Effect Of MiR-128a On Glioblastoma Subtype Transition And Its Mechanism

Glioblastoma(GBM)is a common malignant tumor of the nervous system,with high malignancy,aggressiveness,and low patient survival.Surgical resection is still the main treatment.Although temozolomide(TMZ)chemotherapy combined with radiotherapy can effectively improve the survival rate of patients after surgery,the resistance to TMZ severely limits clinical treatment effect.Therefore,long-term use of TMZ resistance severely limits its clinical effectiveness how to solve the problem of resistance to TMZ is very important.The American Cancer Genome Atlas(TCGA)classifies glioblastoma into four subtypes:classic subtype(Classical),mesenchymal subtype(Mesenchymal,Mes),neural subtype(Neural)and proneural subtype(Proneural,PN).Among them,PN subtype shows less invasiveness,better prognosis and more sensitive to radiotherapy and chemotherapy.While Mes subtype appears infiltrating growth,higher invasiveness,worse prognosis and insensitivity to radiotherapy and chemotherapy.Under certain conditions,the two subtypes of Mes subtype and PN subtype can be converted to each other.Moreover,the conversion of Mes subtype to PN subtype can improve the sensitivity to temozolomide(TMZ)and improve patient prognosis.Therefore,the adjusting of glioblastoma subtype transition has become a research hotspot.MicroRNA(miRNA)is an endogenous,short-stranded and non-coding RNA with a length of about 14-22 nucleotides.MiRNAs can be differentially expressed in the varieties of tumor cells and tissues,miRNAs mainly regulates the translation of the target gene mainly by binding to the 3'UTR of the target gene mRNA,thus affecting the biological behavior of tumor cells.It has been discovered that miRNAs can regulate GBM subtype conversion by regulating target genes whereas the types of miRNAs and their mechanisms are still unknown and essential for further research.ObjectivesTo screen the miRNA that affects glioblastoma subtype conversion and clarify the role of miRNA in glioblastoma subtype conversion.To predict the target genes of the miRNA by bioinformatics methods.To verify whether the miRNA could bind to the 3'UTR region by luciferase experiments and whether miRNA regulates the transcription and translation of the target genes.It will provide experimental basis and theoretical basis for studying the mechanism of miRNA in glioblastoma subtype conversion.Method1.Bioinformatics was applied to screen the miRNAs which were differentially expressed in PN and Mes subtypes.2.Selecting miR-128a as the research object,miR-128a mimics,miR-128a inhibitor and scrambles were transfected U251 cells respectively and the transfection efficiency was verified by Real-time PCR.After confirming the transfection to U251 cells successful,Mes subtype biomarker expression in three groups of U251 cells was detected by Western Blot.3.The target gene CTDSP2 of miR-128a was screened and predicted by bioinformatics.The correlation between miR-128a and CTDSP2 expression was further verified by Starbase database.4.Luciferase reporter plasmids encoding the wild-type(wt)or mutant(mut)3'UTR region of CTDSP2 mRNA were constructed,and co-transfected to 293FT cells with miR-128a mimics and scrambles respectively.miR-128a targeted regulating CTDSP2 was verified by dural luciferase reporter assay.5.Real-time PCR and Western Blot were used to detect the effects of miR-128a on CTDSP2 mRNA and protein expression.Results1.22 miRNAs were screened that were differentially expressed in PN and Mes subtypes by bioinformatics.Ten of miRNAs were highly expressed in the PN subtype and lowly expressed in the Mes subtype,while the other twelve miRNAs were lowly expressed in the PN subtype and highly expressed in the Mes subtype.2.Western Blot results revealed that,compared with the scrambles group,Mes subtype markers such as CD44,VIM and YKL-40 expression decreased in miR-128a mimics group(P<0.05).CD44,VIM expression increased in miR-128a inhibitor group(P<0.05).miR-128a could inhibit the expression of Mes subtype markers CD44,VIM and YKL-40.3.Bioinformatics predicted that there were multiple binding sites in the 3'UTR region of miR-128a and CTDSP2 mRNA.Starbase database predicted that miR-128a was negatively correlated with CTDSP2 expression(r=-0.163,P<0.05).4.Dural luciferase reporter assay suggested that,compared with the scrambles group,miR-128a mimics led to the reduction of the CTDSP2 3'UTR-wt luciferase activity and the difference was statistically significant(P<0.05),there was no significant change in CTDSP2 3'UTR-mut luciferase activity(P<0.05),which indicated that,which indicated that miR-128a directly targeted CTDSP2 3'UTR in GBM.5.RT-PCR and Western Blot demonstrated that,compared with the scrambles group,overexpression of miR-128a inhibited the expression of CTDSP2 mRNA and protein(P<0.05).After down-regulation of miR-128a,CTDSP2 mRNA and protein expression increased(P<0.05).These results supported that miR-128a could inhibit CTDSP2.Conclusion1.miR-128a can inhibits glioblastoma to transit to Mes subtype.2.miR-128a can target the CTDSP2 3'UTR to negatively regulate the transcription and translation of CTDSP2 gene.