| BackgroundMalignancies are one of the top leading causes of human death worldwide,and their mortality and morbidity increase annually,which has become a major public health problem threatening human health.Therefore,exploring the mechanism underlying tumorigenesis and potential therapeutic targets have great clinical significance for improving the survival and quality of life of patients.Circular RNAs(circ RNAs)are a class of single-stranded RNAs with a covalently closed loop structure generated by back-splicing,and have a broad range of regulatory functions.Emerging evidence has demonstrated that circ RNAs are aberrantly expressed in cancers,and promote the tumorigenesis and progression of cancers via regulating cell proliferation,metastasis,apoptosis and angiogenesis,but there are still a large number of functional circ RNAs to be elucidated.Because of the large number,complex structures and diverse biological functions and mechanisms(including interacting with mi RNAs and RNA binding proteins,translation into peptides,m~6A modification,etc.)of circ RNAs,there is an urgent need to develop a visualization tool for displaying circ RNA and predicting its potential mechanisms.In addition,current studies have mainly focused on exploring the differential expression profiles and functions of circ RNAs in certain cancer,but the common and specific expression patterns of circ RNAs in multiple cancers are still unclear.AimsIn this study,we aim to develop the simple and user-friendly circ Plot package and web application for circ RNA visualization,and predict potential mechanisms(including interacting with mi RNAs and RNA binding proteins,translation into peptides,m~6A modification and secondary structure,etc.)of circ RNAs.At the same time,we plan to investigate the expression patterns of circ RNAs across different cancer types by transcriptome sequencing and bioinformatics analysis,and then identifiy circ RNAs with tissue-and cancer-specific expression patterns,and preliminarily explore the potential mechanism underlying the biogenesis of tissue-and cancer-specific circ RNAs.Finally,we aim to validate the analytic results by real-time quantitative PCR(q PCR),then determine the candidate circ RNA hsa＿circ＿0072309 and elucidate its biological functions in cancer cells.Therefore,our study will deepen our understanding of circ RNA,and benifit the development of new biomarkers for cancer diagnosis and new targets for cancer therapy.Methods1.Development of the circ Plot package and web application for circ RNA visualization.(1)The circ Plot package was built based on the functions provided in ggplot2 and ggforce packages,and our in-house scripts.(2)The circ Plot web applicatoin was developed using shiny and shinydashboard packages.(3)The putative spliced sequence of circ RNAs,RNA binding protein binding sites and m~6A modification sites were obtained from the circ Base,star Base and m~6A-Atlas databases,respectively.(4)The interactions between circ RNAs and mi RNAs were predicted by mi Randa.(5)The RNA binding protein binding sites and m~6A modification sites within circ RNAs were analyzed by bedtools.(6)The ORF(Open Reading Frame)and IRES(Internal Ribosome Entry Site)elements within circ RNAs were predicted by in-house script and IRESfinder,respectively.(7)The secondary structure of circ RNAs were predicted by RNAfold.(8)The detailed information and function annotation results of circ RNAs were integrated into the circ Plot web application.2.Transcriptome sequencing and data quality control.(1)A total of 140 pairs of tumor and corresponding normal tissues of 8 cancers were collected(including 22pairs of esophageal squamous cell carcinoma,20 pairs of glioblastoma,20 pairs of colorectal cancer,18 pairs of lung squamous cell carcinoma,17 pairs of lung adenocarcinoma,16 pairs of hepatocellular carcinoma,16 pairs of thyroid cancer and9 pairs of gastric cancer)(2)Total RNA of tumor and paired normal tissues were extracted.(3)Agarose gel electrophoresis,Nano Drop and Agilent 2100 bioanalyzer were utilized to assess the quality of extracted total RNA.(4)The qualifed RNA was then subjected to transcriptome sequencing.(5)Fast QC and fastp were used to evaluate the quality of raw data,the clean data were obtained by filtering reads with low quality,and further assessed by fastp.3.Identification and characterization of circ RNAs.