the basis of glioma recurrence. Because the presence of blood-brain barrier, resulting in the existing anticancer drugs as well as normal brain tissue and irreversible side effects, are also with little success. Relevant statistics show that malignant glioblastoma after combined treatment, the average survival period is still less than 12 months, 2-year survival rate of less than 10%. Therefore, the current treatment of glioma is still recognized as the world's problems.At present the cause is not clear that the glioma is the main reason its difficult to cure. Studies have shown that the incidence of gliomas involving the development of a variety of genes, the complex multi-stage process, including activation of oncogenes and tumor suppressor gene inactivation. Thus, reveal the molecular genetic mechanisms for gliomas to clarify the pathogenesis of glioma and guidance based gene therapy in glioma is of great significance.Human EMMPRIN gene is located in 19q13.3, is about 60kb, contains seven exons and six introns, encodes a 248 amino acid protein. Sameshima and other studies have found that normal brain cells in EMMPRIN mRNA expression was significantly lower than the glioma cells, and mRNA and protein expression levels and positively related to malignant glioma.However, inhibition of EMMPRIN gene expression in glioma cells, glioma cell proliferation and apoptosis in impact, has not been reported in this issue focused on the EMMPRIN gene on glioma cell proliferation and apoptosis impact, is designed to provide the basis for gene therapy of gliomas.PurposeTo investigate effect of EMMPRIN gene siRNA specifi expression on human glioblastoma U251 cells proliferation,apoptosis.MethodsU251 cells were cultured in DMEM medium containing 10% heat-inactivated fetal calf serum and 1% penicillin and streptomycin. Two copies of small interfering RNA were designed to be homologous to wild-type EMMPRIN cDNA sequence and synthesized, as well as one copy of small interfering RNA of negative control. Small interfering RNA was delivered into U251 cells in the form of complex with LipofectamineTM2000 reagent. After 48h of transfection, cells were collected and analyzed for EMMPRIN mRNA and protein expression by RT-PCR and Western blot, respectively. The inhibitory effects of EMMPRIN-siRNA on U251 cells proliferation were assayed with MTT test. Flow cytometry was used to investigate the apoptosis and distribution of cell cycle of U251 cells.Human EMMPRIN gene is located in 19q13. 3, long approximately 60kb, including 7 explicit the son, and six introns, coding produce 248 amino acid composition of proteins. Sameshima etc, the study found EMMPRIN mRNA in gliomas in cells express was much higher than that of normal brain cells and mRNA level and protein level with the degree of expression level and malignant glioma, there is a correlation degree in malignant glioma pleomorphism with the highest level of mother cell tumor (these GBM) in expression for the highest, between degeneration glioma, a low level astrocytomas expression is relatively low. But, restrain EMMPRIN genes in the expression of gliomas cellular proliferation and apoptosis gliomas cells have affected, as yet see report. This topic research on EMMPRIN genes on glial cell proliferation and the influence of apoptosis, for the purpose of gliomas gene therapy provides the basis.ResultsTransfection of U251 cells with small interfering RNA of EMMPRIN can induce the inhibition of EMMPRIN mRNA and protein expression. MTT test showed U251 cells proliferation were significantly inhibited by 36.1﹪and 36.4﹪after transfection with small interfering RNA of EMMPRIN. U251 cells apoptosis were not inhibited by transfection with small interfering RNA of EMMPRIN according to flow cytometry test. The proportion of S phase cells decreased significantly in U251 cells after transfection with small interfering RNA of EMMPRIN.ConclusionDown-regulating the expression of EMMPRIN gene by siRNA can inhibit prolifetation and induce apoptosis in human glioblastoma U251 cells.Therefore,a siRNA-targeted EMMPRIN gene strategy would be a protential and ideal target for gene therapt of glomas. |
