Identification And Research Of A Novel MiRNA About Glioma

Objective Glioblastoma multiforme(GBM)is a common central nervous system tumor with high malignancy and aggressive characteristics.At the same time,GBM is highly heterogeneous and is under a very poor prognosis.It is especially important to perform precise diagnosis and treatment of specific molecular subtypes or targets.The transforming growth factor-?(transforming growth factor-?TGF-?)pathway plays an important role in tumorigenesis through its tumor suppressor or promotion mechanism.In recent years,studies have suggested that TGF-? affects the signal transduction process through microRNA(miRNA)regulation.There is an automatic regulation feedback loop between miRNA and TGF-? signaling pathway,which affects the biological process of tumor cells.We used a small molecule inhibitor of transforming growth factor-beta receptor(TGF?R)for drug treatment of freshly excised clinical tissue samples.And compared the dosing group with high-throughput sequencing.The expression of miRNA differed from that of the control group.In the sequencing results,we unexpectedly discovered some novel miRNAs and there were significant differences between the treatment and the control group.This study aimed to screen and identify novel miRNAs that were found to be significantly dissimilar,to elucidate the relationship between a novel miRNA in the regulation of TGF? signaling pathways and its role in gliomas.Methods Validation analysis of novel miRNAs was conducted by high-throughput sequencing combined with stem-loop qRT-PCR and bioinformatics methods.Based on the human glioblastoma cell line U87,the PDX model of primary cells and glioblastoma,target gene prediction,dual luciferase reporter gene assay,qRT-PCR and Western blot screening to verify target genes,mimics(mimics)or lentiginous transfection for in vitro functional mechanism studies and in vivo animal experiments.Result Sequencing analysis showed that there were considerable differences in the expression of 27 novel miRNAs.The results of stem-loop qRT-PCR showed that novel_miR56 was significantly altered as a novel miRNA and regulated by TGF-? pathway.Through the target gene prediction and analysis of biological processes in which novel_miR56 may be involved,a comprehensive analysis of our screened PRAS40 as a possible target mRNA for novel_miR56.By luciferase reporter gene assay,qRT-PCR and Western blot,the results show that in glioma cells,novel_miR56 can regulate the mRNA and protein levels of PRAS40 by specifically binding to the 3'UTR of wild-type PRAS40 mRNA.The change confirmed that PRAS40 is a natural target gene of novel_miR56.In vitro proliferation experiments showed that over expression of novell-miR56 promoted tumor growth and acted on autophagy and affected proliferation through mTOR signaling pathway.In vivo animal experiments showed that the size of the novel_miR56 over expression group and the growth rate of the control group were significantly increased,and the survival time was short.Immunohistochemistry results showed that the novell-miR56 over-exposure group had a higher degree of malignancy,and novel_miR56 promoted tumor development.Conclusion Our study demonstrates that in the glioma,novel_miR56 acts as oncomiR,and we determined that the downstream target of the TGF?-novel_miR56 pathway is PRAS40 mRNA,which is a negative regulator of mTOR kinase and thus affects autophagy.In vitro and in vivo experiments showed that novel_miR56 promoted cell proliferation while attenuating the level of autophagy in cells.Our study also serves as a new mechanism to explain the role of TGF? signaling in the promotion of glioma.