Influence Of Small Interfering RNA Targeting MMP-9 Gene On Growth And Invasion Of U251 Cells In Vivo And In Vitro

Glioma is the most common primary malignant tumor in the central nervous system. Owing to the feature of its invasive growth, fast cell proliferation and high recurrence rate, glioma is hard to be removed completely by the currently available combined therapeutic approaches, such as surgical resection, postoperative radiotherapy, chemotherapy and immunotherapy etc. Its high recurrence rate seriously affects the life quality and overall survival of the patients, and the clinical prognosis is poor.As the knowledge of tumor biology and molecular genetics increased, it has been shown that the development of gliomas, just the same as the tumors in the other sites of the body, results from the activation of proto-oncogenes and inactivation of tumor suppressor genes, and involves multiple genetic and molecular alterations. Therefore, seeking more genes associated with the gliomagenesis, a comprehensive understanding of the molecular pathology of gliomas., and optimizing treatment strategies and developing novel therapeutic approaches for gliomas on this basis, have become an important research project for gliomas. It can be said that as one of the hot spots in the study of gliomas, the gene therapy for correcting the aberration of genetic events in gliomas has been the forefront focus in medical research field.In the present study, we focused on the abnormal MMP-9 activity in gliomas and the influence of specific small interfering RNA (siRNA) targeting MMP-9 gene on growth and invasion of human brain glioblastoma U251 cells in vitro and in vivo. The study was divided into 3 parts.In the first part, the protein expression of MMP-9 were studied in 49cases of gliomas and 4 normal brain tissues and their correlation with degree of malignancy of gliomas by immunohistochemistry. The relationship between the MMP-9 expression and the invasion in various grade of gliomas was also studied.In the second part, RNAi technology was used to observe its inhibitory effect on the growth of human glioblastoma U251 cells. siRNA targeting MMP-9 was transfected into U251 cells mediated by oligofectamine. MMP-9 mRNA expression were detected by PCR after transfection. The expression of MMP-9 Protein was also studied by Western blotting and immunofluo- rescence staining after transfection. The phenotypic change of U251 cells including proliferation, cell cycle and invasive ability after RNAi trasfection was studied by MTT assay, flow cytometry, Matrigel 2D , 3D growth method and Transwell invasive model.In the third part, subcutaneous U251 glioma model was established in nude mice. Every 4 days the mixture of oligofectamine and siRNA was injected into the tumors and the tumor volumes were measured. On 28nd day after the first injection, tumors were resected. The expression of MMP-9 were detected by immunohistochemistry in tumors of each group.ResultMMP-9 expression increased correspondingly to the ascending of tumor grade. In immunohistochemical study, the positive stained-cell rate of MMP-9 increased with the degree of malignancy of tumors, and their differences of positive rate and expression level between the low-grade and high-grade tumors were statistically significant.RT-PCR and Western blotting revealed significantly lowered mmp-9 expression at both RNA and protein levels in transfected U251 cells, which exhibited a significantly lower growth rate after transfection as shown by cell growth curve and MTT assay and flow cytometry analysis (P<0.001). Cell invasive capability assay demonstrated the infected cells by MMP-9siRNA showed a lower ability than others in 2-dimensional and 3-dimensional model and matrigel-coated transwell model.Subcutaneous U251 glioma model was established in nude mice for in vivo study. As compared with the control group, the tumors in mice treated with siRNA targeting MMP-9 grew slowly and the difference of tumor volumes became significant since the 4th day after the first time of siRNA therapy until the 28nd day tumors were resected. Meanwhile, MMP-9 expression were downregulated as shown by immunohistochemistry.Conclusion1. MMP-9 overexpression can lead to result in cell proliferation and invasion. Meanwhile, in the gliomas from gradeâ… to gradeâ…£, MMP-9 were significantly elevated with the malignant grade of the gliomas.2. Using RNAi technology, siRNA targeting MMP-9 mediated by oligofectamine efficiently knocks down the expression of MMP-9 in human glioblastoma U251 cells, results in decrease of cell proliferation activity and invasive ability , arrests cell cycle.3. The established subcutaneous U251 glioma models in nude mice are treated with siRNA targeting MMP-9. the tumor growth is inhibited. The findings of in vivo study is in accordance with those in vitro study.4.MMP-9 can be the candidate genes for gene therapy of malignant gliomas. RNAi is a new gene silencing technology with high efficiency, which will have potential application prospects in clinics.