Differential Expression And Bioinformatics Analysis Of Circular RNA In Osteosarcoma And Normal Bone Tissue

Background:Osteosarcoma is one of the life-threatening cancers that commonly occur in children and adolescents.The incidence rate of osteosarcoma worldwide is approximately 3 to 4 per million new cases.Although surgery and neoadjuvant chemotherapy have made great progress,the prognosis of most patients is still poor.Even after standardized chemotherapy and radical resection of the primary lesion,there is still a high risk of local recurrence or lung metastasis.For patients with metastatic disease or tumor recurrence,the clinical outcome is even worse.With the advancement and development of medicine,people's understanding of osteosarcoma has gradually deepened.The progression of osteosarcoma is extremely complicated.Therefore,it is of profound significance for early diagnosis,avoidance of chemotherapy resistance,reduction of lung metastasis and improvement of clinical prognosis to elucidate the molecular mechanism of osteosarcoma occurrence and development as well as to identify effective biomarkers or therapeutic targets in osteosarcoma.In recent years,it has been found that circularRNA is closely related to the development of midbrain,Parkinson's,Alzheimer's disease,and tumors.However,the relationship between significant changes in circRNA expression and osteosarcoma has not been elucidated.CircularRNA is a type of endogenousRNA,which is characterized by covalent closed-loop structures,nonpolar or polyadenylate tails.Compared with traditional linearRNA,circRNAs do not have free ends and therefore cannot use many classicalRNA degradation pathways.CircRNAs may have a longer halflife in vivo,which may be due to the stability and non-dividing properties of these molecules.This makes it possible to utilize circularRNA as a biological marker to assist in the diagnosis of related diseases.In recent years,many studies have shown that various biological functions of circRNA are closely related to the biological characteristics of malignant tumors such as rectal cancer and pancreatic cancer.Recently developed high-throughput sequencing can capture almost all transcripts expressed in theRNA samples.These advantages ofRNA-seq make it important for annotating newly sequenced genomes and detecting new genes and subtypes of species with incompletely annotated genes.There are not enough studies on the expression and function of cyclicRNA in osteosarcoma.Therefore,circularRNA sequencing was performed in 3 cases of osteosarcoma and 3 cases of normal bone tissue using high-throughput sequencing technology.The circularRNA expression profile of osteosarcoma was constructed,and the differential expression circularRNA in osteosarcoma was calculated.Among the differentially expressed circularRNAs,five circularRNAs were selected and their relative expression levels in 10 cases of osteosarcoma and normal bone tissue were measured by realtime PCR to verify the results obtained by high-throughput sequencing.Through the bioinformatics analysis of the differentially expressed circularRNA,it is speculated that it may play a role in the occurrence and development of the disease,so as to lay a certain foundation for clarifying the molecular biological mechanism of osteosarcoma and to use circularRNA as the entry point initial exploration of the diagnosis and treatment of osteosarcoma.Methods:For sequencing experiments,the Illumina TruSeqTM Stranded TotalRNA Library Prep Kit method was utilized for specific library construction.TotalRNA was extracted from the tumor tissues of 3 patients with osteosarcoma and 3 normal bone tissues adjacent to the cancer,and the concentration and purity of the extractedRNA were detected using Nanodrop 2000.The integrity ofRNA was detected by agarose gel electrophoresis,Agilent 2100 was used to determine RIN value,and a sequencing library was constructed to transform the library into single strand DNA molecules,amplifying in situ into clusters and use the Illumina Hi Seq sequencer to sequence 150 cycles of circRNAs to obtain double-ended reads after quality control.These reads were compared to the reference genome /transcriptome(Hg19)using Tophat2 software,circRNAs were detected and identified using CIRCexplorer2 software,and the identified circRNAs were annotated using the Ensembl gene,Known Genes gene,and Ref Seq gene databases.CircRNAs were detected and identified using CIRCexplorer2 software,and the identified circRNAs were annotated using the Ensembl gene,Known Genes gene,and Ref Seq gene