Effect And Mechanism Research Of MiRNA-132 On The Malignant Biological Behavior Of Glioblastoma

Background and Objective:Glioma is the most common primary intracranial malignant tumor in adults,most of which is glioblastoma multiforme(GBM)with the highest malignancy.GBM is strongly invasive,highly recurrent,and has a poor prognosis.Improving the survival of GBM patients is still a task to be solved in clinics.In recent years,with the development of molecular target therapy and the discovery of glioma-related micro RNAs,the role of micro RNA in the occurrence and development of glioma and its value in clinical diagnosis,treatment,and prognosis have attracted more attention,and become one of the new directions of exploring GBM therapeutic target.In this study,by analyzing our previous clinical cases and the data of GBM patients in the TCGA database,miRNA-132,a less studied and controversial micro RNA in GBM,was selected as a new molecular entry point,to study the relationship between miRNA-132 and GBM molecular subtypes and its impact on patient prognosis,to clarify its role in regulating tumor cell proliferation and self-renewal,and to explore related downstream targets.The purpose of the study was to enrich the molecular mechanism of miRNA-132 in GBM and provide data support and a feasibility basis for the clinical diagnosis,treatment,and drug development.Materials and Methods:1.The relationship between miRNA-132 and GBM molecular subtypes and patient prognosis.(1)Searching the data of GBM patients in the TCGA database(including gender,age,GBM molecular subtype,overall survival time,and miRNA-132 expression level);(2)Studying the differences in the expression levels of miRNA-132 in different molecular subtypes;(3)Kaplan-Meier survival analysis was used to compare the effect of different miRNA-132 expression level on the prognosis of GBM patients;(4)The Cox regression model was established to analyze the value of miRNA-132 in judging the prognosis of GBM patients.2.The effect of miRNA-132 on GBM cell proliferation and self-renewal and other malignant biological behaviors.(1)Using human glioblastoma cell lines U87 and U373 as experimental modal,monolayer cells and neurospheroids were cultured and isolated,and RT-q PCR was used to detect and analyze the expression differences of miRNA-132 in monolayer cells and neurospheroids;(2)By transfecting miRNA-132 mimic and miRNA-132 antisense to simulate miRNA-132 overexpression and functional inhibition,the CCK-8 method,flow cytometry,and western blotting were used to explore the effects of miRNA-132 on GBM cell proliferation,apoptosis and drug resistance;(3)Using neurosphere culture and clone formation experiments to study the effect of miRNA-132 on the self-renewal and other "stem" characteristics of GBM cells.3.The exploration and validation of miRNA-132 downstream targets.(1)Using GCBI Gene Analysis Cloud Laboratory to perform gene chip differential analysis on GBM cells transfected with miRNA-132 in the GEO database,to study the effect of miRNA-132 on the gene expression profile of GBM cells;(2)Using GO analysis and enrichment analysis to study the effect of miRNA-132 on GBM cell signaling pathways;(3)Scanning commonly used miRNA prediction databases to screen the downstream targets of miRNA-132;(4)Using dual-luciferase report experiment to verify the regulation of miRNA-132 on target genes;(5)Constructing target gene expression vector to study the effect of target gene on GBM cell proliferation and self-renewal;(6)Collecting GBM clinical-pathological specimens,and studying the relationship between miRNA-132 and target genes in tumor entities and the impact on the clinical prognosis of patients by RT-q PCR,Western blotting,Kaplan-Meier survival analysis,and other methods.Results:1.The expression level and proportion of miRNA-132 in classic GBM were higher than other subtypes;the survival time of patients with high miRNA-132 expression was lower than those with low expression;miRNA-132 was an independent risk factor for poor prognosis in GBM patients.2.The expression of miRNA-132 in GBM neurospheroids was significantly higher than that of monolayer cells;transfection of miRNA-132 mimic promoted the proliferation of GBM cells and inhibited TMZ-induced apoptosis,while transfection of miRNA-132 antisense could inhibit its proliferation and increase TMZ-induced apoptosis;transfection of miRNA-132 mimic increased the size and number of GBM neurospheroids and promoted clone formation,while transfection of miRNA-132 antisense could inhibit spheroidization and clone formation.3.Overexpression of miRNA-132 could affect the gene expression profile of GBM cells and induce the activation of various downstream pathways such as the glioma signaling pathway;PTBP2 was a downstream target gene of miRNA-132,miRNA-132 could inhibit the expression of PTBP2 in GBM monolayer cells and neurospheroids,and it was more prominent in neurospheroids;clinical case analysis showed that compared with normal brain tissue,the expression of miRNA-132 was up-regulated in GBM tissue specimens,while the expression of PTBP2 was down-regulated,and the two showed a linear negative correlation;survival analysis showed that patients with high expression of miRNA-132 had a poor prognosis,while patients with high expression of PTBP2 had survival benefits.Conclusions:1.The expression of miRNA-132 was up-regulated in GBM,and its high expression was an independent risk factor for the poor prognosis of patients.2.miRNA-132 played the role of GBM promoting factors by promoting the proliferation and self-renewal of GBM cells and inhibiting cell apoptosis.3.PTBP2 was a downstream target regulated by miRNA-132;miRNA-132 promoted the malignant biological behavior of GBM cells by targeted inhibiting the expression of PTBP2.4.PTBP2 could partially inhibit the proliferation and self-renewal of GBM cells and benefit patients’ survival.5.This study not only enriched the molecular mechanism of miRNA-132 in regulating the malignant biological behavior of GBM but also provided a new perspective for the clinical treatment and the target development of GBM.