MicroRNA-148a Promotes Human Glioma Cell Migration And Invasion By Targeting DYNLL2

Objective:To study the effects of mir-148a on the biological characteristics of glioblastoma(GBM)cells by targeting recombinant human recombinant human kinetic protein LC8 type 2(Recombinant Human Dynein Light Chain LC8 Type-2,DYNLL2)and its possible mechanisms,and may serve as a basis for further interpreting the molecular mechanisms of GBM development and applying mir-148a as a new target for GBM diagnosis and treatment.Methods:1.Screening of possible target genes of mir-148a by Bioinformatics.Three commonly used professional software for miRNA target gene prediction of miRNA,TargetScan,and miRBase were used to screen the potential target genes of mir-148a,and the genes with high cross matching repetition rate were regarded as candidate genes.DYNLL2 is one of the candidate target genes for mir-148a.2.qRT-PCR and Western blot were used to detect the expression level of DYNLL2 in glia cells and glioma cells,and observe whether mir-148a affects the expression of DYNLL2..The DYNLL2 gene expression level between normal glia cells and glioma cells,was compared,and DYNLL2 gene expression level was further analysed in mir-148a overexpression and mir-148a inhibition glioma cells.3.The double fluorescein reporter gene experiment was used to verify whether DYNLL2 was a mir-148a target gene.a recombinant plasmid containing the detected gene-Luc/Rluc was prepared,and the reporter gene and the target gene with a Luc/Rluc marker were co-transfected,and the fluorescence intensity was detected by GENios Pro microplate reader or equipment with similar detection function.4.Establishing mir-148a and DYNLL2 overexpression stable cell line in glioma cell line DYNLL2 and mir-148a overexpressing lentivirus were infected glioma cells.Through the screening by puromycin,mir-148a and DYNLL2 overexpression stable cell line were established in glioma cells respectively qRT-PCR was used to verify the expression levels of mir-148a and DYNLL2.5.Scratch assay and Transwell invasion assay were used to analyse,the migration and invasion of glioma cells.Results:1.A potential target gene for mir-148a was successfully identified.312 mir-148a potential candidate target genes were screened out by microrna.org,TargetScan,and miRBase,and DYNLL2 was one of mir-148a potential candidate target gene.2.The gene expression of DYNLL2 had significantly difference between glia cells and glioma cells.Compared to normal glia cells,the expression of DYNLL2 in glioma cells was significantly decreased(P<0.05).DYNLL2 was downregulated in mir-148a overexpression glioma cells and upregulated in mir-148a inhibition glioma cells.3.The double fluorescein reporter gene experiment verified that DYNLL2 was a target gene of mir-148a.A luciferase reporter gene plasmid was successfully constructed,co-transfected with the mir-148a overexpressing lentivirus in 293T cell,and the fluorescence intensity was detected by GENios Pro microplate reader.The results showed that the intracellular fluorescence intensity was weakened,which proved that mir-148a could directly act on the DYNLL2-3'UTR target sequence and reduce the expression of DYNLL2.4.Successfully construction of DYNLL2 overexpressing stable cell lines.Successfully construction of DYNLL2 overexpressing lentiviral vector,and U87 and U251 cell lines that stably overexpressing DYNLL2,and U87 and U251 stable cell lines that simultaneously overexpressing mir-148a and DYNLL2 were obtained through screening puromycin.qPCR verification showed that overexpression of mir-148a inhibited the gene expression of DYNLL2,thus confirming the regulation effect of mir-148a on DYNLL2.5.Effects of overexpressing DYNLL2 on the biological function in glioma cells.After infection of DYNLL2 lentivirus,U87 and U251 cells were performed scratch test and Transwell invasion assay.The results showed that the invasion ability of U87 and U251 cells were decreased after overexpressing of DYNLL2.Overexpression of DYNLL2 and mir-148a at the same time,the inhibitory effect.on migration and invasion ability was restrained in U251 and U87 glioma cells.Conclusions:1.DYNLL2 is a target gene for mir-148a.2.microRNA-148a promotes migration and invasion of glioma cells by targeting DYNLL2.