Study Of The Effects Of MiR-29s Down-Expression On DNMT3A/3B And The Mechanisms In Gliomas

Objectives:Although recent studies have indicated the importance of miR-29a/b/c, the three members of the miR-29family, as tumor-suppressive miRNAs by showing their low expression levels in multiple extracranial tumors, their expression patterns in glioma cells and their roles in the tumorigenesis and progression of gliomas remain un-known. DNA methyltransferase3A and3B (DNMT3A/3B) are important de novo DNA methyltransferases of mammalian cells which participate the epigenetic modulation processes by catalyzing the methylation of certain DNA fragments. However, their overexpression can lead to the excessive methylation of the promoter region of some important tumor suppressive genes, and thereby epigenetically block their transcription-al activity and facilitate the onset of tumors. Despite the clues from several extracranial tumors that miR-29a/b/c can specifically silence DNMT3A/3B, their exact relationships in glioma cells as well as whether lack of miR-29s results in the expression changes of DNMT3A/3B deserve further elucidation. Using human glioma tissues, non-tumoral control brain tissues and human glioblastoma cell lines U87MG and U251as the objects, we set out in the current study to probe the above issues by:①detecting the expres-sion changes of miR-29s in gliomas of various pathological grades,②confirming the legitimate of DNMT3A/3B as the natural targets of miR-29a, b and c in glioblastoma cells and screening the target regions of these miRNAs on the corresponding3’ UTRs,③observing the impacts of miR-29s Mimics and inhibitors on DNMT3A/3B expres-sion in glioblastoma cells and exploring the underlying mechanisms. Through this study, we aim to explain how the malfunction of the miR-29a/b/c-DNMT3A-DNMT3B pathway induced by miR-29a/b/c low expression contributes to the onset and malignant progression of gliomas, and provide laboratory data to assess the feasibility of the new gene therapy and molecular targeting strategies which takes the above pathway as the center of the intervention.Methods:①The miR-29a/b/c expression levels of the60glioma tissues of vari-ous pathological grades and10non-tumoral brain tissues were detected by tissue mi-croarray based locked nucleotide probe in situ hybridization.②Three miR-29a/b/c transfection groups and a Scr control group were established for U87MG and U251by transfecting the cells with the mimics of miR-29a/b/c and a nonsense scrambled se-quence, respectively. The parallel blank controls were also established with the trans-fection-naive cells. The miR-29s expression levels of all the experimental groups were quantified by stem-loop qRT-PCR.③The legitimacy of DNMT3A/3B as the target of miR-29s as well as the target regions in their3’ UTR were predicted by bioinformatics and verified by luciferase assays.④The expression levels of DNMT3A/3B mRNAs and proteins were measured by qRT-PCR and Western blot to observe the modulating effects of miR-29a,b and c and explore the underlying mechanisms.⑤miR-29a/b/c inhibitors were introduced to the U87MG and U251cells to see whether these inhibitors could affect the expressions of DNMT3A/3B mRNAs and proteins, so as to further demonstrate the above results and unveil the role of miR-29a/b/c downregulation in the DNMT3A/3B overexpression of glioma cells.Results:①Compared with the non-tumoral control brain tissues, the LI%of miR-29a/b/c of the glioma groups were significantly lower, and decreased along with the elevation of the tumor’s malignancy grades. All the differences are statistically sig-nificant.②Stem-loop qRT-PCR results showed that the miR-29s levels of the miR-29a/b/c transcription groups were significantly higher than those of the blank and Scr con-trol groups.③Bioinformatic analysis predicted that both DNMT3A and3B contained a target region for miR-29a/b/c in their3’UTRs. Luciferase assays demontrated that the wild type DNMT3A/3B3’UTRs containing the intact targets of miR-29s could mediate the suppressive effects of the miRNA on reporter gene expression whilst the mutated type with the first6nucleotides of the target regions deleted could not, and therefore confirmed that the both DNMT3A and DNMT3B mRNAs are targets of miR-29a/b/c.④qRT-PCR and Western blot results demonstrated that the expression levels of DNMT3A/3B mRNAs and proteins were significantly higher in the miR-29a/b/c tran-scription groups than in the blank and Scr control groups.⑤Compared with those of the corresponding blank and Scr control groups, the DNMT3A/3B mRNA and protein expression levels of the miR-29s inhibitor transcription groups were significantly high-er.Conclusions:①The abnormally low expression of miR-29a/b/c is a prevalent phenomenon in glioma cells, and its expression level decreases along with the tumor’s malignancy grades. MiR-29s low expression is a potential cause of the tumorigenesis and progression of gliomas, and it can be used as an important auxiliary marker to as-sess the bio-behavior of the tumors and the prognosis of the patients.②Mimics trans-fection can efficiently increase the miR-29s levels in glioblastoma cells.③DNMT3A and3B are natural targets of miR-29s in glioma cells. Binding the target regions in the3’UTRs of DNMT3A/3B with their seed sequences in nucleotide complementary man-ner, miR-29s could induce the degradation of DNMT3A/3B mRNAs, and effectively knock down the expression of the target genes.④miR-29s inhibitors can substantially upregulate DNMT3A/3B expression in glioma cells, further suggesting that lack of miR-29a/b/c is responsible for the overexpression of DNMT3A/3B in glioma cells.