Construction Of CeRNA Regulatory Network Based On The Screening Of Differentially Expressed CircRNAs In Colorectal Cancer Using Comprehensive Analysis Of RNAsequencing

Objective With the change of dietary structure of Chinese residents,the incidence of colorectal cancer(CRC),one of the malignant tumors,has been increasing year by year.In recent years,it has been found that the development of colorectal cancer is closely related to circularRNA(circRNA).In this study,we applied high-throughput sequencing technology to screen circRNAs associated with colorectal cancer and construct a differentially expressed circRNA-regulated ceRNA network to explore potential biological markers associated with colorectal cancer.To provide a theoretical basis for studying the biological functions and specific molecular regulatory mechanisms closely related to colorectal cancer.Methods Cancer and paracancer tissues(about 5-10 cm from the tumor site)were collected from 20 pairs of colorectal cancer patients who underwent surgery in the general surgery department of Erdos Central Hospital,and information related to age,gender and pathology of the patients from which the specimens were collected.The DNBSEQ platform was used to perform second-generation high-throughput sequencing,and the raw sequencing data were screened and quality assessed to identify the differentially expressed circRNAs in cancer and paracancer tissues.circRNAs with a greater differential foldchange(FC)> 2 and Q-value <0.05 were also selected as candidates with Log foldchange(FC)> 2 and Q-value < 0.05 as limiting conditions.circRNAs,construct specific ceRNA regulatory networks using mi Randa,RNAhybrid algorithm,and visualize circRNA-miRNA-mRNA regulatory networks using cytoscape(version 3.9.1)software,select key circRNAs and ceRNA networks for functional analysis,and combine the selected key circRNAs were combined with circ Bank database and UCSC genome browser to visualize and annotate them for analysis.The expression of key circRNAs in cancer and paracancer tissues,their correlation with clinicopathological features were analyzed.Statistical analysis was performed using SPSS26.0 software with P < 0.05 being the difference and statistically significant.Finally,the mechanism of action of key circRNA in colorectal cancer patients was predicted.Results We identified 138,330 circRNAs,615 circRNAs were significantly differentially expressed,209 circRNAs were up-regulated,and 406 circRNAs were down-regulated by DNBSEQ platform.A total of 3280 miRNAs were identified,32 miRNAs were significantly differentially expressed,11 miRNAs were up-regulated and 21 were down-regulated.19514 mRNAs were identified,759 mRNAs were significantly differentially expressed,138 mRNAs were up-regulated and 621 mRNAs were down-regulated.Using mi Randa,RNAhybrid algorithm to derive the interaction energy value(MEF value)between circRNA and miRNA,targeting relationship,targeting sites,and finally screening the differentially expressed circRNA-miRNA-mRNA relationship pairs to construct a colorectal tumor-related specific circRNA-regulated ceRNA network.Combined with the results of second-generation high-throughput sequencing,the ceRNA network was screened by Cytoscape software,and two key circRNAs with both ceRNA activity and significant differential expression were obtained,namely,has＿circ＿0001591 with up-regulated expression and hsa＿circ＿0051165with down-regulated expression.G O functional analysis of ceRNA network showed that it was mainly enriched in intracellular composition,bioregulation,metabolic processes,signaling,protein binding,and Pathway enrichment results showed that altering insulin levels by regulating ceRNA network increased the likelihood of colorectal cancer carcinogenesis for has＿circ＿0001591 and hsa＿circ＿0051165,respectively.Analysis of has＿circ＿0001591 in combination with clinicopathological parameters of colorectal cancer patients revealed that upregulation of has＿circ＿0001591 expression was associated with negative CD31 and CD34 in colorectal cancer patients(p < 0.05),and the difference was statistically significant.has＿circ＿0051165 expression downregulation was associated with the presence or absence of lymph node metastasis in colorectal cancer patients(p < 0.05),and the difference was statistically significant.The difference was statistically significant.has＿circ＿0001591 acted as a molecular sponge adsorbed mi R-497-5p acted on genes,mainly involved in the regulation of blood volume by renal aldosterone,nicotinic response,endogenous coagulation pathway,involved in acetylcholine-gated channel complex,type 4 collagen,half-bridge granule production,and the associated KEGG signaling pathway analysis The results showed that mainly involved in the complement pathway,neuroactive ligand-receptor interaction pathway,affecting the development of colorectal cancer.hsa＿circ＿0051165 antagonism novel-hsa-mi R200-5p acts on genes,mainly involved in the regulatory role of pseudopods,involved in the composition of the cytosol,cytoplasm,insulin-like growth factor binding protein,and the associated KEGG signaling pathway analysis showed that mainly through the Fox O signaling pathway,thus regulating colorectal carcinogenesis.Conclusions(1)This study found that there are significantly different circRNAs,miRNAs,and mRNAs in colorectal cancer,which are interrelated to form CeRNA networks,and their molecular functions and biological processes are predicted by bioinformatics technology,providing new ideas for the pathogenesis of colorectal cancer.(2)This study found that has＿circ＿0001591 and hsa＿circ＿0051165 may play an important role in the formation of colorectal cancer and are expected to be new diagnostic markers and therapeutic targets for colorectal cancer.has＿circ＿0001591 expression upregulation was associated with CD31 and CD34 negativity in colorectal cancer patients(p < 0.05),and the difference was statistically significant.It is suggested that hsa＿circ＿0001591 may play an inhibitory role in the invasion of blood vessels by colorectal cancer.The down-regulation of has＿circ＿0051165 expression was associated with the presence or absence of lymph node metastasis in colorectal cancer patients(p < 0.05),and the difference was statistically significant,suggesting that hsa＿circ＿0051165 may play a role in promoting the invasion of lymph nodes by colorectal cancer.(3)This study also predicted the possible regulatory mechanisms of has＿circ＿0001591and hsa＿circ＿0051165 in colorectal cancer: has＿circ＿0001591 acts as a molecular sponge adsorbing mi R-497-5p and is involved in colorectal carcinogenesis through the complement pathway,neuroactive ligand-receptor interaction pathway development,hsa＿circ＿0051165antagonizes novel-hsa-mi R200-5p and affects Fox O signaling pathway involved in colorectal cancer development.