(1)CIRCexplorer2 was applied to identify circ RNAs,and circ RNAs that annotated in the circ Base database were kept for subsequent analysis.(2)The expression levels of circ RNAs were normalized to RPM(Reads Per Million mapped reads).The junction ratio was used to represent the relative expression between circ RNA and cognate linear transcript.(3)Based on the annotation information provided by the circ Base database and the CIRCexplorer2,the genomic regions,splicing signal motif,putative spliced length,number and genomic position of exon,host genes,and alternative circularization of circ RNAs were analyzed.(4)The introns flanking back-spliced junction site of circ RNAs were extracted.Based on the repetitive element annotation in the UCSC database,the number of repetitive elements contained in flanking introns were analyzed by bedtools software.(5)The profiles and genomic characteristics of ubiquitous circ RNAs that expressed in all samples were analyzed.4.Identification of tissue-specific circ RNAs and investigation of regulatory mechanism underlying its biogenesis.(1)Circ RNAs with median RPM expression greater 0.1 in at least one tissue were kept.(2)The tissue specificity index of circ RNAs were calculated using Yanai’s method,and then the circ RNAs were classfied into tissue specific,intermediately specific and ubiquitous according to the tissue specificity index.(3)The tissue specificity index of circ RNA parental genes were calculated,then the tissue specificity of circ RNAs and its parental genes were compared.(4)The tissue specificity index of RNA binding proteins were calculated,the Pearson correlation coefficients between circ RNAs and RNA binding proteins were computed.5.Identification and validation of differentially expressed circ RNAs in cancers.(1)Comparison of the total abundance and number of circ RNAs that identified in tumor and adjacent normal tissues of different cancers.(2)The differentially expressed circ RNAs and linear transcripts in each cancer were identified by DESeq2.(3)The putative mechanisms of circ RNAs dysregulation were preliminarily investigated by comparing the expression changes between circ RNAs and their host genes,calculation of the junction ratio of circ RNAs,and anslysis the correlations between circ RNAs and RNA binding proteins.(4)The differentially expressed circ RNAs in each cancer type were extracted and compared to identify the dysregulated circ RNAs that specific to certain cancer as well shared by multiple cancers,the putative biogenesis mechanisms of these circ RNAs were investigated.(5)Additional 100 pairs of tumor and adjacent normal tissues of 8 cancers were collected,then the primer sets that with one primer acrossing the back-spliced junction site were designed.The relative expression of circ RNA candidates in 8cancers were measured by q PCR.6.The biological functions of hsa＿circ＿0072309 in cancer cells.(1)Total RNA of each tumor cell line was extracted,and then the relative expression of hsa＿circ＿0072309 in different cancer cell lines were detected by q PCR.(2)RNase R digestion assay was applied to detect the stability of hsa＿circ＿0072309.(3)The back-spliced junction site of hsa＿circ＿0072309 was amplified using divergent primers that flanking junction site,and then validated by Sanger sequencing.The sequence of junction site was confirmed.(4)The subcellular location of hsa＿circ＿0072309 in parental cancer cells were determined by cell nucleus/cytoplasm fractionation followed by q PCR assays,and fluorescence in situ hybridization assays.(5)The lentiviral plasmid to overexpress hsa＿circ＿0072309was constructed and the lentivirus particles were prepared.The cell lines with relative low hsa＿circ＿0072309 expression were selected to construct hsa＿circ＿0072309 overexpression stable cell lines.(6)Total RNAs of the stable cell lines were isolated and the relative expression of hsa＿circ＿0072309 was determined by q PCR,and then the junction site of hsa＿circ＿0072309 was confirmed by Sanger sequencing.(7)The subcellular location of hsa＿circ＿0072309 was further validated in hsa＿circ＿0072309 overexpression stable cells by cell nucleus/cytoplasm fractionation followed by q PCR assays,and fluorescence in situ hybridization assays.(8)MTT and clone formation assays were utilized to detect the influence of hsa＿circ＿0072309 overexpression on cell proliferation.