DNMT1 Mediates Temozolomide-resistance Via Regulating MiRNA-20a Expression In Glioma Cells

BackgroudGlioma is the most common and most malignant primary form of the central nervous system brain tumor.It grows rapidly and displays a high degree of proliferation,invasiveness and recurrence.Its therapeutic schemes still represent a poor prognosis for patients,although the postoperative use of radiotherapy and chemotherapy concurrently has become the current standard regimen for treatment,which has been reported to significantly improve the overall survival.Although temozolomide(TMZ)is first line chemotherapy for glioma patients,the development of chemoresistance can result in an unsatisfactory outcome of TMZ chemotherapy.The potential mechanism of TMZ resistance in glioma has not been well defined.Elucidation of mechanism of tumour resistance to TMZ has helped steer therapeutic strategies attempting to overcome resistance.The miRNAs play an important oncogene or tumor suppressor gene role through regulating diversity target genes.In recent years,The primary focus of these studies had found miRNAs also play an important role in glioma chemoresistance.The miRNAs are endogenous 19-25 nucleotides non-coding RNAs that regulate the expression of target genes by degrading target messenger RNA(mRNA)transcripts and inhibiting mRNA translation via binding to complimentary sequences in the 3'-untranslated regions(3'-UTRs),sometimes 5'-UTRs,of their target mRNA.The miR-20a is a member of the miR-17-92 gene cluster,which also includes miR-17,5p?miR-18a?miR-19a/b?miR-20a and miR?92a-16.The effects of miR-20a on the proliferation,migration,and invasion of tumor cells have been studied for a long time.The miR-20a can promote the glioma cell growth,proliferation,invasion,inhibit the apoptosis of glioma cells and also play a role of oncogene in glioma.The preliminary study of this experiment showed that miR-20a in glioma was at the high expression negatively regulated its target gene LRIG1 relating to the TMZ resistance in glioma.Moreover,LRIG1 was demonstrated to be a tumor suppressor gene and may be related to glioma chemotherapy resistance,such as was observed in our present study,in a manner that could be reversed by miR-20a.The genetic regulation of miRNAs is similar to the regulation of mRNAs and involves specific transcription factors or proteins that interact with the promoter.Accumulating researches have demonstrated DNA methylation can regulate the expression of nearly half of miRNAs,and about half of these miRNAs exist abnormal methylation in a number of human malignant tumors.Aberrant methylation of promoter regions influences the expression of miRNAs,followed by subsequent deregulation of various signaling pathways.DNA methylation mediated by DNA methyltransferases(DNMTs)is one of major mechanisms that govern the epigenetic regulation of the genome.DNA methylation is maintained by DNMTs(such as DNMT1,DNMT3a and DNMT3b)via the transfer of a methyl group to the 5-carbon in the cytosine of a CpG dinucleotide.DNMTs associated with chemoresistance have also been found in various cancers,however,the effect of DNMTs regulation on glioma TMZ resistance remains unclear.In summary,this paper aims to find the relationship between DNMT1 expression and methylation level of CpG island locating in miR-20a promoter region,to explore the association of Aberrant methylation and miR-20a expression,to research DNMT1 regulating miR-20a expression and influence on its target genes LRIG1 expression and their downstream signaling pathways activation,we investigate whether there is a link between DNMT1 and miR-20a expression related to the potential molecular mechanisms of TMZ resistance in glioma cells,as well as look for a new effective method to ameliorate clinical outcome and overcome chemotherapy resistance.ObjectivesThe aim of this study was to identify the association of DNMT1 and miR-20a,to explored whether they were related to the potential molecular mechanisms of TMZ resistance in glioma cells.MethodsChemotherapeutic resistance in U251 human glioma cells was initiated by the stepwise revulsion with TMZ and drug resistance genes expression analysis.Through a TMZ-resisitance U251 cell line model and a xenograft tumor model were used to investigate DNMT1 regulates miR-20a expression and its downstream signaling pathways activation in vivo and in vitro for TMZ resisitance activity.Results(1)A stable drug-resistant cell line U251/TMZ induced by the stepwise revulsion with TMZ was established and verified successfully,which expressed high levels of MDR1?MRP5?LRP1?Bcl-2.(2)The expression of DNMT1 was lower in U251/TMZ cells compared with U251 cells,the expression of miR-20a was higher in U251/TMZ cells compared with U251 cells.(3)DNMT1 regulates miR-20a and LRIG1 expression via regulating methylation level of miR-20a promoter region.(4)The miR-20a-LRIG1 axis is the downstream signaling target for DNMT1 involved in the TMZ resistance of gilom cells.(5)The DNMT1-miR-20a-LRIG1 axis regulates EGFR/AKT/mTOR signaling pathways activation.ConclusionsDNMT1 regulates miR-20a and LRIG1 expression via regulating methylation level of miR-20a promoter region,DNMT1 mediated TMZ-resistance in glioma cells through regulating MiR-20a/LRIG1/EGFR/AKT/mTOR signaling pathways activation in vivo and in vitro.