The Study Of Cell Proliferation And Apoptosis By Small Interfering RNA Targeting Survivin On The Glioma Cell

Background:Glioma is a kind of the most frequent brain tumors and it accounts for 40%-50% of morbidity of brain tumors. In terms of glioma treatment, it remains the most lethal malignant tumor despite surgery, radiation and chemotherapy. Molecular targeting in the treatment of cancer is nowadays becoming a promising approach based on research progress on pathogenesis and molecular mechanism of glioma.The survivin gene was recently identified by hybridization screening of human genomic libraries with the cDNA from a factor Xa receptor,effector cell protease receptor-1 (EPR-1), Survivn is a member of IAP family. The length of survivin gene is 1417 kb, and it is located on human chromosome 17q25.3, encoding a 16.4-kDa protein, which is a unique structure of the cytoplasmic protein,comprising 142 amino acids. It is the IAP with the minimum molecular weight and the strongest anti-apototic function by far. Survivin is generally expressed in embryonic tissues, but is either undetectable or expressed at a very low level in terminally differentiated adult tissues, whereas it is overexpressed in the majority of human cancers. Studies have found that survivin has something to do with the incidence, development, recurrence and prognosis of tumor. Inhibiting its expression could significantly promote tumor apoptosis, including glioma cells, While no significant effect on normal tissues, which is expected to become an ideal target for gene therapy for glioma.RNA interference (RNA interference, RNAi) is a ubiquitous phenomenon in organism and cells inherent to against the foreign gene as a self-protection. The members of the family of RNA enzymes III called dsRNA-specific endonuclease (dsRNA specific endonuclease, Dicer)was first be degraded into 21-23 small nucleotides (nucleotide, nt) after endogenous or exogenous double-stranded RNA (dsRNA) introducted into cells, firstly to be degraded into 21～23 nucleotides (nucleotide, nt) a small piece of that small interfering RNA (Small interfering RNA, siRNA) by members of the family of RNA enzymes III called dsRNA-specific endonuclease (dsRNA specific endonu-clease, Dicer), then to use siRNA as a template, a specific site, a specific sequence of intervals degradation with corresponding mRNA. And thus lead to post-transcriptional gene silencing (post transcriptional gene silencing, PTGS).RNAi has many advantages such as high efficiency, high specificity,high stability and amplification effect. The so-called multiplication effect is that the mRNA fragments, degraded or not,can be used as primers, in the role of RNA-dependent RNA polymerase, the synthesis of additional dsRNA. New dsRNA go into the next round of RNAi loop. as a new blocking Gene expression techniques, At Present RNAi has become widely used as a tool of functional genomies signaling transduction research and gene therapy. The cancer-specific gene intervention has opened up a new way for the clinical treatment.Therefore, the study of gene induced apoptosis and inhibit the proliferation in glioma cells by RNA interferencing (RNAi) survivin may well be a good way of glioma gene-targeted therapy.Objective:To observe the apoptosis and proliferation of glioma by shRNA interference with survivin gene in U251 cell line. And preliminary study the relationship of survivin and minichromosome maintenance protein 2 (minichromosome maintenance proteins 2, MCM2). It provide a theoretical and experimental basis for targeting survivin gene therapy gliomas, Survivin gene for gene therapy may become meaningful treatment goals of glioma therapy, so as to overcome the chronic problem of glioma to provide research-based.Methods:Survivin shRNA plasmid PG-sur (pGenesil-suvrivin) and nonsense shRNA plasmid PG was transfected respectively into U251 by Lipofectamine2000. the non-transfected group is control. Experimental control is the nonsense sequence transfected group. Experimental group is the specific sequence transfected group.Expression of mRNA and protein of survivin was investigated by RT-PCR and Western blot respectively. Flow Cytometry (FCM) was adopted to analyze quantitatively the cell cycle and apoptotic cells in each group. The inhibitory rate of cell proliferation was detected by MTT assay. TUNEL methods were used to observe the morphologic characteristics of the cells in each group. Expression of mRNA and protein of MCM2 (minichromosome maintenance proteins 2) was investigated by RT-PCR and Western blot respectively in each group. Application of SASv8.0 software analysis experimental data obtained.Results:Cells were harvested 48h after transfection, subjected to reverse transcription-polymerase chain reaction (RT-PCR) and Western blot experiments to determine the shRNA vector having the strongest efficiency in inhibiting survivin expression.48h after transfection, a large number of fluorescent cells are observed by Inverted fluorescence microscope in each group. AFCS was used to detect the transfection effect of vectors in various groups. Transfection efficiency of each group were about 85%.1 Flow cytometry analysis showed that transfected U251 cells occurred significant apoptosis (F=20.85, P=0.002<0.01),The percentage of cell apoptosis was about 37.9113±6.8134%.2 The result of TUNEL methods analysis showed that the number of apoptosis cells were increase in the cells transfected by PG-sur plasmid.3 MTT assay results showed that after transfection 24h,48h no significant difference in cell proliferation, the cell proliferation significantly reduced after transfection 72h,96h.4 PI staining detected by flow cytometry-cycle analysis revealed that transfection of U251 cells with pGenesil-survivin resulted in a significant increase in the G2/M fraction, suggestive of cell cycle arrest. 5 After transfected U251 cells with the disturbance sequence, the expression of MCM2 mRNA and protein was markedly reduced.Conclusion:1 Survivin gene in glioma cell line U251 has an anti-apoptosis2 Survivin gene in glioma cell line U251 is able to promote the proliferation of tumor cells3 RNAi targeting suvrivin gene can be inhibited U251 cells induced apoptosis and cell cycle arrest, which may be a new strategy of targeted gene therapy glioma;4 Over-expression of MCM2 and Survivin was not an isolated individual incident in glioma,the two gene may be play a synergistic role in theoccurrence and development of glioma;...