(9)Transwell migration and wound healing assays were employed to check the effect of hsa＿circ＿0072309overexpression on cell migration.Results1.The circ Plot package was designed to visualize circ RNA coding potential(IRES elements and ORF),mi RNAs and RNA binding proteins binding sites,m~6A modification sites as well secondary structure.In addition,we predicted the potential mechanisms of 108735 circ RNAs that recorded in circ Base database,and developed a user-friendly and easy-to-use circ Plot web applicatoin for circ RNA visualization.2.The data of all samples were qualified and subjected to subsequent analysis.A total of 60182 circ RNAs that annotated in the circ Base database were identified in these 280 clinical samples,and were demonstrated to have following characteristics:1)92.43%of circ RNAs were expressed at lower levels than cognate linear transcripts.2)98.84%of circ RNAs were originated from the exonic regions.3)80.92%of exonic circ RNAs contained at least 2 exons.4)73.63%of genes were found to produce at least two circ RNA isoforms and generally express a predominant circ RNA with high abundance.5)The circ RNAs that expressed in all samples had higher abundance,and its abundance was correlated with their host genes.Alternative circularization was the main contributor to the diversity of circ RNAs,and the formation of circ RNAs was regulated by cis-acting elements.3.A total of 439 tissue-specific circ RNAs were identified,of which 144(32.80%)circ RNAs were derived from tissue-specific genes,and 359(81.78%)circ RNAs were closely associated with RNA binding proteins,implying that the tissue specificity of circ RNAs were mainly regulated by tissue-specific RNA binding proteins.4.By comparing the circ RNAs identified in tumor and adjacent normal tissues,it was found that the total abundance and number of circ RNAs were downregulated in tumor tissues.A total of 1409 differentially expressed circ RNAs were identified,in which 1040(73.81%)circ RNAs were exclusively dysregulated in certain cancer,369(26.19%)circ RNAs were significantly altered in at least two cancers.The dysregulation of circ RNAs in tumors was attributed to the aberrant expressoin of its parental genes and RNA binding proteins.The circ RNAs that significantly altered in at least 7 cancer types were verified by q PCR,and the results demonstrated that hsa＿circ＿0072309 was consistently downregulated in other 7 cancers except for glioblastoma.Therefore,hsa＿circ＿0072309 was selected for subsequent functional investigations.5.The results of RNase R digestion,Sanger sequencing,cell nucleus/cytoplasm fractionation assays,and fluorescence in situ hybridization assays confirmed that hsa＿circ＿0072309 was endogenous circ RNA and was predominantly distributed in the cytoplasm.We constructed the hsa＿circ＿0072309 overexpression stable cell lines in lung cancer,esophageal squamous cell carcinoma,colorectal cancer and liver cancer cells,and found that hsa＿circ＿0072309 inhibited cell migration of cancer cells,but had no significant effect on the cell proliferation.Conclusions1.The circ Plot package and web application were developed for displaying the detailed information and putative mechanisms(interacting with mi RNAs and RNA binding proteins,translation into peptide,m~6A modification and secondary structure)of 108735 circ RNAs,which provide a useful tool for circ RNA research.2.The transcriptome sequencing was conducted on clinical samples collected from West China Hospital,and the results of bioinformatics analysis revealed that the expression of circ RNAs were closely associated with its parental genes and RNA binding proteins.Besides,the expression landscape of circ RNAs in multiple cancers was characterized and a number of circ RNAs were found to be commonly dysregulated across cancers,providing candidates for elucidating the functions of circ RNAs in cancers.3.Hsa＿circ＿0072309 was consistently downregulated in esophageal squamous cell carcinoma,lung adenocarcinoma,lung squamous cell carcinoma,colorectal cancer,gastric cancer,thyroid cancer and hepatocellular carcinoma.The results of in vitro assays demonstrated that hsa＿circ＿0072309 inhibited the migration of lung cancer,esophageal squamous cell carcinoma,colorectal cancer and liver cancer cells,which laid a foundation for further study on the roles of circ RNAs in cancer metastasis. |
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