| Background:Glioma is the most common primary malignant tumor in the central nervous system,accounting for 30%of all primary central nervous system tumors,accounting for 80%of the central nervous system malignancies.More than half of the gliomas are GBM(glioblastoma multiforme,GBM).At present,it is generally recommended for patients with glioma at home and abroad to use surgery plus radiotherapy and chemotherapy,but the median survival time of GBM is still only about 15 months.Although the continuous development in recent years,technological improvements,equipment replacement surgery chemotherapy technology,but still the treatment of glioma is just passable,still not achieved a major breakthrough,and clarify the etiological factor of glioma is still unclear.The study of the molecular mechanism of glioma development is one of the hot topics in academic circles.In 2013,the researchers analyzed the glioma genome,combined with the related results on glioma research in recent years,identified the current glioma common mutation gene:PTEN TP53,EGFR,PIK3CA,PIK3R1,NF1,RBI,IDH1 and PDGFRA,common channel:p53,Rb,PI3K etc..In addition to studying functional genes,non coding RNA has gradually become a research hotspot in various biological fields including cancer.Studies have shown that non coding RNA(micRNA,lncRNA and so on)are involved in the occurrence and development of tumors(including gliomas).In addition,the mechanism that the non coded RNA acts on the encoding gene is the focus of the present research.Long-non coding RNA(lncRNA)is a class of non coding RNA with a length of more than 200 nucleotides and does not have the function of protein encoding.According to the different location of LncRNA relative to the protein coding gene,it can be divided into 5 types:sense,antisense,bidirectional,intronic and intergenic.According to the function of lncRNA,it can be divided into 4 categories:Signal molecule,ecoymolecule,Scaffoldmolecule.In the human genome,less than 2%of the sequences encoded proteins,about 7%of the non transcriptional regions.More than 90%of the genome sequences were transcribed to generate non coding RNA.For a long time,people have been thinking that non coded RNA is "unfunctional garbage"or "dark matter".However,a lot of recent studies show that non coding RNA is abnormally expressed in many kinds of tumors,and plays an important role in tumor occurrence,proliferation,invasion,metastasis and prognosis.Recently,a number of studies have shown that lncRNA is involved in the development glioma.In 2012,researchers at Stanford University School of Medicine published an article in"Genome Biology","Transcriptional profiling of long non-coding RNAsand novel transcribed regions regions," They carried out the first large cancer lncRNA expression profiling,analyzed 64 tumor samples(including gliomas)with high throughput RNA-seq sequencing,and identified 1065 differentially expressed lncRNA among various tumor types.At present,lncRNAs related to glioma mainly include LncRNA HOTAIR,H19,CDKN2B-AS,MEG3,ADAMTS9-AS2,CRNDE,TSLC1-AS1 and so on.At present,the study of glioma related lncRNA is mainly to explore the abnormal expression and preliminary effect.The detection of lncRNA for glioma is still less.In particular,the difference of lncRNA expression profile,function and mechanism is still unclear.In addition,the relationship between the differentially expressed lncRNA and the known genes?protein and channel of glioma,and the application of lncRNA in clinic still need further experiments to prove..In order to identify the expression profile,function and mechanism of glioma related lncRNA,we used high-throughput sequencing technology to analyze the expression difference of lncRNA and mRNA in glioma tissue specimens and normal brain tissues.We combined the information of glioma related genes,proteins and channels,and screened out differentially expressed lncRNAs by bioinformatics,and constructed a co-expression network to identify target lncRNA-PAR5.We further verified the expression of PAR5 in glioma tissues and glioma U87 and U251 cell lines by qRT-PCR method,and confirmed the relationship between the expression level of PAR5 and the clinical characteristics and molecular markers of glioma.Further,we used transfection to change the expression of PAR5,observe its effect on the phenotype of glioma cells,and explore the potential functional mechanism of PAR5 through RIP and Pulldown experiments.Objectives:To investigate whether there are differentially expressed lncRNAs in glioma tissues compared with normal brain tissues.Based on the existing lncRNA databases,to analyze the overall differential expression of differentially expressed lncRNA in glioma tissues.Moreover,to analyse the expression of newly discovered lncRNAs.To carry out bioinformatics analysis of differentially expressed lncRNAs in glioma.To screen the potential lncRNAs related to glioma,according to genes,proteins,and signal channels related to glioma.Moreover,to test the selected lncRNA by qPCR experiment.To