Screening Of Differentially Expressed CircRNAs And Integrated Analysis Of CircRNAs Regulator Network In Breast Cancer

Part one: Screening and validation of tissue-specific circRNAs in breast cancerBackgroundBreast cancer is one of the most common cancers among women in the world,with an estimated 1.7 million incident cases each year.Epidemiological studies have shown that obesity,physical inactivity,alcohol consumption,age at menarche,menopause age,family history of breast cancer,advanced maternal age at the first birth,and the estrogen or progestin use are associated with the risk of breast cancer in women.However,the fact of family aggregation and the fact that only a few people suffering from the same exposure indicate that genetic factors are essential to breast cancer etiology.CircRNAs were found in viroids,viruses and tetrahymena decades ago,but they were initially considered to be by-products of aberrant RNA splicing or splicing errors due to their low expression.In recent years,studies have found that circRNA is closely related to the occurrence and development of diseases.But the relationship between circRNAs and breast cancer is still in its infancy.In this study,we used the chip technology to screen the differentially expressed circRNAs in breast cancer and evaluated the value of them as biomarkers for the diagnosis of breast cancer.MethodsThis study used a two-stage case-control design.In the first stage,four pairs of breast cancer tissues and adjacent normal-appearing tissues were collected and compared.The Arraystar Human circRNA Array was used to construct a genome-wide circRNA profile.We screened the differentially expressed circRNAs using the criteria of fold change?FC?>2 and P <0.05.In the second stage,50 pairs of breast cancer tissues and adjacent normal-appearing tissues,42 pairs of breast cancer plasma and healthy control plasma,and 3 types of breast cancer cell lines and one normal cell line were used to validate the expression of circRNAs identified from the first stage.We used the quantitative real-time polymerase chain reaction?qRT-PCR?to detect the expression of circRNAs.We also explored the relationship between differentially expressed circRNAs and the clinical characteristics of patients with breast cancer.The Receiver Operating Characteristic?ROC?curve together with the area under curve?AUC?were used to analyze the diagnostic value of specific circRNA.ResultsBased on the chip screening,we identified 1155 differentially expressed circRNAs.Among them,715 were upregulated and 440 were downregulated in breast cancer tissues.Six candidate circRNAs were selected for further validation.Data from the second stage showed that the expression level of hsa_circ₁03110,hsa_circ₁04689 and hsa_circ₁04821 were elevated in breast cancer tissues,whereas hsa_circ₀06054,hsa_circ₁00219 and hsa_circ₄06697 were downregulated.By plotting the ROC,we found that hsa_circ₁00219 had the highest diagnostic accuracy,with an AUC of 0.78?95% CI: 0.69-0.88?,the sensitivity of 0.69?95% CI: 0.54-0.81?,and the specificity of 0.71?95% CI: 0.56-0.83?.When we combined the hsa_circ₀06054,hsa_circ₁00219 and hsa_circ₄06697 as the biomarker,the AUC was 0.82?95% CI: 0.73-0.90?.There was no significant difference in the expression of circRNAs in plasma between breast cancer patients and normal healthy controls.The hsa_circ₁04821 was found to be up-regulated in three types of breast cancer cell lines?MCF-7,MDA-MB-231 and MDA-MB-468?.ConclusionCircRNA is differentially expressed in breast cancer and adjacent normal-appearing tissues,and can be used as a new class of biomarkers for human breast cancer.Part two: Gene Function analysis of differentially expressed circRNAs in breast cancer BackgroundRecent evidence has indicated that circRNAs can regulate gene expression by acting as a miRNA “sponge” or as a competitive endogenous RNA?ceRNA?.Aberrant expression of circRNAs affects the normal physiological function of human body and is related to the development of many diseases.In this study,we sued the bioinformatics analysis to predict the function of the differential circRNAs and explore their mechanisms in the carcinogenesis of breast cancer.MethodsBased on the differential expression profile constructed in the first stage,we performed the gene ontology?GO?analysis and Kyoto encyclopedia of genes and genomes?KEGG?Pathway analysis to predict the function and signal pathway of target genes.We also used the Arraystar software to predict and annotate circRNA-miRNA interactions.ResultsGO analysis indicated that the highest enriched term corresponding to up-regulated transcripts were “transmembrane receptor protein tyrosine kinase signaling pathway?ontology: biological process?”,“protein binding?ontology: molecular function?” and “synapse?ontology: cellular component?” and the highest enriched term corresponding to down-regulated transcript were “single-organism developmental process?ontology: biological process?”,“cytoplasm?ontology: molecular function?” and “protein binding?ontology: cellular component?”.The KEGG pathway analysis showed that the Hippo signaling pathway and the WNT signaling pathway were related to the upregulated circRNAs,and the RAP1 signaling pathway and the RAS signaling pathway were related to the downregulated circRNAs.Each circRNA can bind multiple miRNAs.ConclusionscircRNAs are involved in cancer-related signaling pathways and can bind multiple cancer-related miRNAs and may be involved in the development of breast cancer.