Effect Of MicroRNA-17 On The Biological Function Of Glioma Cells And Its Mechanism

Glioma is one of the most prevalent and malignant tumors of the brain,they are rarely curable,and the prognosis for patients with highgrade gliomas is poor,especially for elderly patients.The mortality rate of patients diagnosed with malignant gliomas was 50%after 1 year,and 25%after 2 years.Glioblastoma multiforme has a worse prognosis in gliomas,and the average survival time is within 1 year after diagnosis.Now the research interest is directed to hunt for molecular markers and the target of glioma in order to improve the treatment efficiency.MicroRNAs(miRNAs),a class of 19-22 nucleotide RNAs,regulate gene expression post-transcriptionally by base-pairing with com-plementary sequences in the 3’-untranslated regions(UTRs)of protein-coding transcripts.This interaction leads to translational repression and in many cases to decreased mRNA levels.Previous studies have shown that a variety of miRNAs are down-regulated or up-regulated in human cancers,including glioma,providing new molecular basis into tumorigenesis.miR-17 plays an important and diverse role in the tumorigenesis,however,the biological functions of miR-17 in glioma remain unclear.The purpose was to clarify the significance of miR-17 in the occurrence and development of glioma,our study may shed light on potential pathogenesis of glioma and may help to develop novel effective therapies for glioma.This study was divided into three parts and main methods and results are as follows.Part I The expression of microRNA-17 in gliomaObjective:To investigate the expression level of miR-17 in glioma and normal brain tissues,and the relationship between miR-17 and tumor malignancy.Methods:The expression pattern of miR-17 in 10 normal brain tissues and 20 glioma tissues was deteched by RT-PCR.Results:The expression level of miR-17 was significantly higher in glioma tissues than that in normal brain tissues.miR-17 expression was significantly higher in the high-grade glioma tissues compared with that in the low-grade glioma tissues.Conclusion:miR-17 is overexpressed in high-grade gliomas,and it may be a potential oncogene in glioma.Part II Effects of microRNA-17 on the biological function of glioma cellsObjective:To identify the functional role of miR-17 on glioma cell proliferation,invasion,apotosis,and tumor growth in vivo.Methods:The expression pattern of miR-17 in normal human astrocyte cells and glioma cells was deteched by RT-PCR.The effects of miR-17 overexpression/silencing on glioma cell proliferation,invasion,apotosis and tumor growth were determined by transfection,MTT,Tanswell,FACS and tumor growth assays.Results:The results showed that miR-17 expression was significantly higher in the glioma cells compared with that in the astrocyte cells.The expression level of miR-17 was upregulated or downregulated in mimic or inhibitor transfected group,respectively.Overexpression of miR-17 dramatically increased U251 cells proliferation,invasion and tumor growth ability,resulted in a decrease of apoptotic cells.However,miR-17 silencing exert an opposite effect.Conclusion:Overexpression of miR-17 may promote glioma cell proliferation,invasion and tumor growth.Part III microRNA-17 promotes glioma cell proliferation and invasion by directly targeting CCND1Objective:To screen the possible target gene of miR-17 and to explore the possible molecular mechanism of miR-17 in glioma.Methods:To analysis the role of miR-17 in the regulation of target gene CCND1 by Western blot,luciferase reporter assay,MTT,Transwell,and FASC assays,and biological characteristics change of glioma cell after CCND1 siRNA transfection,to make clear the mechanism of miR-17 and it’s target gene in genesis and development of glioma.Results:The gene CCND1 harbored a potential miR-17 binding site predicted by three bioinformatics software(TargetScan,miRanda and miRWalk).Overexpression of miR-17 increased the protein levels of CCNDland the luciferase activity carrying the wild-type 3’-UTR.Ablation of CCND1 reversed the oncogenic roles of miR-17 mimic.Conclusion:CCND1 is a target gene of miR-17,upregulation of miR-17 plays an important role in the glioma progression.