Aberrant expression of a five‐microRNA signature in breast carcinoma as a promising biomarker for diagnosis

Abstract Background Breast cancer (BC) is the most common malignancy among females with dismal quality of life in patients. It has been proven that epigenetic factors, especially microRNAs, are involved in breast carcinogenesis and progression. This study aimed to assess the expression and clinical performances of a five‐microRNA signature (miR‐127‐3p, miR‐133a‐3p, miR‐155‐5p, miR‐199b‐5p, and miR‐342‐5p) in breast cancer and adjacent normal tissues to identify a potential biomarker for BC and investigate the relationship between their expression and clinicopathological features of BC patients as well. Methods In this case‐control investigation, we recruited 50 pairs of tumor and matched non‐tumor surgical specimens from patients diagnosed with BC. Expression levels of miR‐127‐3p, miR‐133a‐3p, miR‐155‐5p, miR‐199b‐5p, and miR‐342‐5p were measured in BC and adjacent normal tissues by RT‐qPCR. Results We found that miR‐127‐3p, miR‐133a‐3p, miR‐199b‐5p, and miR‐342‐5p were significantly down‐regulated, while miR‐155‐5p was significantly up‐regulated in BC tumor tissues compared with the corresponding adjacent normal tissues. The decreased expression of miR‐127‐3p, miR‐133a‐3p, miR‐342‐5p, and up‐regulation of miR‐155‐5p showed a significant correlation with disease stage. We also found a significant down‐regulation of miR‐127‐3p, miR‐199b‐5p, and miR‐342‐5p compared in HER‐2‐negative patients. Our results indicated that miR‐155‐5p had a higher expression level in HER‐2‐positive patients. Receiver operating characteristic (ROC) curve analysis demonstrated that all these five microRNAs can serve as potential biomarkers to distinguish between tumor and non‐tumor breast tissue samples. Conclusions The present findings suggested that dysregulation of this five‐miRNA signature might be considered as a promising and functional biomarker for BC diagnosis.


| INTRODUC TI ON
Breast cancer (BC) is the most commonly diagnosed cancer among women worldwide. Although early detection techniques and therapy methods have been improved, BC is still the leading cause of cancer death in females, and it is estimated that 627 000 women died from BC in 2018. 1,2 BC starts as a local disease but it might probably metastasize to distant organs such as bone, lung, regional lymph nodes, liver, and brain. Accordingly, early diagnosis is crucial to select the most appropriate treatment for patients with BC. 3 Mammography is the gold standard screening method in BC which helps to find early signs of BC. Furthermore, other imaging techniques such as MRI, CT scan, PET scan, and elastography could be utilized as beneficial methods to detect BC. Nevertheless, all these tools have their own limitations including being expensive, radiation risks, and more importantly, lack of specificity which count them as inefficient screening methods. 4,5 In this regard, numerous efforts have been strived to find better diagnostic and therapeutic tools for patients with BC. By having high sensitivity, being noninvasive, and more specificity, biochemical biomarkers, including proteins, DNAs, microRNAs (miRNAs, miRs), and long non-coding RNAs (ln-cRNAs), are recently considering as easily accessible markers with promising potentials for the detection of BC at early stages. 6,7 miRNAs are a class of small non-coding RNAs with a length of about 22 nucleotides which regulate gene expression at the post-transcriptional level. 8 miRNAs are the key player of multiple crucial biological processes such as proliferation, differentiation, and apoptosis.
Unsurprisingly, aberration in miRNAs expression has been demonstrated in various types of cancers. miRNAs may play a crucial role a class of oncogenes or tumor suppressor genes in cancer. [9][10][11] In BC, miRNA profiling has allowed for the identification of biomarker signatures associated with the diagnosis, staging, progression, and prognosis. [12][13][14][15][16] The aim of the present study was to analyze the expression levels of miR-127-3p, miR-133a-3p, miR-155-5p, miR-199b-5p, and miR-342-5p in BC patient tissues and normal counterparts. Furthermore, we investigated the correlation between alterations of these miRNAs with clinical phenotypes and HER-2 expression status of the patients.

