MiR-21 Targeting PDCD4 Apoptosis Of Glioma Cells

Gliomas are the most common malignant primary brain tumors.Despite the advances in surgery,radiation therapy and chemotherapy,the prognosis of patients with gliomas have not improved significantly over the past 20 years.Understanding the regulation of signaling pathways involved in glioma migration,invasion and apoptosis is important for the development of more efficient tumor therapies.MicroRNAs(miRNAs) are a class of endogenous small non-coding RNAs that regulate the stability or translational efficiency of target messenger RNAs.A number of miRNAs have been identified from different human tumors and have been demonstrated crucial roles in cellular processes,including proliferation, differentiation,metabolism and apoptosis.Previous study showed that miR-21 is abberant expressed in human glioblastoma cells and miR-21 knockdown in cultured glioblastoma cells triggers activation of caspases and leads to increased apoptotic cell death.In this thesis,we focused on the potential involvement of miR-21 and its targets in gliomas' apoptosis,miRanda,TargetScan and Pictar were used to analyze miR-21's possible target genes.PDCD4,MTAP,SOX5,NTF3,JAG1,RHOB,MAPK10 and NFIB related to gliomas and apoptosis were selected for luciferase activity assay. Result showed that PDCD4,MTAP and SOX5 3'UTRs carry putative miR-21 regulatory elements.To confirm there are miR-21 binding sites in the PDCD4,MTAP and SOX5 3'UTRs,we further identified the binding site with miRanda and compared the nucleotides in different species.We cloned these 3'UTRs and their relevant mutants into luciferase report vector pcDNA3.1-Luc.Luciferase activity assay showed that PDCD4,MTAP and SOX5 carry putative miR-21 binding sites and are regulated by miR-21. We investigated the relationship between miR-21 and PDCD4 in glioma cell lines and tissues,found that PDCD4 correlates inversely with miR-21 amounts in glioma cell lines and tissues.To further study if PDCD4 is down-regulated by miR-21 in glioma cells,miR-21 was knocked down with 2'-O-Me-anti-miR-21 in T98G.. Result showed that miR-21 inhibition lead to a corresponding increase in endogenous PDCD4 protein levels,relative to the effect of the scrambled control.To insure that 3'UTR of PDCD4 contains a functional binding site for miR-21 in T98G,we cloned PDCD4 3'UTR and its relevant mutant with mutation of predicted miRNA binding site into downstream of the GFP reporter at pcDNA3.1-GFP and transfected them into T98G which possess high level of endogenous miR-21.Results showed that the putative miR-21 binding site within PDCD4 3'UTR is responsible for miR-21-mediated translational repression of GFP in T98G.To investigate the biological function played by miR-21 and its target PDCD4 in glioma apoptosis,we set up an artificial system.Results showed that over-expression miR-21 not only down-regulates PDCD4 but also participates other pathways to act as an anti-apoptosis factor.In summary,we report that PDCD4,MTAP and SOX5 carry putative miR-21 binding sites;PDCD4 correlates inversely with miR-21 amounts in glioma cell lines and tissues;miR-21 down-regulated PDCD4 at translational level in T98G; over-expression miR-21 not only down-regulates PDCD4 but also participates other pathways to act as an anti-apoptosis factor.Together,these results might be fundamental for the comprehension of glioma apoptosis and suggest that miR-21 would be a useful chemotherapeutic target for gliomas.