MicroRNA-411 represents an innovative bio-marker in breast cancer detection

Background: MicroRNA-411 (MiR-411) has been reported to play an important role in tumorigenesis. This study was aimed to investigate the diagnostic performance of serum miR-411 in breast cancer. Methods: The serum level of miR-411 was determined in breast cancer patients using quantitative real-time PCR (qRT-PCR). Chi-square was applied to evaluate the association between miR-411 expression and clinical characteristics. The diagnostic value of serum miR-411 for breast cancer was estimated using receiver operator characteristic (ROC) analysis. Results: Serum miR-411 in patients with breast cancer was markedly decreased compared with healthy controls (P < 0.001). The level of miR-411 was correlated with clinical stage (P = 0.019), histological grade (P = 0.014), and lymph node metastasis (P = 0.036). ROC curve showed that serum level of miR-411 could discriminate between breast cancer patients and healthy controls, with the AUC of 0.796, combing with the sensitivity of 82.1% and the specicity of 83.2%. The cut-off value of miR-411 for breast cancer diagnosis was 1.245. Conclusions: MiR-411 plays inhibitory roles in aggressive progression of breast cancer. Serum miR-411 may be a potential non-invasive biomarker for breast cancer diagnosis.


Background
Breast cancer is one of the most common cancers among women, with severe mortality [1]. The mainly cause of breast cancer death is tumor metastasis [2]. Despite of great progress made in treatments, numbers of breast cancer patients die from the metastatic disease [3]. The therapeutic effects are signi cantly associated with tumor stage at diagnosis [4]. Early detection remains a major challenge for breast cancer [5]. Biomarkers detection provides an effective approach for early diagnosis of breast cancer. The commonly used biomarkers for breast cancer diagnosis include CEA (carcinoembryonic antigen), CA153 (carbohydrate antigen 153), HER-2/Neu, estrogen receptor (ER), progesterone receptor (PR), epidermal growth factor receptor (EGFR), and vascular endothelial growth factor receptor (VEGFR) [6]. Unfortunately, the low speci city and sensitivity limit their clinical application in breast cancer screening [7][8][9]. As a consequence, new biomarkers are urgently needed in early detection and screening of breast cancer. MicroRNAs (miRNAs) are a novel class of small non-coding RNAs that play regulatory roles in gene expression at the post-transcriptional level [10,11]. MiRNAs take part in regulation of various important cellular processes, such as differentiation, migration, and apoptosis [12]. Abnormal expression of miRNAs may contribute to pathological mechanisms of human disease, like cancer [13]. The expression pro les of miRNAs are stable in body uids and archived tissue samples which can be detected by non-invasive methods, suggesting that detection of miRNAs may provide a promising way for cancer diagnosis [14][15][16] served as an oncogene in hepatocellular carcinoma by promoting cell proliferation of the cancer cells [17]. However, until now, the serum level of miR-411 and its diagnostic performance in breast cancer remained unidenti ed.
In the current study, we sought to investigate diagnostic performance of serum miR-411 in breast cancer. The serum levels of miR-411 in breast cancer patients were detected, as well as its association with clinical characteristics. In addition, receiver operating characteristic (ROC) analysis was performed to determine the diagnostic value of miR-411 in breast cancer.

Patients and serum samples
The present study was approved by the the Ethical Committee of the hospital and all participants provided written informed consents in advance. 107 patients who were pathologically diagnosed with breast cancer at Harrison International Peace Hospital were enrolled in the study. None of the patients had received surgery, chemotherapy, or radiotherapy before blood collection. The control blood samples were collected from 95 age-matched healthy volunteers who were without malignancy history and in ammatory diseases. Clinicopathological features of patients were summarized in Table 1. Note: ER: estrogen receptor; PR: progesterone receptor; HER-2: C-erbB-2.
Page 5/12 5 ml whole blood was collected from each participant in a serum separator tube containing EDTA. Samples were left to clot at room temperature for 30 min and then were centrifuged at 4,000 rpm for 10 min at 4℃. Serum specimens were stored at -80℃ until further use.
RNA extraction and quantitative real-time ploymerase chain reaction (qRT-PCR) Total RNA was extracted from serum using the standard TRIZOL LS (Invitrogen, CA, USA) method according to the manufacturer's protocol. Total RNA concentration and integrity were determined with an ultraviolet specrophotometer (Beckman, CA, USA) and a digital gel image analysis system (Bio-Rad, CA, USA).
Total RNA samples were reversely transcribed (RT) to cDNAs using primers speci c to miR-411 target. Speci c cDNAs were then ampli ed by real-time quantitative RT-PCR using SYBR Premix Ex Taq

Statistical analysis
All statistical analyses were carried out with SPSS 18.0 software and GraphPad prism 5. Data were presented as mean ± SD. MiR-411 expression levels were compared using Students's t test. The relationships between miR-411 expression and clinicopathological factors were analyzed using chisquare test. Receiver operating characteristics (ROC) curves were established to evaluate the diagnostic value of serum miR-411 in breast cancer. P < 0.05 was considered statistically signi cant.

