The Regulatory Role And Mechanism Of Long Non-coding RNA NEAT1 In Glioma Proliferation And Apoptosis

BACKGRUONDGlioma is one of the most common malignant primary tumor in central nervous system,accounting for about 60% of malignant primary brain tumors.Although the treatment options of surgery,chemotherapy and radiotherapy continue to improve,the overall survival rate of glioma patients is still very low,so there is an urgent need for new and effective treatments method.Numerous previous studies have shown that long non-coding RNA(lncRNA)plays an extremely important role in the occurrence and progression of a variety of cancers,including glioma.The research results show that the long non-coding RNA NEAT1(Nuclear Paraspeckle Assembly Transcript 1)regulates cancer growth by affecting cell proliferation,migration,invasion and drug resistance.In the early stage,we analyzed the expression data of glioma oncogenes in the TCGA database and found the correlation between the long non-coding RNA NEAT1 and the histological malignancy of gliomas.Next,we assessed whether the aberrant expression of long non-coding RNA NEAT1 has a potential impact on the median survival of patients by using the TCGA database.Long non-coding RNA(lncRNA),more than 200 nucleotides in length,involves multiple biological regulatory processes.A large number of studies have shown that lncRNA can control the expression of a variety of genes,and may contribute to the diagnosis and prognosis of cancer.LncRNA functions are very diverse and can affect epigenetic,transcription,and post-transcriptional translation at the gene level.Previous data showed that long non-coding RNA is abnormally expressed in cancer cells and is related to cancer progression,including effects on apoptosis,adhesion,metabolism,proliferation,and metastasis.Many studies have found that lncRNA can participate in the regulation of biological processes by competing endogenous RNA(ce RNA)way.This regulatory mode is a very important route for regulating cancer progression,also known as molecular sponges,which regulate downstream genes through the interaction between lncRNAs and miRNAs.The long non-coding RNA NEAT1 is up-regulated in many tumors,such as laryngeal cancer,leukemia,and ovarian cancer.Generally,there is more than one binding site for non-coding RNA structures,and they play different roles.Moreover,the exact regulatory mechanism of NEAT1 in glioma is not yet fully understood.In-depth research and analysis of the impact of NEAT1 on glioma is of great significance for us to propose further treatment options.PURPOSES1.Screen and determine the abnormally expressed lncRNA molecules in glioma tissues and cells;2.Explore the effect of lncRNA NEAT1 on the proliferation and apoptosis of gliomas through in vivo and in vitro experiments;3.Reveal the regulation relationship between NEAT1/miR-324-5p/KCTD20 through the cell function regression experiment;METHODS AND RESul TS1.The TCGA database was analyzed by bioinformatics to observe the expression and survival of lncRNA NEAT1.The results showed that lncRNA NEAT1 expression was significantly down-regulated in II-III grade compared with GBM sample group.And the median survival time of high expression lncRNA NEAT1(High NEAT1)is less than that of low expression group(Low NEAT1).The expression of NEAT1 in glioma tissues and cell lines was determined by RT-PCR experiment,and the results showed that compared with normal brain tissues and NHA cells,NEAT1 expression levels in tumor tissues and cells were increased.2.Cell Counting Kit-8(CCK-8),colony formation,Ed U and Western blot analysis were used to detect the effect of NEAT1 on cell biological behavior,flow cytometry assay was used to analyze NEAT1 function on cell cycle and apoptosis.These results indicated that NEAT1 knockdown can inhibit proliferation of glioma cells,and cause glioma cell cycle arrest and increase cell apoptosis rate.3.In order to more effectively verify the effect of NEAT1 on the proliferation of glioma cells,we established a nude mouse tumorigenesis animal model through directional intracranial in situ injection,and found that the down-regulation of NEAT1 significantly inhibited the proliferation of tumors,and the reduction of NEAT1 expression can improve the survival time of the mouse model.4.Numerous studies have shown that NEAT1 can regulate the progression of tumors through competitive binding way.Therefore,in order to verify the possible regulatory relationship between NEAT1 and miR-324-5p,we have further carried out bioinformatics analysis,online website target prediction analysis,RNA immunoprecipitation experiment(RIP)and luciferase reporter analysis.The results of the RIP experiment found that compared to the negative control group anti-Ig G,the anti-AGO2 enriched NEAT1-AGO2 complex in the experimental group was significantly increased.5.Online prediction sites Targetscan,miRDB and miRWalk found that 6 target genes are related to miR-324-5p by using the Venn diagram.We verified the knockout efficiency and found that compared with the other five target genes,KCTD20 has the highest knockout efficiency.The up-regulation of miR-324-5p expression can inhibit the luciferase activity of WT-KCTD20,further verifying that KCTD20 is a potential downstream target gene of miR-324-5p.6.The regulatory relationship between NEAT1/miR-324-5p/KCTD20 can be valued by cell function regression experiments,and then respectively transfected si-NEAT1,miR-324-5p inhibitor and si-KCTD20 into glioma cells.These results showed that cotransfection of miR-324-5p inhibitor and si-NEAT1 could partially reverse the knockdown effect of NEAT1,and western blot assay showed that si-NEAT1 could inhibit the expression of KCTD20,miR-324-5p inhibitor can promote the expression of KCTD20.The effect of NEAT1 knockdown was reversed when co-transfected with si-NEAT1 and miR-324-5p inhibitor,and this reversed result is consistent with cell proliferation and apoptosis assays.We concluded that NEAT1 regulates the expression of KCTD20 by competitively binding miR-324-5p,thus it can reduce cell proliferation and increase apoptosis when NEAT1 expression is downregulated.CONCLUSIONSCurrent research results indicated that the expression of long non-coding RNA NEAT1 is significantly up-regulated in glioma tissues and glioma cell lines,and it is a tumorpromoting factor for glioma.The expression level of lncRNA NEAT1 may be used as a basis for evaluating the survival and prognosis of glioma patients.Cell function experiments showed that down-regulating the expression of NEAT1 or up-regulating the expression of miR-324-5p can inhibit the proliferation of glioma cells.LncRNA NEAT1 affects the tumor suppressor effect of miR-324-5p by competitively binding(ce RNA)miR-324-5p,thereby increasing the expression of the downstream target gene KCTD20 and promoting the progression of cancer.In combination with the above results,NEAT1 and miR-324-5p are involved in the regulation of the progression of glioma through competitive binding pattern,and the NEAT1/miR-324-5p/KCTD20 axis is identified as a new regulatory axis and human glioma Potential therapeutic target.