The Anti-tumor Dffects Of RNAa Mediated P21^WAF1/CIP1 Activation In Human Glioma Cells

Glioma is the most refractory malignancy in human central nervous system. The cause of disease, pathogenesis and the effective intervention of this tumor are still ambiguous. In recent years, people increasingly pay their attention to gene therapy of glioma. It is become the prevalent issue to pursue pathogenesis related genes, penetrate the molecular pathology and on this basis looking for new strategies and targets to treat glioma.P21WAF1/CIP1(p21) gene is an inhibitor of cyclin-dependent kinase(cdk) and the most widely active gene of cell cycle inhibitor currently known which involved in variety of regulatory functions, such as cell proliferation, differentiation, aging and apoptosis. Survivin was discovered as a IAPs family members recently. Survivin involves in cell mitosis and apoptosis process. Survivin plays an important role in the progression of tumors and the prognosis of patients making it is a significant target for combined modality therapy, especially gene therapy in human’glioma.RNA activation (RNAa), refers to describe the phenomenon of gene activation at the transcriptional level mediated by some small non-coding RNA(non-coding RNA, ncRNA) molecules. This is a new ideas and ways of gene therapies to treating tumors by utilizing RNAa to activate the tumor suppressor gene. It has been confirmed experimentally that the increased expression of p21by specifically activating gene expression may inhibit the growth of human renal carcinoma, bladder cancer, lung cancer, liver cancer and other tumors, but no study has been reported about intracranial glioma.The purpose of this study is to explore the relationship between the expression of p21and survivin in glioma cells and the histological grade of glioma; as well as to investigate the effectiveness and related mechanisms about cell proliferation, apoptosis and cell cycle distribution by utilizing saRNA to activate the expression of p21, thereby to discuss the possible new target and new methods for clinical anti-glioma treatment. The study is divided into two parts.The first part Aim:To investigate the expression and localization of p21and survivin protein, and the relationship between the expression of p21and survivin and histological grades in human glioma; Methods:The expression of p21and survivin of I-IV grade human glioma was examined with immunohistochemical technique. The relationship of the expression of p21and survivin with the histological grades of the glioma was analyzed. Results:The positive staining of p21protein positioned in the nuclei of the glioma cells, it was brown or tan. There were significant differences between the positive expression rates of p21and different grade of glioma. With higher malignancy, the expression level decreased. The positive staining of survivin protein positioned in the cytoplasm, occasionally been seen in nuclei of the glioma cells, it was pale yellow, brown, yellow or tan coloring. There were significant differences between the positive expression rates of survivin and different grade of glioma. With higher malignancy, the expression level increased. Conclusions:The decreased expression of p21in glioma was related to the malignancy of the tumor. The increased expression of survivin in glioma was related to the malignancy of the tumor.The second part Aim:to investigate the effectiveness about cell proliferation, apoptosis and cell cycle distribution by utilizing saRNA to activate the expression of p21. Methods:Small double-stranded RNA molecules(dsP21) which complementary with p21promoter DNA sequences was designed to transfect glioma cell line SHG-44. Real-time PCR and Western blot analysis were conducted to detect p21and survivin mRNA and protein respectively. Cell proliferation was examined by MTT assay. Apoptosis and cell cycle distribution were detected by flow-cytometric analysis. Results:1. The up-regulated expression of p21mRNA and protein were statistically significant and the down-regulated expression of survivin mRNA and protein were statistically significant in glioma cells compared to blank and negative control groups by detection of real-time PCR and Western blot analysis at72h after transfection with dsP21.2. Since the third days after transfection with dsP21, the proliferation of human glioma cells were significantly depressed.3. The early and late stages of apoptosis were increased in human glioma cells at72h after transfection with dsP21. Analysis of cell cycle distribution revealed that dsP21transfection increased an accumulation in the G0/G1phase and decreased an accumulation in the S phase in human glioma cells. Conclusions:Up-regulation expression of p21by RNAa significantly inhibited cell proliferation, promoted the rates of apoptosis, leaded to G0/G1arrest and remarkably depressed the expression of survivin in human glioma SHG-44cell line. P21activation by RNAa had anti-tumor activity in vitro in human glioma SHG-44cell line. These results suggest that RNAa could be used for human glioma treatment by targeted activation of tumor suppressor genes.