Knockdown Of Long Non-coding RNA CRNDE Promotes MiR-136 Expression And Inhibits Migration And Invasion Of Glioma U251 Cells

ObjectiveMalignant gliomas are the most common and deadly primary malignant brain tumors of the central nervous system(CNS)and are characterized by invasive growth and early metastasis.Studies have shown that the metastasis and invasion of malignant tumors are regulated by long-chain non-coding RNA and microRNA.In this study,we investigated the relationship between long non-coding RNA CRNDE and microRNA miR-136 in glioma cell line U251 and their effects on migration and invasion of glioma cell U251.The aim is to find new ideas for the clinical diagnosis and treatment of glioma.MethodsBioinformatics methods were used to analyze the differentially expressed lncRNA in human glioblastoma and paracancerous tissues in the TCGA database,to map heat maps and to predict potential binding sites for CRNDE and miR-136.Small interfering RNA(siRNA)was used to specifically knock down CRNDE in glioma cell U251.The knockdown efficiency and the expression of miR-136 after knockdown were detected by qRT-PCR.The experiment was divided into negative control group(NC),small interfering RNA group(si-CRNDE),miR-136 inhibitor group(miR-136 inhibitor)and co-transfected small interfering RNA and miR-136 inhibitor group(si-CRNDE+ miR-136 inhibitor),Wound healing assay and Transwell assay were used to detect the migration and invasion ability of the four groups of cells.Western blot was used to detect the expression levels of epithelial-mesenchymal transition(EMT)-related proteins in four groups of cells.Results(1)Analysis of the TCGA-GBM dataset,a total of 1013 lncrRNA differentially expressed in glioblastoma tissue and paracancerous tissues,wherein CRNDE is higher in glioblastoma tissues than in paracancerous tissues.expression.The starBase v2.0 database analysis revealed potential binding sites for CRNDE and miR-136-5P.(2)The expression of CRNDE in glioma U251 cells was significantly lower than that in the NC group after transfection of small interfering RNA,indicating that knockdown was successful,and the expression of miR-136 was significantly higher than that of the NC group.(3)Wound healing and Transwell assay results showed that compared with the NC,the wound healing area and invading cells number of the si-CRNDE group was significantly smaller,while the wound healing area and invading cells number of the si-CRNDE+miR-136 inhibitor group was not significantly different;compared with the si-CRNDE group,the wound healing area and invading cells number of the siCRNDE+miR-136 inhibitor group was significantly larger,which rescued the effect of knocking down CRNDE on cell migration and invasion ability.(4)Western blot showed that compared with the NC group,the expression levels of N-cadherin and vimentin were decreased and the expression level of E-cadherin was increased in the si-CRNDE group,while the proteins expression levels of the siCRNDE+miR-136 inhibitor group was not significantly different;compared with the siCRNDE group,the expression levels of N-cadherin and vimentin were increased in the si-CRNDE+ miR-136 inhibitor group,and the expression level of E-cadherin was decreased,which rescued the effect of knockdown of CRNDE on the expression of EMT-related proteins.Conclusion(1).CRNDE is highly expressed in glioblastoma tissues compare with paracancerous tissues,CRNDE and miR-136 have potential binding sites.(2).Knockdown of CRNDE can inhibit the migration and invasion of glioma U251 cells by up-regulating the expression of miR-136,and affect the expression of EMTrelated proteins,and this effect can be reversed by miR-136 inhibitor.