Study On The Differential Expression Of CircRNAs And Its Functional Role In Brain Glioma

Glioma is the most common primary central nervous system tumor.The current treatment for glioma include surgery,radiotherapy and temozolomide based chemotherapy.Even after surgery,chemotherapy and radiotherapy,the prognosis of high grade gliomas,especially glioblastoma is still very poor.With the development of medical science,people have a deeper understanding of glioma.The occurrence and development of glioma is a complex process.It is of great significance to clarify the molecular mechanism of the development of glioma and to find new biomarkers for early diagnosis,prognosis and treatment.In recent years,scholars have found that circular RNAs are closely related to the tumorigenesis and development of many tumors.Circular RNA is a class of single stranded,closed circular non-coding RNA,which has neither the polarity of the 5 '-3' nor poly A tail.Because of its special stable structure,it can not be degraded by RNA enzyme,and it is conservative in evolution.Recent studies have found that circRNAs may play an important role as competitive endogenous RNA(ce RNA)or mi RNA sponge.Based on these characteristics of circRNAs,the study of circRNAs may provide new ideas for the development of drugs,and provide a new direction for the study of the evolution of life.With the development of high-throughput sequence technology,researchers use this technology to screen out differentially expressed circRNAs in tumors,and further study reveals part of differentially expression circRNAs in the pathogenesis of tumor.These studies are mainly focused on liver cancer,colon cancer,cervical cancer,and so on.The expression and function study of circRNAs in glioma is less.Therefore,we use high-throughput sequencing of circRNA expression in 5 cases of glioblastoma and5 cases of normal brain tissue,screen the circRNAs expression profile of glioblastoma,and compares the differential expression of circRNAs in glioblastoma.By analyzing the differential expression of circRNAs,we speculate the role of circRNA in tumorigenesis and development of glioma.This work lay a solid foundation for elucidating the molecular biological mechanism of glioma.According to the biological information of the differential expression of circRNAs analysis,we focus on one circRNA-circBRAF to verify its expression in 68 cases of different grades glioma and 13 cases of normal brain tissues,then analysis correlation with prognosis of glioma patients.We also use the plasmid vector to construct enhance expression plasmid,and transfected to glioblastoma cell line U251,and do a series offunctional experiments to verified the biological characteristics of glioma cell lines after enhance the expressing the circRNA.To lay a foundation for the diagnosis and treatment of glioma with circRNAs as the starting point in the future.Part I Differential expression and bioinformatics analysis of circRNAs in glioblastoma and normal brain tissueObjective: To construct the expression profile of circRNAs in glioblastoma and normal brain tissue.To analyze the possible role of differential expression circRNAs and to provide the basis for the subsequent study of circRNAs related to the tumorigenesis and development of glioma.Methods: 5 cases of glioma and 5 cases of normal brain tissue to extract total RNA,using circRNA Enrichment Kit(cloud-seq,USA)with truseq stranded total enrichment in circRNAs,RNA library prep Kit(Illumina San,Diego,CA,USA)RNA pretreatment and construct the sequencing Library,the library of degeneration single stranded DNA molecules on Illuminaflowcell capture and in situ amplification for cluster circRNAs sequencing for 150 cycle by Illumina HiSeq sequence,after obtaining double end reads control after using STAR software,the reads compared to the reference genome and transcriptome(Hg19),using DCC software for circRNAs detection and identification,and using circBase and circ2 Trait database to comment the identification of circRNAs.Standardized log2 conversion of the original junction reads for each sample is performed using total reads.And uses student t-test to calculate the significant difference between the two groups,get circRNAs which the significant difference in fold change ?2 times and P?0.05.GO and KEGG pathway analysis to study the potential function of different expression circRNAs,and predict the interaction of circRNAs-microRNA with microRNA target gene prediction software TargetScan and Miranda to construct circRNAs-microRNA network diagram by using Cytoscape software.Results: There are 50386 circRNAs candidates expressed in all of the 10 tissues,of which there are 1411 circRNAs significant differences in the expression between the two groups.There are 206 circRNAs up-regulated expressed in the GBM group,and 1206 circRNAs down-regulated expressed in the GBM group.GO analysis showed that the up-regulated expressed circRNAs in the GBM group in biological pathways(BP)involves 35 terms,cell component(CC)31 terms,and molecular function(MF)12 terms.Down-regulated expression of circRNAs in BP involves 663 terms,CC146 terms,MF150 terms.KEGG pathway analysis revealed that there are no enrichment of in the up-regulated expression of circRNAs,while the down-regulated expression of circRNAs had a total of 70 signaling pathways.CircRNAs-microRNA interaction network diagram shows that a circRNAs can correspond to multiple microRNA,and a microRNA can also interact with multiple circRNAs.Conclusion:(1)CircRNAs are abundant in GBM and normal brain tissue in circRNAs,and there are a large number of differentially expressed circRNAs,these differentially expressed circRNAs may be closely related to the tumorigenesis and development of glioma.(2)GO and KEGG pathway analysis showed that differentially expressed circRNAs may be involved in signal transduction and biological processes of tumor cell proliferation and tumor related differential expression,generally reflect the circRNAs play an important role in the tumorigenesis and development of glioma.(3)The circRNAs-microRNA network diagram suggests that circRNAs and microRNA have complex regulatory relationships,and the specific regulatory mechanisms need to be further studied.Part II Expression of circ BRAF in different grades glioma and its correlation with prognosisObjective: To compare the expression of circBRAF in gliomas of different grades,and to analyze the correlation between the expression of circ BRAF and prognosis.Methods: qRT-PCR is used to detect the expression of circBRAF in 68 different grades glioma tissues and 13 normal brain tissues,the correlation between the expression level of circ BRAF and the prognosis of these patients is study by statistical analysis.Results: The expression level of circ BRAF in normal brain tissues is significantly higher than that in glioma tissues,and its negatively correlated with tumor grade(p<0.01),survival analysis showed that the expression level of circ BRAF are positively correlated with progression free survival(PFS)and overall survival(OS).Conclusion: The expression levelof circ BRAF decreased with the increase of tumor grade,and its positively correlated with the prognosis of patients,so we speculated that the decrease of circ BRAF expression may promoted the tumorigenesis of glioma.Part III The functional study of circ BRAF in gliomaObjective: To investigate the function of circBRAF in glioma cell line.Methods: To construct the circBRAF overexpression plasmid,and transfected the plasmid to U251 cell,than detected the plasmid transfection efficiency by realtime PCR.The functional experiment carred out to tested the biological characteristics changes of U251 cells after the overexpression of circ BRAF.Results: The expression of circ BRAF is significantly increased after transfection of circ BRAF overexpression plasmid with U251 cells.Transwell migration assay showed that after transfected overexpression plasmid,the migration ability of the cells is significantly decreased.The invasion of Transwell showed that after transfected overexpression plasmid,the invasion ability of the cells is significantly decreased.Conclusion: Overexpression of circBRAF can significantly inhibit the migration and invasion of glioma cell line U251.