investigate the expression of lncRNA-PAR5 in glioma tissues and glioma cell lines U87 and U251.To study the relationship between PAR5 and the pathological grade of glioma and the prognosis of glioma.To investigate the effect of PAR5 on the viability,proliferation and migration of glioma cells by plasmid transfection of PAR5.Moreover,to explore the potential mechanism of PAR5 in glioma.Methods:The glioma tissues and normal brain tissues,from patients in Department of Neurosurgery of the First Affiliated Hospital of Kunming Medical University,since January 2013 to December-2013,were collected and stored in liquid nitrogen.Trizol method was used to extract RNA from specimens and the quality of RNA was detected by qPCR.4 cases of glioma and 2 normal brain tissues were sequenced with high throughput sequencing.The results of sequencing were preliminarily screened and analyzed.KEGG and GO bioinformatics were used to analyze genes differentially expressed in glioma tissues.The co-expression of lncRNA and mRNA was analyzed.Further screened the lncRNAs,according to the information from existing lncRNA database and genes,proteins and signaling pathways released to glioma.The consistency of the sequencing results was verified by qPCR experiments on 10 selected lncRNA,and lnGRNA-PAR5 was finally established as the target for follow-up experiments.The expression levels of PAR5 were determined in clinical amples and U87,U251 cells using real-time reverse transcription quantitative polymerase chain reaction(qRT-PCR)analysis.The effects of PAR5 on cell proliferation,migration and invasion were determined using in vitro assays.RNA immunoprecipitation(RIP)and RNA pull-down assays,as well as the evauation of the expression of various oncogenes were carried out to reveal the underlying mechanisms.Results:Tissue's total RNA,purity and integrity of 4 cases of glioma and 2 cases of normal brain have reached the sequencing requirements;mRNA expression of glioma and normal brain tissue:total of 2432,of which 450 were up-regulated(FC value more than 2 times and P<0.05 391),1982 down-regulated(FC value more than 2 times and 1409 P<0.05);lncRNA expression of glioma and normal brain tissue:total of 488,of which 171 were up-regulated(FC value more than 2 times and P<0.05 149),317down-regulated(FC value more than 2 times P and<0.05 264).KEGG gene differential expression analysis results:there are 490 genes and biological systems are linked,then 266 associated with human diseases,160 related to cellular processes,266 related human diseases,20 directly related to glioma,and information processing related to the 27 differential expression genes;The results of GO analysis of differentially expressed genes:1834 were related to cell components,937 were related to molecular function,and 2553 were related to biological processes.The results of KEGG and GO analysis and glioma related genes,proteins and channels were analyzed,and 10 lncRNA were screened.The result of the 10 selected lncRNAs' qPCR quantitative detection in 20 cases of gliomas and 10 normal brain tissues:The expressions of lncRNA 1,2,3,4,5 and 6 in glioma tissues were significantly down regulated(P<0.05),and the expressions of lncRNA7,8,9 and 10 in glioma tissues were significantly up-regulated(P<0.05),and the change times were consistent with the sequencing results.The target lncRNA4 is lncRNA-PAR5 based on the results of the experiment and the literature review.We found that PAR5 was significantly downregulated in glioma tissues and cell lines.Furthermore,PAR5 expression was negatively correlated with tumor size,World Health Organization(WHO)grade and Karnofsky performance score(KPS).Patients with low PAR5 expression in tumors had a worse overall survival compared to those with higher expression.Finally,in vitro restoration of PAR5 expression inhibited human glioma cell proliferation,invasion and migration by binding to EZH2 and regulating oncogene expression.Conclusions:There are differential expression lncRNAs in the glioma tissue than in the normal brain tissue;The down-regulation differential expression lncRNAs are more than that up-regulation;There is still a class of lncRNAs that are not found in the current database-The differentially expressed genes obtained from high-throughput sequencing are associated with glioma related genes,channels and proteins;The co expression network of lncRNA and mRNA is associated with glioma;The expression of PAR5 is significantly lower in glioma and glioma U87 and U215 cell lines;The expression of PAR5 is closely related to the clinical characteristics of glioma size,WHO pathological grade and KPS score;The prognosis of glioma patients with low expression of PAR5 is worse;in vitro experiments,overexpression of PAR5 can inhibit U87 and U251 glioma cell proliferation,migration and invasion;PAR5 plays a role of tumor suppressor gene through EZH2,and PAR5 may be a potential target for clinical glioma 's submolecular grading,prognosis and treatment. |
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