Study On The Mechanism Of MicroRNA-195-5p In Malignant Progression Of Glioma

Glioblastoma multiforme(GBM)is the most prevalent malignant primary brain tumor in humans and ranks highly among all cancers.Due to the tumor’s concealed onset and rapid progression,it is difficult to detect it early.Low resection rates and a high recurrence rate characterize the aggressive growth of tumors.Due to the blood-brain barrier’s inherent existence and the tumor’s obvious resistance to radiotherapy and chemotherapy,clinicians are frequently powerless.Therefore,we urgently need to understand the molecular mechanism of malignant progression of glioma through extensive and in-depth research,and find new therapeutic targets to break through the current dilemma of diagnosis and treatment of glioma.Current research indicates that the progression of glioma is closely associated with various epigenetic phenomena,including histone modification,DNA methylation,chromatin remodeling,and abnormal expression of non-coding micro RNA(mirna).Exploring the biological function of specific micro RNAs in glioma tumor development and occurrence will provide theoretical support for the discovery of new therapeutic targets.MiRNAs are small endogenous non-coding RNAs.The seed region of miRNA,2-8nt from the 5′ end,binds to the 3′ untranslated region(3′UTR)of target messenger RNAs(m RNAs)through an imperfect match,which leads to m RNA destabilization and/or translational inhibition.Numerous studies have shown that miRNAs most often block the translation of m RNA into proteins.The expression levels of these proteins are involved in the construction of biological characteristics of many tumors,including tumorigenesis,invasion,angiogenesis,immune microenvironment,EMT,cancer stemness,metabolism and drug resistance.Dysregulation of miRNA expression levels has been found to affect tumors of various origins,including breast,colon,lung,liver,and pancreatic cancers,as well as gliomas.Studies have shown that miRNA is an important regulator of the malignant progression of glioma,and based on the mechanism of action of miRNA,we are constantly trying to find effective therapeutic targets.MiR-195-5p,a miRNA derivative from the 5p arms of miR-195 precursors by Dicer cleaving,had been found to correlate with almost all types of human cancers,which included respiratory system,digestive system,urinary system,nervous system,motor system,reproductive system and endocrine system.miR-195-5p targeted many oncogenes,and was thus implicated in various tumorigenic processes such as migration,invasiveness,proliferation and chemoresistance.However,the its role in glioma is rarely reported,and its molecular mechanism is still unclear.Cyclin D2(CCND2)is an important cell cycle regulator and a key member of the CCND2-CDK4(DC)complex.CCND2 is a downstream target of c-Myc and RUNX1/ETO oncogenes.Hyperactivation of proto-oncogenic signaling by these oncogenic factors accelerates cell cycle progression in part through increased CCND2 transcription.Studies have found that CCND2 is overexpressed in a variety of tumors.It associated with tumor invasion,metastasis and prognosis in patients with colon cancer and gastric carcinoma.Furthermore,the stability of CCND2 m RNA is also regulated by micro RNAs in thyroid carcinoma,NSCLC,prostate cancer,and oral squamous cell carcinoma;insufficient expression or lack of those micro RNAs also results in elevated CCND2 protein levels.In this study,the bioinformatics analysis firstly showed miR-195-5p and CCND2 had great potential in the malignant process of glioma.Then RT-q PCR was used to detect the expression level of miR-195-5p between human glioma cells and normal human astrocytes.To validate the tumor suppression ability of miR-195-5p,we analyzed cell proliferation,invasion and migration by cell counting kit-8(CCK8),scratch wound healing assay and transwell assay.Further,we conducted target gene validation between miR-195-5p and CCND2 by RT-q PCR,western blot and dual luciferase reporter gene assays.Finally CCND2 was identifed as a target gene of miR-195-5p.In addition,miR-195-5p activated PI3K/AKT/mTOR signaling pathway by inhibiting CCND2.Part Ⅰ:Screening of glioma prognosis-related miRNAs and prediction of their target genes based on bioinformatics Objective:To screen out the differentially expressed miRNAs associated with the prognosis of glioma by bioinformatics.Methods:1.A dataset(GSE32466)containing