| BACKGROUND AND OBJECTIVEThe distinction between breast cancer and benign breast diseases with nipple discharge remains an important diagnostic challenge. Diagnosis of patients with nipple discharge is generally difficult due to the lack of non-invasive methods for breast cancer detection. An expedient and cost-effective diagnostic tool is needed to complement galactography and exfoliative cytology for detection of benign or malignant breast diseases with nipple discharge. The purpose of this study was to predict the potential usefulness of tumor markers in nipple discharge and to investigate the relationship of tumor markers and clinical characteristics with breast cancer. The aim of this study was to assess the diagnostic values of glycoprotein tumor markers (i.e., CEA, CA153 and CA125) in milk and nipple discharges in the prediction of different breast diseases diagnoses. Novel tumor biomarkers for better cancer detection and diagnosis are urgently needed. The purpose of this study is to reveal and validate the differential expression of miRNAs in nipple discharge and identify miRNAs as novel potential biomarkers for primary breast cancer.METHODSThree hundred thirty-six patients with nipple discharge and a control group of 56 healthy parturient participants were enrolled in the present study. Nipple discharge samples were preoperatively collected from the patients, and milk was collected from the colostrum of the parturient participants. Patients were divided into two groups according to postoperative pathological results:96 cases in breast cancer group and 240 cases in benign group.The samples were assayed for the CEA, CA153, CA125, CA199, CA724 and AFP levels. Cutoff values were determined for the detection of breast diseases using ROC curves. The relationships of clinical characteristics with breast cancer were investigated by multivariate analysis with a logistic regression model. The initial screening of miRNAs expression was performed with Axon GenePix 4000B microarray scanner using a novel approach to label miRNAs. The expression levels of miRNAs selected from the initial screening were examined with quantitative real-time polymerase chain reaction (qRT-PCR) in 21 validation samples (8 cancer patients and 13 individuals with benign tumor). Independent T-test was used to detect significant correlation between miRNA expression level and breast cancer. Different subtypes of breast cancer cell line and normal breast cell line MCF-10A as control were selected to be examined for the expression level of miR-4732-5p. Quantitative real-time polymerase chain reaction (qRT-PCR) was done to validate data in clinical breast tissue including breast cancer tissue and pericarcinous tissue. A series of comprehensive experiments for the target miRNA were done, such as lipofection transfection, overexpression of miRNA, Methyl Thiazolyl Tetrazolium Assay(MTT Assay), Annexin V-FIFC/PI flow cytometry analysis.The research team conducted the effect of miRNA on migration,proliferation and apoptosis of different subtypes of breast cancer cell lines. According to predicting outcome of downstream gene of target miRNA, we confirmed the expression levels of downstream genes in breast cancer cell lines and pairing breast cancer tissue.RESULTS1.In the nipple discharges,the CEA and CA153 levels in the breast cancer group were significantly greater than those in the benign group (all ps<0.001), and cutoff values of 263.3 ng/mL and 1235.3 U/mL, respectively, were established. However, the expression of CA125 did not differ significantly between the breast cancer and benign groups. It showed significant differences in levels of nipple discharge CEA (p<0.001) and CA1530=0.014), but not CA125, CA199, CA724 and AFP among two groups.2. The levels of CEA, CA153 and CA125 were significantly different between the nipple discharges and the milk (all ps< 0.001). The median levels of CEA, CA153 and CA125 were elevated by 38.7-,3.9-and 7-fold, respectively, in the patients with breast cancer compared with the healthy lactating controls. All of the differences between the breast cancer group and healthy controls were significant (all p< 0.001). The levels of CEA and CA125 in the nipple discharges from the patients with benign lesions were significantly higher than those of the healthy controls.3. Microarray profiling demonstrated that 3 miRNAs were down-regulated in breast cancer patients compared with benign group. The qRT-PCR analysis further verified that one miRNAs (miR-4732-5p) might serve as potential tumor biomarkers for breast cancer detection.4. The expression levels of miR-4732-5p in different breast cancer cell lines were lower than the breast epithelial cell line MCF-10A:The expression levels of miR-4732-5p was measured in eight different subtypes of breast cancer cell line (MCF-7, MDA-MB-453, MDA-MB-157, MDA-MB-231, ZR-75-1, SK-BR-3, BT549 and T47D) and MCF-10A by qRT-PCR. All of breast cancer cell lines expressed lower levels of miR-4732-5p than MCF-10A.5. The expression levels of miR-4732-5p in pairing breast cancer tissue and pericarcinous breast tissue were different:In all pairing breast tissue samples, about 78.6% of breast cancer tissue showed lower levels of miR-4732-5p than pericarcinous breast tissue. According to postoperative pathological outcome, clinical stage was higher, the level of miR-4732-5p was higher in all breast cancer patients. Patients with lymph node metastasis had higher levels of miR-4732-5p in breast cancer tissue. Patients with more metastatic lymph nodes has higher expression levels of miR-4732-5p.6. miR-4732-5p had the effect on cell viability of breast cancer cell lines:In different subtypes of breast cancer cell lines(MD-MB-231 and T47D), overpression of miR-4732-5p had inhibiting effect on cell migration and proliferation. But it had no effect on breast cancer cell apoptosis.7. According to downstream gene prediction of miR-4732-5p, target genes such as ALKBH5, ANKRD17, BCLAF1, CPSF6, NHLH1, PPM1E, and PSMA5 were detected in different subtypes of breast cancer cell lines and pairing breast cancer tissue and pericarcinous breast tissue. ALKBH5, NHLH1, PPM1E and PSMA5 were potential downstream genes of miR-4732-5p. miR-4732-5p and downstream genes had negative correlation.CONCLUSIONS1. Differences in the apparent expression levels of CEA, CA153 and CA125 in patients with nipple discharge and healthy persons were validated. Nipple discharge from breast surgery department was validated to be pathological nipple discharge.2. Tumor marker analysis of nipple discharge fluid for CEA and CA153 would enhance the accurate assessment and treatment planning for patients with nipple discharge.3. CA125 did not seem to be useful for breast cancer detection. Measurements of CA199, CA724 and AFP in nipple discharge are not of great clinical value.4. One miRNA (miR-4732-5p) might serve as potential tumor biomarkers for breast cancer patient with nipple discharge. Nipple discharge with breast cancer expressed lower levels of miRNA than benign breast lesions.5. miR-4732-5p showed low expression levels in different subtypes of breast cancer cell lines and pairing breast cancer tissue. miR-4732-5p had inhibiting effect on cell migration and proliferation.SIGNIFICANCE1. An effective diagnostic tool was built for accurate stratification of nipple discharge patients for further work up and treatment. Levels of CEA and CA153 in nipple discharge help to establish the diagnosis of different breast diseases. The method was widely used in clinical diagnosis. It initially identified and validated miRNAs in nipple discharge and investigated the potential of miR-4732-5p as a novel breast cancer biomarker. Analysis of the fluid from discharge would likely shed light on the underlying malignant or benign cause of nipple discharge.2. It evaluated the levels of glycoprotein and miRNAs in nipple discharge patients undergoing surgery and correlate the tumor marker levels with the underlying breast conditions. The research showed the impact of nipple discharge test on the evaluation of abnormal nipple discharge. It was validated the difference between milk (physiological nipple discharge) and pathological nipple discharge.3. The presence of miRNAs in nipple discharge showed a deep knowledge of the correlation between breast cancer and specific miRNA level in nipple discharge. miR-4732-5p served as a potential tumor biomarker that can assist in screening for breast cancer. miRNA level in nipple discharge may be a predictor of malignant breast cancer. |
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