The Study Of MiR-218-5p Inhibiting Gliomagenesis By Targeting On HMGB1 Pathway

Background : Glioma is the most common intracranial malignant tumor,accounting for approximately 80% of primary brain and central nervous system malignancies.With high invasiveness,easy to relapse,high mortality and other characteristics.Although great advances have been made in microsurgical techniques,neuroimaging techniques,and chemoradiotherapy regimens in recent years,the prognosis of patients with gliomas,especially high-grade gliomas,is still poor.Therefore,further insights into the mechanisms of glioma development are needed to explore new glioma molecules and targeted therapies.With the deepening of miRNA research in recent years,its role in the development of various tumors,including gliomas,has become increasingly prominent.MiR-218-5p is frequently down-regulated in various cancers and correlates with clinical stage,prognosis,and metastasis.The role of miR-218-5p in the development of glioma and its molecular mechanisms still need further study.Objective:1.By analyzing differentially expressed miRNAs in gliomas,miR-218-5p was selected as the study object to analyze the expression of miR-218-5p in glioma tissues and the relationship between miR-218-5p and the prognosis of glioma patients.2.To study the effect of miR-218-5p on the proliferation,invasion,migration and apoptosis of glioma cells by using U251 cells as the research object.3.To investigate the molecular mechanism by which miR-218-5p inhibits glioma growth by predicting and verifying that the target gene of miR-218-5p is HMGB1.The effect of miR-218-5p on glioma growth in vivo and the targeting relationship between miR-218-5p and HMGB1 were verified by animal experiments.Methods:1.To screen differentially expressed miRNAs in gliomas using bioinformatics methods and detect the expression of miR-218-5p in gliomas by real-time PCR.The relationship between miR-218-5p expression levels and clinical prognosis in gliomas was analyzed.2.Using U251 as the research object,miR-218-5p was overexpressed by transfection of miR-218-5p mimic,and the effects of miR-218-5p on the proliferation,invasion,migration and apoptosis of U251 cells were examined using CCK-8 assay,cell scratch healing assay,transwell chamber assay,and flow cytometry,respectively.3.Predict and screen the target gene HMGB1 of miR-218-5p using bioinformatics methods.The expression of HMGB1 in glioma tissues and cells was detected by qRT-PCR.Dual-luciferase target gene reporter assay was used to verify the targeting relationship between miR-218-5p and HMGB1.4.Subcutaneous glioma transplantation model in nude mice and intracranial orthotopic glioma model in Wistar rats were established.MiR-218-5p was overexpressed by intratumoral injection of agomir-218-5p,tumor size was observed by cranial energy spectrum CT scan,pathological changes were observed by HE staining,and the expression of HMGB1 as well as proliferation-related proteins was detected by Western blot and immunohistochemistry.Results:1.The miRNA of interest was selected as miR-218-5p by GEO data,and the results of qRT-PCR analysis showed that miR-218-5p was differentially expressed in glioma and normal brain tissue and down-regulated in glioma.The expression level of miR-218-5p in gliomas is correlated with the prognosis of glioma patients,and low expression of miR-218-5p indicates poor prognosis of glioma patients.2.CCK-8 cell proliferation assay showed that miR-218-5p was able to inhibit the proliferation of glioma cells.3.The results of cell scratch healing assay and transwell chamber assay showed that miR-218-5p could inhibit glioma cell invasion and migration.4.The results of apoptosis by flow cytometry showed that miR-218-5p could induce apoptosis in glioma cells.5.Predictions indicate that HMGB1 may be a target gene of miR-218-5p,and HMGB1 is significantly upregulated in gliomas.The results of dual luciferase target gene validation experiments showed that miR-218-5p may regulate gene expression by binding to sites on the 3'UTR of HMGB1 mRNA.6.In vivo results in animals showed that miR-218-5p targeted inhibition of HMGB1 expression in vivo inhibited the growth of subcutaneous gliomas in nude mice.Conclusions:miR-218-5p was down-regulated in glioma tissues and inhibited proliferation,invasion,and migration of glioma cells and induced apoptosis by targeting the regulation of HMGB1 expression.Targeted regulation of HMGB1 by miR-218-5p inhibits glioma growth in vivo.