the sequencing results of 24 cases of GBM tumors was extracted from the GEO database,and 12 cases of primary GBM and 12 cases of recurrent GBM were compared using GEO2R(an online analysis tool based on the miRNA differential analysis method).Notably,the data set contains 12 GBM patients who have undergone recurrence monitoring.This uncommon paired study eliminates the analysis error brought on by individual differences.2.Using the differentially expressed miRNAs as a basis,we use the miRNA data set from the China Glioma Database(CGGA)to determine if there are significant differences in expression and to identify miRNAs associated with the prognosis of gliomas.3.Using multiple miRNA target gene prediction tools,such as miRDB,miRDIP,miRmap,miRPath DB,miRTar Base,miRWalk,Target Scan,micro T-CDS,and Tar Base,to predict the screened miRNAs,the intersection of eight or more prediction results is considered to be a reliable prediction target.4.Using the online analysis software DAVID,all predicted target genes were added to the GO and KEGG databases for enrichment analysis,and their biological functions and potential signaling pathways were predicted.5.All predicted target genes were added to the STRING database,and a protein-protein interaction network diagram was created.6.The cyto Hubba and MCODE Cytoscape plug-ins were used to analyze the preceding network diagram and identify the key genes.7.GEPIA2 database(an online analysis tool based on TCGA database)and Glio Vis database(a brain tumor database dominated by glioma)were used to analyze the expression and prognosis of key genes in glioma.8.On the basis of the TCGA and CGGA databases,gene probe enrichment analysis(GSEA)was used to analyze the enrichment of key genes,and significantly enriched signal pathways were identified.9.Using the HPA database to examine the expression of essential genes in glioma.10.Using single cell sequencing databases(GBMseq and Cancer SEA)to analyze the situation of characteristic miRNAs and key genes at the level of single cell gene expression,and confirming that miRNAs and target genes are associated with tumor cells.Results:1.Compared with primary GBM,recurrent GBM significantly expressed 26 miRNAs,of which 11 were up-regulated and 15 were down-regulated.2.Using CGGA database analysis,it was found that miR-195-5p and miR-223-3p were significantly down-regulated in recurrent GBM(compared with primary GBM),and the low expression level of both miRNAs was related to the poor prognosis of GBM.3.It is predicted that there are 28 target genes of miR-195-5p and 14 target genes of miR-223-3p.4.GO and KEGG enrichment analysis of the predicted target genes showed that:(1)GO-CC: the cells were located in protein kinases and transferases;(2)GO-MF: The molecular functions are mainly DNA transcription inhibition and RNA polymerase II specificity;(3)GO-BP: biological processes are mainly positive regulation of cell catabolism and negative regulation of cell development;(4)KEGG: The signal pathway is mainly enriched in PI3K-AKT signal pathway,abnormal regulation of tumor cells during transcription and regulation signal pathway of pluripotent stem cells.5.The confidence score is 0.7,which is highly credible,and the interaction network with 14 proteins is obtained.6.The top 10 key genes were screened by cyto Hubba plug-in,and two subnets were obtained by MCODE plug-in.The possible target genes of miR-195-5p are CCNE1,CCNT2 and CCND2),and the possible target genes of miR-223-3p are FOXO3,FOXO1 and POU2F1.7.Based on the analysis of TCGA and CGGA databases,it was found that FOXO1 and CCND2 were significantly differentially expressed in glioma,and high expression was related to poor prognosis.8.Based on the glioma data set,GSEA analysis showed that FOXO1 and CCND2 were mainly concentrated in PI3K-AKT-mTOR signal pathway.9.Immunohistochemistry of tumor samples in HPA database suggested that FOXO1 was highly expressed in normal cerebral cortex,but not in glioma cells.However,the expression of CCND2 was obvious in tumor cells and weakened in normal cells.10.Based on GBM single cell sequencing database,it was found that miR-223-3p and miR-195-5p were almost undetectable in tumor cells,while FOXO1 and CCND2 were overexpressed in tumor cells.Conclusions:Based on bioinformatics analysis,it is found that the low expression of miR-223-3p and miR-195-5p is related to the occurrence and development of