Myc Modulates The Biological Behavior Of Glioma Cells By Inhibition Of RUNX3 Through MiR-4295

Objective: Glioma is the most common malignant tumor in the brain,accounting for 40%-45% of the entire nervous system tumor,with strong invasive ability.Most studies suggest that gliomas originate from glial cells or progenitor cells of the neuroectoderm,but the evidence for the origin is inconclusive.Although there are currently treatments such as surgery,radiotherapy and chemotherapy,gliomas are prone to recurrence and the degree of malignancy is gradually increasing,which still plagues patients and clinicians.Therefore,research on glioma has continued over the years.Currently,the WHO classification scheme is widely accepted and remains to be a practical and effective means to classify gliomas.However,this system is solely reflected by histologic visual criteria and prone to subjective inter-observer variation.Researchers need to find more direct ways to prevent disease,improve the accuracy of diagnosis,and the effectiveness of treatment.MicroRNAs(miRNAs)are endogenous,non-coding,single-stranded,small-molecule RNAs of about 18 to 25 nucleotides in length,which regulate gene expression at post-transcriptional levels,with high levels of conservation,timing,and organization.Their specific characteristics: The miRNA binds to the 3' non-coding region of the target mRNA completely or incompletely,thereby causing degradation of the target mRNA or inhibiting transcriptional translation,thereby regulating the expression of the target gene.miRNAs can participate in a range of important biological behaviors such as cell differentiation,cell proliferation,apoptosis,and stress response.The discovery of miRNA not only broadens our understanding of the mechanism of gene regulation,but also provides a novel perspective for studying the regulation of tumor development.Increasing data shows that many specific miRNAs are associated with cancer,such as lung cancer,breast cancer,and astrocytoma.Although miRNA is a non-coding protein,it can regulate the expression of downstream proteins through various pathways.It has many special and complex functions,especially the regulation of the biological behavior of tumor cells,and has a great influence on the proliferation,migration and invasion of tumor cells,apoptosis and differentiation.Different miRNAs have different functions: some are oncogenes,and some are tumor suppressor genes.Even with the same miRNA,different regulatory mechanisms can occur in different tumors,thus having different functions.More and more reports on glioma research indicate that many types of miRNAs play different roles in their formation and development,and directly affect the biological behavior of human glioma.Therefore,the study of the regulation mechanism between miRNAs has greatly promoted the development and treatment of glioma.The Runt-related gene family includes RUNX1,RUNX2,and RUNX3.The RUNX gene is known as an important regulator of cell fate and,under certain conditions,has a dual role as a tumor suppressor gene or a dominant oncogene.The different roles of this family of transcription factors in cancer are largely related to regulating growth and differentiation.RUNX3 is under-expressed in primary glioblastoma,whereas overexpression of RUNX3 in glioma cells significantly inhibits cell invasion and metastasis.Various evidences suggest that miR-4295 may target USP28 in non-small cell lung cancer,while high expression of USP28 promotes cell proliferation.However,there is no direct evidence that there is a potential regulatory pathway involved in miR-4295/RUNX3 in gliomas.Considering the research status domesticly and abroad,this study aimed to verify the targeted binding and binding sites of miR-4295 and RUNX3,and to clarify the mechanism of miR4295 inhibiting the expression of RUNX3;to study the effect of miR-4295 on apoptosis and cell cycle of glioma cells;to observe its anti-tumor effect on nude mice glioma;to further study the targeted binding and binding sites of N-myc and miR-4295,and elucidate the regulation mechanism of N-myc on miR-4295;finally,to study N-myc Modulation of the miR-4295/RUNX3 signaling pathway.The results of this study can not only confirm the negative regulation of RUNX3 expression by N-myc binding to miR-4295,but also affect the biological behavior of glioma cells,and provide a new target for improving the efficacy of glioma.Methods: 1.Real-time PCR was used to detect the expression levels of miR-4295 and RUNX3 in normal brain tissues,glioma tissues and cells.2.Double luciferase reporter gene expression analysis verified the direct binding of microRNA-4295 to RUNX3 and its key binding sites,as well as the direct binding of N-myc to the promoter region of miR-4295.3.MTT assay,cloning and transplantation models were used to detect the proliferation of glioma cells.Flow cytometry was used to detect apoptosis and cell cycle of glioma cells.Western blot was used to detect the expression of RUNX 3. The effect of miR-4295 on malignant biological behavior of glioma cells and its related mechanisms were clarified.4.Western blot was used to detect the expression of RUNX3,Realtime PCR was used to detect the expression of miR-4295,and the effect of N-myc on the biological behavior of glioma cells and its related mechanisms were clarified.5.ChIP confirmed that N-myc binds to the promoter region of the miR-4295 precursor.Results:1.MiR-4295 is up-regulated in glioma tissues and cells.The expression level of miR-4295 was correlated with in tumour grade2.MiR-4295 induces glioma tumourigenesis in vitro and in vivo.3.The cells infected miR-4295 exhibited a significantly increased apoptosis. Knocking down miR-4295 in both U87 and U251 cells significantly inhibited cell cycle progression.4.MiR-4295 directly targets RUNX3 and inhibits its expression.5.RUNX3 was down-regulated in glioma tissues.6.N-myc bound to the promoter of miR-4295,and promotes miR-4295 expression7.N-myc reduced RUNX3 expression in glioma.Conclusions: 1.MiR-4295 was up-regulated in glioma tissues;In addition,the expression level of miR-4295 was correlated with glioma pathological grades.2.MiR-4295 inhibited cell apoptosis and regulated G0/G1 transition,and could promote tumourigenesis in glioma in vitro and in vivo.3.MiR-4295 recognized and regulated RUNX mRNA through specifically binding to its 3'UTR.4.N-myc inhibited RUNX-3 partly through binding to the promoter of pri-miR-4295.5.N-myc/miR-4295/RUNX3 may play a potential role in tumorigenesis and progression of glioma.