| Glioma is the most common malignant tumor of central nervous system with rapid growth,low survival rate,and high mortality,especially to the patients who could not completely remove the cancer.Despite great efforts have been made in the past several decades toward improvement of combined therapies for glioma,the prognosis of patients remains grim.Better understanding the molecular mechanism of glioma genesis and finding genes associated with the pathogenesis of glioma will certainly lead to the development of novel targeted therapies for glioma.Micro RNAs(miRNAs)are a new class of endogenous,small(19?22nt)non-coding single-stranded RNA molecules that negatively regulate protein-coding genes by base pair matching with the 3'untranslated region(UTR)of m RNA,resulting that the translation of target gene is suppressed or the m RNA of target gene is degradated.Recently,miRNAs,the relatively new group of non-coding RNA,have been proven to play an essential role in the regulation of many physiological and pathological processes such as cancer.Mi RNAs can affect tumor development through a variety of mechanisms.Abnormalities of miRNAs expression are now known to be involved in glioma genesis and may function as oncogenes or tumor suppressors.To identify critical miRNA as regulatory role in glioma,the gene signature of miRNAs in 8 glioma tissues and their corresponding non-tumor tissues were screened using high-throughput Agilent human microarray.miR-433 expression was aberrantly decline and was selected for further validation.Quantitative PCR was then conducted to confirm the miRNA array by detecting miR-433-5p and miR-433-3p expression levels in 12 glioma tissue and 6 brain tissues.The results showed that miR-433-5p and miR-433-3p was low expressed in glioma tissue comparing to brain tissue(P<0.05).The same results in glioma cell lines were also found.In our study,overexpression of miR-433-5p and miR-433-3p in U251 and U87glioblastoma cells were used to study miR-433-5p and miR-433-3p impact on biological characteristics of the human brain glioma cell lines.Cell proliferation was measured by MTT assay and colony formation,showed that cell proliferation was significantly inhibited after transfection of miRNA-433-3p mimics(P<0.05).Cell apoptosis was assessed by Annexin V-FITC/PI double staining flow cytometry,showed that cell apoptosis was significantly increased after transfection of miRNA-433-3p mimics(P<0.05).Cell cycle was assessed by PI staining flow cytometry,showed that cell cycle was arrest in G0/G1 phase after transfection of miRNA-433-3p mimics(P<0.05).Cell invasion and migration was assessed by transwell,showed that cell invasiveness and migration capability were significantly reduced after transfection of miRNA-433-3p mimics(P<0.05).But there was no significant difference in biological characteristics of glioma cells after transfection of miR-433-5p mimics(P>0.05).The bio-informatic prediction and dual-luciferase analysis showed that cAMP response element binding protein(CREB)is a direct target gene of miRNA-433-3p(P<0.05).The expression of CREB was tested by Real-Time PCR and Western blot after transfection of miRNA-433-3p mimics in glioma cells.Overexpression of miR-433-3p by miRNA-433-3p mimics was shown to down-regulate CREB,as well as its downstream target genes PCNA,BCL2,Cyclin D1,MMP-9(P<0.05).In nude mice harboring subcutaneous U87 xenografts,overexpression of miR-433-3p suppressed the growth of tumor xenografts after transfection of miR-433-3p mimics in vivo(P<0.05).The immunohistochemistry staining showed that the expression of CREB significantly decreased in miR-433-3p mimics group(P<0.05).In our study,the tumor suppressive effect of miR-433-3p was validated in vivo and in vitro,showed miR-433-3p estoration of its function inhibits glioma cell proliferation,invasion and migration capability,promotes apoptosis and suppress the growth of tumor xenografts,which could be partly due to its inhibitory effect on CREB protein expression in glioma cells.These findings indicated the low expression of miR-433-3p is related to malignant progression of glioma cell,highlighting it may be a potential therapeutic target for glioma. |
PDF Full Text Request