Study On MicroRNA Screening And Verification And Functional Prediction Of Breast Cancer In Nipple Discharge And Blood

Breast cancer is one of the most common malignant tumors in women.Early diagnosis of breast cancer is an important clinical issue.miRNA has been shown to be present in a variety of body fluids and remains stable,facilitating its application as tumor marker.At present,the study of miRNA in breast cancer patients with nipple discharge and blood is scarce.Part I Screening,verification and functional prediction of breast cancer associated microRNAs in nipple dischargePurpose:1.To confirm the presence of miRNAs in nipple discharge.2.To screen and verify the differential expression of miRNAs in benign and malignant nipple discharge.3.To verify the stability of miRNA in the presence of physiological nipple discharge-milk.4.To investigte the possible mechanism of miRNA by bioinformatics.Materials and methods:1.The nipple discharge was collected from 3 patients with breast cancer and 3 patients with benign tumor.The differentially expressed miRNAs were screened by miRNA microarray.2.Differential expression of miRNA was detected by real-time quantitative PCR in nipple discharge of 8 patients with breast cancer and 13 patients with benign tumor.3.Maternal milk was placed at 4℃ for 0-72 hours.The expression of miRNAs were detected by real-time quantitative PCR.4.Targetscan and miRDB software were used to predict the target gene of miRNAs.Then GO analysis,KEGG pathway enrichment and protein interaction analysis were performed.Results:1.Microarray results showed that,compared with benign lesions,three miRNAs(miR-4484,miR-K1 2-5-5p,and miR-3646)were up-regulated in malignant nipple discharge,and the other three miRNAs(miR-4732-5p,miR-4687-3p,and miR-Sl-5p)was down-regulated.2.PCR analysis confirmed that the expression of miR-4484,miR-K12-5-5p and miR-3646 was up-regulated in malignant nipple discharge,while the expression of miR-4732-5p was down-regulated.3.The expression level of miR-3646 and miR-4732-5p in milk was relatively stable within 72 hours at 4℃.4.Numerous carcinoma-associated genes,including ADIPOQ,CPEB1,DNAJB4,EIF4E,APP and BCLAF1,were revealed to be putative targets of miR-3646,miR-4484 and miR-4732-5p.Bioinformatics analysis associated miR-3646 with the Rapl and TGF-β signaling pathways,miR-4484 with the ErbB,estrogen and focal adhesion signaling pathways,and miR-4732-5p with the proteoglycan signaling pathway.Notably,protein-protein interaction analysis identified that numerous predicted targets of these miRNAs were associated with one another.In addition,the target genes of the miRNAs were identified to be under the regulation of a number of transcription factors(TFs).Conclusions:We confirmed the up-regulation of miR-4484,miR-K12-5-5p and miR-3646,and the down-regulation of miR-4732-5p expression in the nipple discharge of breast cancer patients.The expression of miR-3646 and miR-4732-5p in milk was stable.Bioinformatics analysis suggests that miRNA may be involved in several cancer-related pathways by regulating tumor-associated proteins.Our study provides a theoretical basis for miRNAs as biomarkers of breast cancer in nipple discharge.Part Ⅱ Screening,verification and functional prediction of breast cancer associated microRNAs in bloodPurpose:1.To screen and verify the differential expression of miRNA in breast cancer and healthy controls.2.To analyze the possible mechanism and prognostic value of miRNA by bioinformatics.Materials and methods:1.The whole blood samples of 6 patients with breast cancer and 6 healthy controls were collected.MiRNA microarray was used to screen the differentially expressed miRNAs.2.The difference of miRNA expression was confirmed by real-time quantitative PCR in the whole blood samples of 15 patients with breast cancer and 13 healthy controls.3.miRWalk database was used to predict the target gene of miRNA,and the DAVID tool was used to find the enrichment pathways of target genes.Prognostic values of miRNA and its target genes in breast cancer were analysed using public databases.Results:1.A total of 171 differentially expressed miRNAs were identified by microarray,including 169 up-regulated and 2 down-regulated miRNAs in breast cancer.2.Five upregulated miRNAs(miR-30b-5p,miR-96-5p,miR-182-5p,miR-374b-5p,and miR-942-5p)were confirmed by real-time quantitative PCR.ROC curve analysis showed that the 5 miRNA could distingguish breast cancer patients and healthy controls.3.Upregulation of miR-30b-5p,miR-96-5p,miR-182-5p,miR-374b-5p and miR-942-5p was observed in the whole blood of early breast cancer patients.4.A total of 855 genes were predicted to be targeted by the five miRNAs,and the one cut domain family member 2(ONECUT2)gene was a shared target of the five miRNAs.Analysis of publicly available data revealed that these dysregulated miRNAs and target genes were associated with the survival of breast cancer patients.Furthermore,the five miRNAs were significantly enriched in numerous cancer-related pathways,including "MicroRNAs in cancer","Pathways in cancer","FoxO signaling pathway’’,"Ras signaling pathway","Rapl signaling pathway","MAPK signaling pathway",and "PI3K-Akt signaling pathway".Conclusions:Our data supported the potential of the five identified miRNAs as novel biomarkers for the detection of breast cancer,and indicated that they may be involved in breast cancer development and progression.