| Glioma is one of the common malignant in human,which has a high mortality rateof malignancies in the central nervous system. The mortality of glioma is the highest inthe central nervous system diseases after stroke, ranking second. Currently, the highestlevel of glioma only have about14.8months for the median survival, and the5-yearsurvival rate is only about9.6%. Investigate the cause of the high mortality rate whichis mainly because of its high degree of atypia, strong biological invasion andchemoradiation therapy resistance. At present, because of the glioma pathogenesis is notyet fully understood, it is not yet found an effective treatment regimen for glioma.Therefore, in-depth study of the pathogenesis of gliomas has become urgent problemfor the treatment of glioma.Resulting carcinogenic tumors is a complex, multi-stage process, these process iscaused by the development of the endogenous and exogenous factors. In recent years,many studies have shown that mutations in DNA provides an important molecular cluesfor tumor development. Especially in post-transcriptional regulatory events, such asRNA editing is becoming the new direction of research human tumors and otherdiseases. ADARs are a family of enzymes which can hydrolyze deamination of adenosine(A) into inosine(I) for the target RNA sequence of post-transcriptional，affectthe pairing of bases in translation, thus completing the RNA editing. This RNA editingis essential and fnely regulated in normal cells.RNA editing on ADARs related research in the field of cancer also focused onADAR2. Present study suggests that ADAR2have closely related with glioblastoma onproliferation and invasiveness. ADAR2by acting on the mRNA of ionotropic glutamatereceptors (AMPAR subtype) in the Q/R site, and then change AMPAR permeability ofCa2+. Reducing the concentration of cytoplasmic Ca2+, thereby inhibiting tumor growthand invasiveness.Related research found that ADAR1(p110) was highly expressedwhen they detected the expression of ADAR2on children’s glioblastoma. They thinkADAR1(p110) and ADAR2may form heterodimers, thus affecting ADAR2editingAMPAR mRNA’s, but ADAR1associated with apoptosis and proliferation studies withglioma have not been reported. The purpose of this experiment is to detect theexpression of ADAR1gliomas as well as proven in after interference the expressionADAR1on the human glioma cell lines, such as changes in the biological behavior ofproliferation and apoptosis, thus proved the role of ADAR1in glioma developmentmechanism played.Part ⅠDetecteded the expression of ADAR1in different types of adult gliomasObjective: To detected the expression of ADAR1in human glioma tissue byqualitative detection of proteins. Methods: Detected the expression of ADAR1in humanglioma and adjacent normal tissues by Western-Blot and immunohistochemistry methodsResults: ADAR1expression in gliomas was significantly higher than its peritumoralnormal brain tissue. Conclusion: The test results found that ADAR1high expression ingliomas, indicating the existence of a certain correlation with the development of gliomas,and related biological functions. This result provides a theoretical and researchfoundation to detect changes in the latter part of the proliferation and apoptosis rate ofglioma cell lines after interference the expression ADAR1in glioma. To further provedADAR1provides a new theoretical foundation and research directions in thedevelopment of glioma and treatment of glioma. Part II Detecteded the expression of ADAR1in various human glioma cell linesObjective: To detected the expression of ADAR1in four kinds of classic humanglioma cell lines.Selected one or more glioma cell lines which expressed highly ADAR1as follow-up study subjects. For a further study of the biological function of ADAR1.Methods: Detected the expression of ADAR1in four kinds of human glioma cell linesby Western-Blot. Results: After detected the expression of ADAR1in four kinds ofglioma cell lines，we found T98G glioma cell lines expressing the minimum amount ofADAR1, and U87, U251glioma cell lines both higher expression levels of ADAR1, theexpression of A172’s second. Conclusion: The test results and related research consistentwith reliability. Therefore, subsequent trials will select U87, U251glioma cell lines forthe trial to ascertain the relationship between ADAR1and cell apoptosis&proliferation.Part III Interference ADAR1expression in human glioma cell linesObjective: With ADAR1shRNA transfected U87, U251glioma cell lines, choosethe best transfection efficiency cells and detect the expression of ADAR1mRNA andprotein after transfection by RT-PCR, Western-Blot techniques. Select the success ofinterfering cell line as the experimental subjects for the next step to detect cellproliferation and apoptosis. Methods: With ADAR1shRNA transfected U87, U251glioma cell lines，the transfection efficiency was observed in transfected after24h,48h,72h by fluorescence microscopy. According to the results we choose the cell lines withthe highest transfection efficiency，and detect the expression of ADAR1mRNA andprotein by RT-PCR, Western-Blot techniques. Results: Observed the cell which wastransfected after24h,48h,72h by fluorescence microscopy. We found that at48h aftertransfection, ADAR1-shRNA transfection efficiency is the highest. We found that aftertransfection72h, green fluorescent protein (EGFP) expression gradually decay, andfound a large number of cell death. Therefore, we detected the expression ADAR1mRNA and protein with the cell lines which was transfected48h. After48hADAR1-shRNA-transfected cells were detected by RT-PCR and Wetern-Blot. Theseresults showed that the expression level of ADAR1-shRNA interference group ADAR1mRNA and protein was significantly lower than the negative control group and blank group. Conclusion: We found that at48h after transfection, ADAR1-shRNA transfectionefficiency is the highest. So the next tests we would choose the cell lines which wastransfected48h as the subjects. To determine the effect of ADAR1on glioma cellproliferation and apoptosis.Part Ⅳ Detection proliferation rate of human glioma cell lines after ADAR1interferenceObjective: Detection proliferation rate of these two human glioma cell lines afterADAR1interferenceby MTT assay. Methods: ADAR1shRNA transfected with gliomacell lines, detection of the proliferation rate of these two cell lines by MTT assay. Results:Detection proliferation rate of these two human glioma cell lines after ADAR1interference12h,24h,48h and72h.The results showed that ADAR1-shRNA comparedinterference group with negative control group and the blank group was significantlylower in cell proliferation. Conclusion: The experimental results showed that duringthe interference ADAR1expression in glioma cell lines, glioma cell proliferation rate inthe experimental group was significantly lower than the control group. So we think afterinterference can reduce the appreciation rate of glioma cells, and the high expression ofADAR1have a closely relationship with high proliferation in glioma. ADAR1highexpression has the ability to promote glioma cell proliferation.Part Ⅴ Detection rate of apoptosis of human glioma cell lines after ADAR1interferenObjective: Detection rate of apoptosis of human glioma cell lines after ADAR1interferen by Flow Cytometry. Methods: Detect apoptosis rate of these two cell lineswhich were transfected with ADAR1shRNA after48h by flow cytometry. Results:Detection proliferation rate of these two human glioma cell lines after ADAR1interference48h. This results showed that ADAR1-shRNA compared interference groupwith negative control group and the blank group was significantly higher in cellapoptosis. Conclusion: The experimental results showed that during the interferenceADAR1expression in glioma cell lines, glioma cell apoptosis in the experimental groupwas significantly higher than the control group. So we think after interference ADAR1can promote the apoptosis rate of glioma cells, and the high expression of ADAR1have a closely relationship with low apoptosis in glioma. ADAR1high expression has theability to reduce glioma cell apoptosis. |
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