Identification,Regulatory Network Construction And Preliminary Verification Of Differentially Expressed Circular RNA In Colorectal Cancer

Objective: For a long time,cancer has been hailed as the "number one killer" of human health.Colorectal cancer?CRC?is the third most common tumor and the second most common malignant tumor in the digestive tract.According to surveillance data from the National Cancer Institute,in 2019,there will be approximately 145,600 cases of colorectal cancer and 51,012 new deaths.In China,the incidence of colorectal cancer has gradually increased over the past few decades,ranking third among men with cancer and second among women.With the improvement of the national economy and changes in dietary structure,the age of onset of colorectal cancer has gradually decreased,and the number of patients with adolescents and young people has increased.However,many colorectal cancer patients have developed advanced malignancy with multiple metastases at the time of diagnosis.If early diagnosis of the tumor can be achieved,the incidence of the tumor can be greatly reduced.At present,the treatment of colorectal cancer mainly uses surgical resection and local ablation as adjuvant chemotherapy drugs.Cancer patients often have a poor prognosis and a high recurrence rate.Less than 40% of patients have a survival period of more than 5 years.In recent years,great breakthroughs have been made in targeted drugs and precise treatments based on tumor-specific screening targets,and they have achieved good results in a variety of tumors.However,the occurrence and development of tumors is a multi-faceted and complicated process,which is affected by many factors.At present,the pathological mechanism of colorectal cancer has not been thoroughly studied,and effective therapeutic targets and biomarkers are lacking.Therefore,it is of great research significance to deeply study its mechanism and to screen out specific therapeutic targets and biomarkers.Non-coding RNA constitutes most of the transcription of the human genome.Studies have found that it can regulate gene expression by affecting transcription,epigenetics,and other methods,and trigger a series of changes in biological processes.Circular RNA?circRNA?is a new type of endogenous non-coding RNA,which exists widely in the eukaryotic transcriptome and is stably expressed in the cytoplasm.The structural feature of CircRNA is that the 5' end and the 3' end are connected by a covalent bond to obtain a closed loop structure.The uniqueness of this structure makes it difficult to be degraded.CircRNA was first discovered in RNA viruses and was thought to be the product of transcription errors for some time.With the development of high-throughput RNA sequencing and single-cell sequencing technology,people found that circRNA exists in mammalian cells in large quantities,suggesting that circRNA may have important biological functions,so a lot of research on circRNA has begun.CircRNA is currently known to be involved in the pathological processes of a variety of diseases including tumors,cardiovascular diseases,Alzheimer's disease,and diabetes.Recent studies have shown that circRNA is differentially expressed in a variety of digestive tract tumors such as gastric cancer,liver cancer,and colorectal cancer.It acts as a microRNA sponge,RNA-binding protein sponge,and even encodes a polypeptide or protein to participate in the proliferation and invasion of tumor cells,even biological processes such as metastasis,apoptosis,and epithelial-mesenchymal transition.The specificity of the CircRNA structure makes it more stable and can be detected in both exosomes and plasma,so it is considered an ideal new biomarker.However,the biological function of most circRNAs in CRC remains unknown.In this study,we identified circRNAs?DECs?that are differentially expressed in colorectal cancer tissues and adjacent tissues,and predicted the function of DECs by constructing a circRNA /miRNA /mRNA regulatory network.At the same time,the biological function of hsa_circ₀001550 in CRC Scientific role to verify.Methods: 1.Screening and identification of dysregular circRNAs: Select two pairs of samples from cancerous tissues and adjacent tissues of patients with colorectal cancer,use the Illumina sequencing platform for deep RNA sequencing,and use the MEM algorithm in Burrows-Wheeler to perform sequencing with the human genome By comparison,differentially expressed circRNAs were identified in cancer and adjacent tissues?