Results
The expression level of serum miR-411 from breast cancer patients and healthy controls QRT-PCR assay was carried out to measure the serum level of miR-411 in 107 patients with breast cancer and 95 healthy controls. As shown in Fig. 1, the expression of serum miR-411 were signi cantly lower in breast cancer than that in healthy controls (P < 0.001).
The association between serum miR-411 expression and clinicopathological characteristics The patients were divided into high expression group (n = 48) and low expression group (n = 59), according to their average expression level of miR-411. To further determine the clinical signi cance of serum miR-411 expression, chi-square test was performed. Results showed that miR-411 was obviously associated with clinical stage (P = 0.019), histoltogical stage (P = 0.014) and lymph node status (P = 0.036). No signi cantly differences were found between miR-411 and other clinicopathological features, including age, tumor diameter, lymph node status, ER status, PR status and HER-2/neu status (all P > 0.05) ( Table 1). It indicated that miR-411 was implicated with the development and metastasis of breast cancer.
The diagnostic value of miR-411 in breast cancer ROC curves which were built based on serum levels of miR-411 in breast cancer patients and healthy individuals were used to evaluate the diagnostic value of miR-411 for breast cancer. The curve showed that serum miR-411 could distinguish breast cancer patients from healthy individuals at the optimal cutoff value of 1.245, with the an area under the curve (AUC) of 0.796, combing with the sensitivity of 82.1% and the speci city of 83.2% (Fig. 2).

Discussion
Breast cancer is a severe threat to health among women worldwide, due to its high incidence rates and low overall survival [19]. Tumor metastasis may be responsible for the dismal clinical outcomes of breast cancer [3]. Unfortunately, the etiology of breast cancer remains unclear, and it is unable to determinate the key factor for tumor progression [20]. Early diagnosis is a pivotal approach to improve outcomes of breast cancer patients. Until now, the commonly used biomarkers for breast cancer diagnosis, such as CEA, CA153, HER-2/Neu, ER, PR, and EGFR, show limited diagnostic effectiveness [21]. Therefore, it is necessary to identify new diagnostic biomarkers for breast cancer patients to improve prognosis.
Growing evidences have suggested that miRNAs may provide an effective tool for cancer diagnosis, due to its stable expression pro les in body uids and tissues specimens, as well as its signi cantly association with tumor progression [22]. In breast cancer, a variety of dysregulated miRNAs were observed, suggesting their important functions in tumor development and progression. MiR-4262 was proved to be a tumor oncogene in breast cancer that its over-expression might contribute to proliferation and invasion of the cancer cells [23]. MiR-421 was down-regulated in breast cancer tissues specimens and cell lines, and its expression patterns showed negative link with metastasis, tumor stage and recurrence. MiR-421 might be a tumor suppressor in breast cancer [24]. Given their functional roles in progression of breast cancer, miRNAs were considered as promising biomarkers for the disease. In the present study, we investigated the diagnostic signi cance of miR-411 in breast cancer.
In this study, we found miR-411 expression was decreased in serum samples collected from breast cancer patients compared with healthy controls. Moreover, the down-regulated serum miR-411 levels were tightly correlated with advanced clinical stage, high histological grade, and positive lymph node metastasis. It suggested that miR-411, as a tumor suppressor, was involved in the progression of breast cancer. The conclusion was consistent with the previous investigations. It was reported that the expression of miR-411 was signi cantly down-regulated in breast cancer patients, and recovery its expression might suppress growth, migration, and invasion of the cancer cells [25,26]. However, the molecular mechanisms for the anti-tumor action of miR-411 in breast cancer remained poorly known. Further researches were still needed.
Breast cancer diagnosis is a challenging research job. The cancer is characterized by heterogeneous, with diverse genetic alterations [27]. In the previous studies, various molecular biomarkers were con rmed for breast cancer. For instances, Zhang et al. reported that plasma long non-coding H19 levels were signi cantly different between breast cancer patients and healthy individuals that might be a potential diagnostic biomarker for the disease [28]. The study carried out by Chen et al. demonstrated that serum levels of DAND5 were positively correlated with aggressive clinical characteristics and low survival rate of breast cancer patients, suggesting its predictive potential in the cancer [3]. Identi cation of genetic alterations might provide an effective approach for early diagnosis and prognosis evaluation of breast cancer. In this study, ROC curve analysis was performed to investigate the diagnostic performance of miR-411 in breast cancer patients. The results revealed that serum miR-411 expression could differentiate breast cancer patients from healthy controls with satisfactory sensitivity and speci city. Despite of the various identi ed molecular biomarkers for breast cancer, few of them were applied in clinic. Therefore, well-designed study with large sample size was still needed to investigate the application value of serum miR-411 for breast cancer diagnosis.

Conclusions
In conclusion, serum miR-411 levels in patients with breast cancer is down-regulated, and its decreased expression correlates with malignant tumor progression. Serum miR-411 may be a potential biomarker for early detection of breast cancer. Ethics approval and consent to participate This study was supported by the Ethics Committee of Harrison International Peace Hospital and also has been carried out in accordance with the World Medical Association Declaration of Helsinki.
The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Figure 1
Serum miR-411 levels in 107 breast cancer and 95 healthy controls. Serum expression level of miR-411 in breast cancer were strongly down-regulated compared with healthy controls. ***: suggested P<0.001.