Regulation Of Human Glioma Cell Apoptosis And Invasion By MiR-152-3p Through Targeting DNMT1-NF2 Pathway

BcakgroundBecause of the complex regulation mechanism of brain glioma and how to conquer the most common primary malignant tumor brain always kept the focus and difficulty of neurosurgeries.Glioblastomas was a type of primary brain tumour characterized by strong aggressivity and malignancy,with median surial only 12 to 18 months due to its resistance to current therapies.Its expression product merlin protein had the key function in regulating tumor cell proliferation,invasion and migration.DNMT was the catalytic role of maintaining methylation,and abnormal expression of DNMT was closely related to tumorigenesis.A lot of damage DNA methylations were charactered as glioma tumor cells rapidly proliferation.MicroRNAs disorders were closely related to the tumor pathogenesis,but how to control the glioma pathogenesis between miRNAs and DNMT1 was not knew,the process between miRNAs and DNMT1 relationship about the apoptosis and invasion of glioma cells need further research.Aims and meaningThis topic around the regulatory mechanism of miR-152-3p and DNMT1-NF2 pathway,and glioma biology behavior occurrence,development influence was revealed.Through the expressions of miR-152-3p,DNMT1,NF2 and methylation in glioma tissues and glioma cells,miR-152-3p,DNMT1,NF2 and the relationships between the pathogenesis of glioma were discussed.Molecular biology study aimed to know whether the miR-152-3p could be targeted to adjust DNMT1 expression or not,and we hoped to find a glioma cell apoptosis and regulatory pathway after the study.MethodsThe project was separated into two parts.One part was the glioma tissues and glioma cells detection of miR-152-3p,DNMT1,NF2 mRNA expression by qRT-PCR and Western blot.DNMT1,merlin expression of normal brain tissues and glioma tissues were detected by immunohistochemical.NF2 gene methylation level of glioma tissues and different kinds of glioma cell lines were detected by MSP and BSP.The other part was to construct DNMT1 siRNA recombinant plasmid and NF2 gene expression vector,DNMT1 siRNA and overexpressions of miR-152-3p,NF2 were transfected in vitro cultured U251 glioma cells.The expressions of miR-152-3p,DNMT1 and NF2 after transfection were examined by qRT-PCRWestrn blot、MSP and BSP.Proliferations of glioma cells were detected by CCK,and the results were proved by immunofluorescence.Apoptosis of glioma cells were detected by fluid cytology and EDU,invasion ability of glioma cells were observed by transwell.ResultsIn the present study,we observed that NF2 was hypermethylated and DNA methyltransferase 1(DNMT1)was markedly increased in GBM tissues and glioma cells,including U251,U87,T98-G and A172 cells.Knockdown of DNMT1 demethylated NF2 gene and resultantly increased the expression of NF2 at transcriptional and translational levels.Aberrant expression of microRNAs plays critical role in human cancers,including GBM.Here,we found that miRNA-152-3p was decreased in GBM tissues and directly targeted DNMT1,leading to the demethylation of NF2 gene and its expression.Furthermore,we demonstrated that both miR-152-3p overexpression and DNMT1 kncokdown significantly induced cell apoptosis and inhibited the invasive activity.And this was also observed after NF2 overexpression.These results indicated that miR-152-3p can inhibit glioma cells proliferation and invasion activities by decreasing DNMT1.ConclusionsAltogether,our results indicated that there was a miR-152-3p/DNMT1 regulatory circuit and that this miRNA acted as a tumour suppressor.These results indicated that miR-152-3p directly targeted DNMT1 and miR-152-3p overexpression lead to NF2 demethylate and increase NF2 expression.NF2 can inhibit glioma cells proliferation and invasion activities by decreasing DNMT1.and restoration of miR-152-3p may be taken as the therapeutic application to treat GBM.