glioma.The target gene predicted by miR-195-5p is CCND2,which has the potential biological effect of regulating PI3K-AKT-mTOR signaling pathway.Part Ⅱ:Effects of miR-195-5p on the proliferation,migration and invasion of glioma cellsObjective:To verify the difference in the expression of miR-195-5p between glioma cell line and normal glial cell line,and its effect on the proliferation,migration and invasion of glioma cells.Methods:1.Real-time fluorescence quantitative polymerase reaction(RT-q PCR)was used to detect the expression level of miR-195-5p in human glioma cell lines(U87,U251) and human astrocyte cell lines(NHA).2.miRNA mimics and inhibitors were used to overexpress or silence miR-195-5p,and CCK-8,scratch test and transwell invasion test were used to verify the effects of miR-195-5p on the proliferation,migration and invasion of glioma cells.Results:1.The expression of miR-195-5p was significantly down-regulated in the U87 and U251 cell lines compared to the NHA cell line,and the difference was statistically significant.2.The results of CCK-8 experiments suggest that the growth and proliferation of cells were suppressed after transfection of miR-195-5p mimic in U87 and U251 cell lines.On the contrary,transfection of miR-195-5p inhibitor to promote cell growth and proliferation.3.The results of the wound healing assay indicated that compared with the blank control group,the wound healing rate of the glioma cells transfected with the miR-195-5p mimic decreased significantly;the cells transfected with the miR-195-5p inhibitor,the wound healing rate increased significantly.4.The results of the transwell invasion assay indicated that compared with the control group,whether it was U87 or U251,the number of tumor cells passing through the basement membrane of the miR-195-5p mimic group was significantly reduced,while miR-195-5p inhibitor group was significantly increased.Conclusions:Overexpression of miR-195-5p can inhibit the proliferation,migration and invasion of U87 and U251 glioma cells..Part Ⅲ:miR-195-5p mediates CCND2 to regulate PI3K/AKT/mTOR pathwayObjective:To verify that CCND2 is the target gene of miR-195-5p.And explore whether miR-195-5p and CCND2 can regulate PI3K/AKT/mTOR signal pathway.Methods:1.The expression level of CCND2 in human glioma cell lines(U87,U251)and human astrocyte cell lines(NHA)was determined using RT-q PCR and western blot. 2.Western blot analysis was used to detect the expression level of CCND2 in glioma cell lines transfected with miR-195-5p mimic and mimic NC(mimic negative control),and dual luciferase reporter gene assay was performed to determine whether miR-195-5p can bind to the 3’UTR of CCND2.3.Using small interfering RNA to knock down CCND2 in glioma cell lines firstly.Then we detected the expression of key proteins in the PI3K/AKT/mTOR signaling pathway by western blot.4.A plasmid pc DNA3.1-CCND2 was constructed and co-transfected with miR-195-5p mimic into U251 and U87 cells.After transfection,proliferation and invasion were evaluated using the CCK-8 and transwell assays,respectively.Furthermore,the relevant rescue experiments were performed to evaluate the protein levels of p-mTOR and p-AKT by western blot.Results:1.The expression of CCND2 in the glioma cell lines U87 and U251 was statistically significantly greater than that of NHA.2.RT-q PCR and western blot revealed that miR-195-5p over-expression could down-regulate the protein expression level of CCND2;The dual luciferase reporter gene assay confrmed that miR-195-5p could bind with CCND2 3’UTR.3.The rescue experiments,CCK8 assays and transwell assays,revealed that the upregulation of CCND2 restored the viability and invasion of the miR-195-5p mimics-transfected glioma cells.4.Western blot results showed that compared with NC group,knockdown of CCND2 expression in U87 and U251 cell lines decreased the phosphorylation level of AKT and mTOR,suggesting that CCND2 can activate PI3K/AKT/mTOR signal pathway.5.The Western blot assays showed that miR-195-5p significantly down-regulated the expression of p-AKT and p-mTOR.It indicated miR-195-5p could block the PI3K/AKT/mTOR signaling pathway.The over-expression of CCND2 could reverse the blocking effect of miR-195-5p on the signaling pathway.Conclusions:By targeting CCND2,miR-195-5p blocked the activation of the PI3K/AKT/mTOR signaling pathway,thereby inhibiting the proliferation,migration,and invasion of glioma cells.