The Inhibitory Effect Of Glioma Growth By Knocking Down MiR-221/222 Expression And Its Possible Mechanism

Gliomas are the most common intracranial tumors which account for 46% of primarybrain tumors.The incidence of malignant gliomas is approximately 5～8 per 10,0000individuals per year.According to the statistics of World Health Organization(WHO),the mortality rate of malignant gliomas is the second in all tumor patients that areunder 34 years old and the fourth in all tumor patients that are 35～54 years old.Because of invasive growth,malignant gliomas can not be totally resected.Moreover,malignant gliomas are resistant to radiotherapy and chemotherapy.In addition,chemo-radiotherapy usually generates toxic and side-effects to normal central nervoussystem,so malignant glioma is a refractory disease in neurosurgery,the prognosis ofpatients with malignant glioma has not been changed significantly over the past threedecades.The study on the pathogenesis and new therapeutic approaches of malignantgliomas is imperative in neurosurgical field..By study on the molecular pathology of gliomas,it is considered that glioma is adisease of multiple gene aberration involved amplification and overexpression ofoncogenes and mutation or deletion of tumor suppressor genes,which results in thederegulation of signaling transduction pathways,then the tumor cells escape from thenormal growth control mechanism and present the malignant phenotype.Recentstudies have discovered microRNAs(miRNAs or miRs),21-25 nucleotides in length,being the largest family of noncoding RNA involved in gene silencing.MiRNAsnegatively regulate protein expression at the post-transcriptional level in a sequencespecific manner through binding to the target mRNA 3'UTRs(3'untranslated regions)and triggering translation inhibition or target mRNA degradation.Some studiesdemonstrated that more than 50% of miRNA genes are located in cancer-associatedgenomic regions or in fragile sites of chromosomal regions.In particular,Throughnegative regulation of gene expression,microRNAs(miRNAs)can function incancers as oncosuppressors,and they can show altered expression in various tumortypes.These evidences suggest that miRNAs may play an important role in thepathogenesis of human cancers.As the same with other cancers,The miRNAexpression profiles of gliomas also demonstrate that some miRNAs are expressed aberrantly in gliomas including upregulation of miR-221 and miR-222.However,thetarget genes regulated by miR-221 and miR-222 inducing the development of gliomasare not clear.Whether knocking down miR-221 and miR-222 is able to reversemalignant phenotype of gliomas or not and if miR-221 and miR-222 can be thepotential targets of glioma therapy.These problems need to be explored.In the presentstudy,the role ofmiR-221 and miR-222 may play in gliomagenesis is investigated.This study is ivided to 5 parts:1 The role of miR-221 and miR-222 playing in the development and progressionof glioblastoma is analyzed by 2′-O-methyl(OMe)-modified antisenseoligonucleotides for knocking down miR-221 and miR-222 in glioblastoma.2 After transfection with As-miR-221/222 into glioblastoma(GBM)cells,thechange of biological characteristics of GBM cells including proliferation activity,cellcycle kinetics,invasive ability and apoptosis are investigated in vitro.3 By bioinformatics analysis,some potential target genes of miR-221 andmiR-222 are obtained and identified by luciferase reporter assay.Then,themechanism of miR-221 and miR-222 regulating glioma cell growth and invasion maybe preliminary clarified.4 The established U251 subcutaneous glioma model in nude mice is treated withAs-miR221/222 and the growth rate and volume of tumors in vivo are compared withthe controls.The correlation of expression of target genes with proliferation,invasionand apoptosis in xenograft tumor samples are investigated.5 The effect of knocking down miR-221 and miR-222 on the radiosensitivity ofglioma cells is studied by Clonogenic assay.The conclusion drawing from the present study is as follows:1)Some miRs are altered expression in the four glioma cell lines includingupregulation of miR-221 and miR-222.Mir-221 overexpression is considered to be anovel molecular signature in gliomas.2)Five genes including p27kipl,PUMA,PTEN,TIMP3,Cx43 are target genesofmiR-221 and miR-222.3)By transfecting As-miR-221/222 into glioma cells,the expression of miR-221 and miR-222 are knocked down,and that results in reduced cell proliferationand invasive ability,arrested cell cycle and increased cell apoptosis.The reversedphenotype of glioma cells is correlated with the upregulation of the target genes ofmiR221/222.4)The growth rate of established U251 subcutaneous glioma model in nude micetreated with As-miR221/222 is slowed down and the tumor volume of treated mice ismuch smaller than that of control mice.MiR-221/222 expression in tumor specimensis knocked down but the expression of their target genes is significantly upregulated.This result in vivo is in concordant with that of in vitro experiment.5)Downregulation of miR-221 and miR-222 by transfection withAs-miR-221/222 can enhance radiosensitivity of glioma cells as determined byclonogenic assay.6)Since miR-221 and miR-222 possess the similar seed sequence,it is importantto co-suppress the expression of miR-221 and miR-222 cluster for optimizing theantiglioma effect.