*** p < 0.05,fold change> 2?.Combine our sequencing data with GSE116589 chip data?n = 2?,select circRNAs that were? up-regulated and significantly different in multiples in cancer tissues as candidate circRNAs,and verify the expression levels in colorectal cancer tissue pairs and cell lines.2.Construction and function prediction of CircRNA/miRNA/mRNA regulatory network: First,we analyzed the possible mechanism of candidate circRNA through the CSCD database.The Starbase database was then used to predict the interaction between circRNA and mi RNA and further analyze the expression of these miRNAs in CRC.MiRNAs down-regulated in CRC were selected as targets for circRNA?Fold change <0.5,p <0.05 and FDR <0.05?.Starbase database was used to predict the target mRNA of candidate miRNAs and mRNAs co-expressed with circRNA were selected as candidate mRNAs.GEPIA database was used to further analyze the expression of candidate mRNAs in CRC tissue pairs,and up-regulated mRNAs were selected as targets of miRNAs.Finally,Cytoscape3.2.1 software was used to construct the circRNA-miRNA-mRNA function regulation network.The protein-protein interaction?PPI?networks of genes regulated by the core circRNA were constructed using the String database.Enrichment analysis of target genes regulated by circRNA was performed using David database to predict the function of circRNA.3.Functional verification of Hsa_circ₀001550 in CRC: The hsa_circ₀001550 silencing and overexpression plasmid was constructed and transfected into two CRC cell lines,HCT116 and SW480.The effect of hsa_circ₀001550 on the proliferation of CRC cells was detected by MTT proliferation experiments;the effects of hsa_circ₀001550 on the colon cancer cell cycle,cell division,and apoptosis were analyzed by flow cytometry.Results: 1.Through RNA-seq sequencing analysis,we screened 76 circRNAs that were significantly differentially expressed in colorectal cancer paired tissues,including 33 up-regulated DECs and 43 down-regulated DECs.These DECs were mainly distributed in NC₀00005.10?chr5,14.5%?,NC₀00009.12?chr9,9.2%?,NC₀00001.11?chr1,6.6%?,NC₀00004.12?chr4,6.6%?,and NC₀00014.9?chr14?,6.6%)on five chromosomes.The sequence length of DECs was between 201 bp and 76919 bp,mainly 200bp-600bp?68.4%?and more than 2000bp?19.7%?.The GO and KEGG enrichment results of the parent genes showed that the functions of the parent genes of these DEGs were mainly concentrated in: tissue and cell differentiation and proliferation regulation;MAPK pathway,G-protein coupled receptor PIK3-AKT pathway,Ras signaling pathway and protein processing and inflammatory response.2.Based on the combined analysis of RNA-seq data and GSE116589 data,we obtained 23 circRNAs differentially expressed in CRC,including 6 up-regulated DECs and 17 down-regulated DECs.The six up-regulated circRNAs were hsa_circ₀030632,hsa_circ₀004887,hsa_circ₀001550,hsa_circ₀001681,hsa_circ₀002970,and hsa_circ₀006528,while the down-regulated circRNAs included hsa_circ₀002947,hsa_circ₀003553,and others.3.The expression levels of Hsa_circ₀030632,hsa_circ₀001550 and hsa_circ₀001681 in six CRC cell lines?HCT116,SW620,SW480,HT-29,RKO,CACO2?were significantly higher than those in normal intestinal epithelial cells HIEC.And compared with adjacent tissues,hsa_circ₀030632,hsa_circ₀001550 and hsa_circ₀001681 were significantly higher expression in colon cancer tissues.4.Analysis of CSCD database predicted that hsa_circ₀030632,hsa_circ₀001550 and hsa_circ₀00168 all have miRNA response elements?MRE?,suggesting that they might play biological roles as miRNA sponges.Therefore,we constructed a circRNA-miRNA-mRNA regulatory network,including 5 miRNAs?hsa-miR-664b-3p,hsa-miR-139-5p,hsa-miR-370-3p,hsa-miR-181a-5p and hsa-miR-181b-5p?and 54 target mRNAs.Hsa_circ₀030632-regulated mRNA is mainly concentrated in pathways of DNA damage signal transduction,regulation of cell cycle G1/S transition,mitochondrial apoptosis,and purine nucleotide synthesis.Hsa_circ₀001681-regulated mRNA is enriched in nuclear transport and viral processes.The mRNAs regulated by hsa_circ₀001550 were mainly concentrated in signal pathways such as cell division and formation and pentose phosphate branch.The core proteins CCT2,HSPD1,TALDO1,RPIA,and PPP1 CA in the PPI network were involved in the enrichment of cancer-related pathways.5.After hsa_circ₀001550 is overexpressed in HCT116 and SW480,the cell proliferation ability was increased,the cell proliferation index was increased,and the level of apoptosis was decreased.After hsa_circ₀001550 is silenced in HCT116 and SW480,the CRC cell proliferation ability was inhibited and the cell proliferation index was reduced.Apoptosis levels increased.Conclusion: 1.CircRNA was differentially expressed in colon cancer and adjacent tissues.The differentially expressed circRNAs were mainly distributed in NC₀00005.10?chr5?,NC₀00009.12?chr9?,NC₀00001.11?chr1?,NC₀00004.12?chr4?and NC₀00014.9?chr14?on five chromosomes.The sequence length of DECs were between 201 bp and 76919 bp,mainly 200bp-600 bp and more than 2000 bp.2.The expressions of Hsa_circ₀030632,hsa_circ₀001550 and hsa_circ₀001681 were significantly up-regulated in colon cancer tissues and cell lines.3.Bioinformatics analysis suggested that hsa_circ₀030632,hsa_circ₀001550,and hsa_circ₀001681 could target miRNAs to regulate downstream mRNA expression through the ceRNA mechanism;at the same time,these three circRNAs might participate in the cell cycle,apoptosis,telomerase activity,and DNA damage Biological processes promote the occurrence,development and pathological process of colon cancer.4.Hsa_circ₀001550 can promote the proliferation of colon cancer cells by regulating the cycle of colorectal cancer cells and inhibit the apoptosis of colon cancer cells.