RNA-seq Profiling Of Serum Exosomal Circular RNAs Reveals Circ-PNN As A Potential Biomarker For Human Colorectal Cancer

Objective:In recent years,more research evidence has shown that circRNA is closely related to tumor genesis and development,and can be secreted from tumor cells to body fluids through exosomes,making it possible for exosomal RNA to serve as a reliable indicator of various cancer diagnoses and prognosis.In this study,differential expression profile of serum exosomes of colorectal cancer(CRC)was preliminarily established,and serum exosomes were extracted and identified to screen out circRNA for the diagnosis of CRC,and the diagnostic efficacy of the discovered circRNA molecules as tumor markers was investigated.Methods:In our previous studies,we performed RNA sequencing(RNA-seq)analysis of ribosomal RNA-depleted total RNA to obtain the differential expression profiles of circRNAs in serum exosomes samples of CRC group and control group.Then,eight circRNAs were selected as candidate moleculars according to their expression in multiple samples.We performed Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis for circRNAs to predict their potential functions.We initially extracted and identified the exosomes in serum samples of CRC group and control group.Transmission electron microscopy(TEM),Western blotting analysis(WB),nanoparticle tracking analysis(NTA)were used to confirme the exosomes we extracted.After obtaining serum exosomes,RNA was extracted.We then designed divergent primers for those circRNAs,we gel-extracted and sequenced those products,confirming that they contained the head-to-tail junction.After treatment with Actinomycin D,RT-qPCR analysis showed that circRNAs were stable in comparison to mRNAs.Then,RT-qPCR was performed to verify the expression of exosomal circRNAs in a traning sample sets(including 88 CRC patients and 88 healthy controls)and the potential diagnostic values were evaluated through ROC curve analysis.The circRNAs molecules whose expression trends were consistent with the sequencing results in the training set entered the validation phase.During the validation phase,differential circRNAs expression was detected in the validation set(including 58 colorectal cancer patients and 58 healthy controls).We also analyzed the correlation between serum exosomes circRNAs expression in colorectal cancer and clinicopathological parameters.The miRNAs binding sites and target mRNAs were predicted by popular target prediction softwares:Targetscan and Miranda.Cytoscape software was applied to construct a circRNA-miRNA-mRNA network map based on data of circRNAs with its miRNAs-binding and predicted miRNAs sites.Results:We performed RNA sequencing(RNA-seq)analysis of serum exosomals circRNAs to obtain the differential expression profiles of circRNAs in CRC patients and healthy controls.And we performed GO and KEGG analysis for upregulated circRNAs to predict their potential functions.TEM was performed to show the typical oval-shaped extracellular vesicles.WB was used to detect exosome surface specific proteins CD9 and TSG101.NTA showed the exosomes with diameters of 50-150 nm.In the training set,the expression levels of circ-PNN(P<0.001)were significantly up-regulated in CRC patients,which was consistent with previous sequencing results.The area under the ROC curve(AUC)of serum exosomal circ-PNN was 0.855(95%CI=0.794-0.904,sensitivity=89.8%and specificity=73.9%).Subsequently,the expression of circ-PNN was further verified in the validation set,The dysregulated expression trend was consistent with the training set,and the diagnostic accuracy of circ-PNN in the validation set,measured by AUC was 0.826(95%CI=0.745-0.890,sensitivity=89.7%and specificity=69.0%).We then built a circRNA-miRNA-mRNA network for circ-PNN using Cytoscape.It was verified that the expression level of hsa-miR-6833-3P、hsa-let-7i-3p and hsa-miR-1301-3P was negatively correlated with the expression level of circ-PNN in CRC patients.Conclusion:In this study,high-throughput circRNAs sequencing results of serum exosomes were analyzed and verified.It was found that circ-PNN was highly expressed in CRC,with good sensitivity and specificity for the diagnosis of colorectal cancer,and could be used as a potential tumor marker for the diagnosis of CRC.