databases.Differential expression analysis was performed using the quasi-likelihood F-tests method of the edge R software package to screen for significant differential expression of circRNA between cancer tissues vs.adjacent tissues in two groups of matched tissues.The threshold was set to | log FC |> 1 and pvalue <0.01.Among the differentially expressed circRNAs,five circRNAs were selected to detect their relative expression in the tumor tissues of 10 osteosarcoma patients and 10 normal bone tissues adjacent to the cancer between the two groups by real-time PCR.The consequences of highthroughput analysis were verified.Go analysis and KEGG pathway analysis were carried out for the source genes of different circRNAs,and the interaction prediction of circRNAs and miRNA was carried out by Miranda,a miRNA target gene prediction software.CircRNAs-miRNA network maps were constructed using Cytoscape software.MiRNAs and genes related to osteosarcoma were collected from databases such as Dis Genet.For miRNAs and genes related to osteosarcoma obtained in the previous step,it can be predicted whether there is a regulatory relationship between them by mi RWlak,and it can select the results shared by mi RWalk,mi Randa,and Targetscan as the final miRNA-gene relationship.Through combining with the analysis of the regulatory relationship between circRNA and miRNA and integrating miRNA as the junction point,the network map of circRNA and disease-related miRNA / gene can be obtained.Results:In 6 samples,36,631 circular RNAs were identified through analysis,corresponding to 8001 genes.Cluster analysis revealed that there was a significant difference in the expression pattern of circRNAs between paracancerous and osteosarcoma tissues.A total of 259 differentially expressed circRNAs were observed in the two groups of tissues,of which 132 circRNAs were up-regulated in tumor tissues of osteosarcoma patients and 127 circRNAs were down-regulated in tumor tissues of osteosarcoma patients.Differential expression of circ?32279,circ?24831,circ?2137,circ?6798,and circ?20403 in osteosarcoma(human source)tissues was verified by q PCR experiments.Compared with the paracancerous tissues,circ?andcirc?were significantly decreased in osteosarcoma.Circ?2137,circ?6798,circ?20403 were significantly up-regulated.Go function enrichment analysis and KEGG pathway enrichment analysis were carried out for the circna hosting gene with significant difference.GO analysis revealed that the differentially expressed circularRNA involved 44 GO biological pathways(BP),19 GO cell components(CC)in biological pathways,and 15 GO molecular functions(MF,Molecular function),a total of 78 GO functions,and KEGG pathway analysis showed that there were 10 KEGG pathways in differentially expressed circularRNA.The study concluded that there are multiple target positions of miRNAs on some circRNA sequences.From the analysis,11 miRNAs and 44 genes related to osteosarcoma were obtained.Six miRNAs out of 11 mRNAs interact with differential circRNAs in the high-throughput gene sequencing analysis.The mi RWlak is used to predict whether there is a regulatory relationship between them,and the results shared by mi RWalk,mi Randa,and Targetscan are selected as the final miRNA-gene relationship,which includes 6miRNAs and 14 genes.The miRNA-circRNA regulatory relationship obtained by routine analysis was utilized to integrate miRNA as the connection point to obtain a network diagram of circRNA and disease-related miRNAs / genes.Conclusions:(1)There is a great amount of circular RNA in osteosarcoma tissue and normal bone tissue adjacent to the cancer.Compared to normal bone tissue adjacent to cancer,a large amount of circularRNA expression in osteosarcoma tissue is significantly up-regulated or down-regulated.It is suggested that a large number of circularRNAs have obvious expression differences in osteosarcoma tissue and normal bone tissue adjacent to the cancer.(2)Go analysis and KEGG pathway analysis of regulated expression circRNAs showed that they may be involved in a variety of tumor related biological processes,such as inositol phosphate metabolism,phosphatidylinositol signal transduction and so on,which proved that cyclicRNA plays an important role in the occurrence and development of osteosarcoma.(3)The circRNA-miRNA-gene network diagram shows that there is an extremely complex regulatory relationship between circularRNA and miRNA and genes.Its role in tumorigenesis and development of osteosarcoma patients and its specific regulatory mechanism need further study.