Study On The Mechanism And Antitumor Effects Of MiR-124 And MiR-137 In Glioblastoma Multiforme

Background:Glioma, also called Neuroglioma, is difficult to treat with high morbidities. Among glioma, malignant glioma and Glioblastoma multiforme(GBM) are the most common and aggressive primary brain tumors with poor prognosis in central nervous system(CNS), which are the most malignant diseases with a huge influence on human health second to pancreatic cancer and lung cancer. The pathogenesis of glioma is still unclear but these reasons could increase the incidence such as genetic factors, virus infection, head trauma, radiotherapy(RT), immunosuppressors, and endocrine and metabolism disorders. Molecular genetics studies of glioma have confirmed that genetics aberrance are potential risk factors if there were proto-oncogene increase in chromosome 7, or tumor-suppressor gene deletion in chromosome 10, or some genes with abnormal amplication and expression(such as EGFR, c-erb B1, H-ras, gli, N-myc, et al), or tumor suppressor genes depletion or loss of their expression. Micro ribonucleic acid(mi RNA) is characterized by small non-coding and single-stranded RNAs with 18-25 nucleotides, which negatively regulate the targeted gene expression through base pairing to the 3’-untranslated region(3’-UTR). mi RNA exists everywhere in organisms with wide physiological functions, especially in the tumorgenesis and tumor chemoresistance. mi RNA is a kind of regulatory molecules which are necessary for organisms with complicated functions. According to their biological functions, tumor-associated mi RNAs are classified as tumor suppressor mi RNAs and onco-mi Rs. mi R-124 and mi R-137 belong to tumor suppressor mi RNAs. Although they are classified as tumor suppressor mi RNAs by some experiments and bioinformatic analyses, the underly mechanisms are still unclear. Therefore, the objectives of the current study are to search for the new targets of mi R-124 and mi R-137 and to enrich their tumor suppressing function and to explore novel therapeutic strategies for chemotherapy resistance. Part 1: Study on the mechanism and antitumor effects of mi R-124 in Glioblastoma Multiforme Objectives: To explore the mechanism and antitumor effect of mi R-124 in GBM and the role in the chemoresistance of Temozolomide(TMZ).Methods: GBM cells such as U87, U251, A172 and T98 G cells were transfected with mi R-124 mimics to detect the overexpression of mi R-124 and apoptosis rates after TMZ administration. RAD51-3’UTR expression plasmid was constructed to perform the dual luciferase report(DLR). The changes of RAD51 m RNA and protein after mi R-124 overexpression in GBM cells were detected by quantitative Real-time Polymerase Chain Reaction(q RT-PCR) and Western blot, respectively. The foci of γH2AX and RAD51 in mi R-124 overexpressed GBM cells administered with TMZ were observed by indirect immunofluorescence staining. The function of RAD51 in TMZ chemotherapy was analyzed on RAD51 downregulated by RNA interference(RNAi). The data between RAD51 expression and survival of GBM patients in GEO Data Sets were downloaded and analyzed. Results: The apoptosis rates of GBM cells were increased with statistic significance in Annexin V-FITC/PI staining after mi R-124 overexpression. Taken together, the decrease of relative luciferase ratio in DLR, RAD51 downregulation in m RNA and protein and the decreased foci in RAD51 indirect immunofluorescence staining after mi R-124 overexpression proved that mi R-124 functioned as a tumor suppressor to induce apoptosis after TMZ administration by direct inhibition of RAD51. Knockdown of RAD51 by si-RAD51-1 or si-RAD51-2 was verified successfully by q RT-PCR and Western blot. After RAD51 interference, the apoptosis rates of GBM cells were increased with statistic significance which were similar to the biological effects after mi R-124 overexpression. Low expression of RAD51 was statistically significant associated with longer overall survival(OS) which was indicated from GEO Data Sets(ID: GSE4271). Conclusions: mi R-124 augments the response to TMZ chemotherapy through regulation of RAD51 in GBM. Part 2:Study on the mechanism and antitumor effects of mi R-137 in Glioblastoma Multiforme Objectives: To explore the mechanism and antitumor effect of mi R-137 in GBM and the role in sensitizing GBM cells to Tumor necrosis factor-related apoptosis- inducing ligand(TRAIL)-induced apoptosis. Methods: GBM cells were transfected with mi R-137 mimics to detect the overexpression of mi R-137 and apoptosis rates after TRAIL administration. The changes of 6 predicted genes’ m RNA and protein after mi R-137 overexpression in GBM cells were detected by q RT-PCR and Western blot, respectively. The potential target gene-XIAP was screened out and XIAP-3’UTR expression plasmid was constructed to perform DLR. Fifteen clinical GBM specimens were collected to analyzed the correlation between mi R-137 expression and XIAP m RNA expression. The lentivirus expressed mi R-137 were packaged and screened by Blasticidine S successfully, and verified by fluorescence observation, q RT-PCR and Western blot. The apoptosis rates were detected after XIAP expression was rescued in GBM cells infected with lentivirus continuously expressed mi R-137. The nude mice models with xenografts were built by injection subcutaneously into the flank with 2×107/cells stably overexpressing mi R-137 and luci as control. About three weeks later, the effect between of mi R-137 and TRAIL plasmid treatment were compared. Results: The apoptosis rates of GBM cells were increased with statistic significance after mi R-137 overexpression. Taken together, XIAP downregulation in m RNA and protein after mi R-137 overexpression and the decrease of relative luciferase ratio in DLR proved that mi R-137 functioned as a tumor suppressor to induce apoptosis after TRAIL administration by direct inhibition of XIAP. Pearson’s correlation analysis showed that mi R-137 expression was inversely correlated with XIAP m RNA expression in clinical GBM specimens. The strain infected with lentivirus of overexpressed mi R-137 was constructed successfully. The expression of XIAP in mi R-137 overexpressed strain rescued the TRAIL sensitization effects caused by mi R-137 in U251 cells. The volume of xenografts before and after TRAIL treatment, XIAP protein level and immunohistochemistry staining indicated that mi R-137 inhibited the GBM tumors’ growth and increased TRAIL-induced apoptosis by directly targeted XIAP. Conclusions: mi R-137 sensitized GBM cells to TRAIL-induced apoptosis by direct inhibition of XIAP.