| Statistical analysis
Data analyses were performed using ABI StepOne Real-Time PCR Software v2.0.2 (Applied Biosystems), and figures were made using GraphPad Prism v.7.0 (GraphPad Software). The results were analyzed by performing t tests in which P < .05 considered statistically significant. To be on the safe side, all experiments were carried out at least two times.

| Expression analysis of a five-miRNA signature in tumor and non-tumor samples
We utilized bioinformatics tools for mining miRNAs, which aim at identifying the most possible miRNAs that potentially cause BC development. Furthermore, we reviewed previous experimental findings that suggested possible role of these miRNAs in BC progression. Finally, five miRNAs including miR-127-3p, miR-133a-3p, miR-155-5p, miR-199b-5p, and miR-342-5p were selected to be evaluated as possible biomarkers in BC patients. The reaction efficiencies for each primer set were calculated on 5-fold serial dilutions of cDNA samples. As shown in Figure S1 and Table S2, the amplification ef-  Figure 1A,B). Unsupervised hierarchical clustering analysis of the relative expression of the differentially expressed miRNAs revealed that this set of markers is able to discriminate between the tumors and the non-tumor breast tissues ( Figure 1C).

| Correlation of miRNAs expression levels with level of malignancy and HER-2 status in human BC
We analyzed whether the expressional evaluation of this five-miRNA signature was correlated with clinicopathological characteristics of BC patients. As compared with the different stages of malignancy of BC, the expression levels of miRNAs including miR-127-3p, miR-133a-3p, and miR-342-5p were significantly lower in stages III compared with stages I and II, except miR-199b-5p which down-regulated non-significantly in higher stages, whereas the miR-155-5p was significantly up-regulated in stages III in comparison with stages I and II ( Figure 2).
In this study, we also investigated the possible association between expression levels of miRNAs and HER-2 status in BC tissues.

| Determination of the biomarker quality for BC
We utilized ROC curve analysis to evaluate the sensitivity and specificity of the expression levels of these miRNAs to discriminate BC tissues from healthy tissues. The calculated area of the miRNAs which are examined in this study suggests that all of these five miRNAs may be suitable as a tumor marker and can potentially serve as an efficient diagnostic biomarker for BC ( Figure 4).

F I G U R E 2
Comparison of miRNA expression level in breast cancer patients with different tumor stages. Note that the expression of miR-127, miR-133a, and miR-342-5p was significantly lower in higher stage. The observed difference in the expression level of miR-199b was not statistically significant. On the other hand, miR-155 was significantly up-regulated in stage III in comparison with stages I and II

| D ISCUSS I ON
Breast cancer is the most prevalent cancer among women worldwide. 19 Numerous studies have been reported that miRNAs have regulatory functions in pathological processes, in particular in the development and progression of tumors. 9 Hence, it is possible that many miRNAs could serve as biomarkers for the diagnosis of different types of cancer, including BC. 16 Integrative computational bioinformatics approaches have been utilized as an effective tool to detect the potential outlier miRNAs in cancer. 20 were culled to be analyzed and confirmed by RT-qPCR.
In this study, a comparison between the expression profile of our candidate miRNAs in breast carcinoma tissue samples and their normal tumor margin was applied. In other words, this study has been applied to investigate whether the candidate miRNAs have the potential to be a novel diagnostic marker. Derived results showed a significant down-regulation in miR-127-3p, miR-133a-3p, miR-199b-5p, and miR-342-5p in tumor tissues of the patients compared with the normal margin tissues, while miR-155-5p has been up-regulated in the aforementioned samples. Interestingly, a correlation between the expression levels of these miRNAs with some clinical characteristics of disease was observed. Collectively, these results suggest that these microRNAs signature might be considered as a tool for early diagnosis in BC. Several studies have shown that miR-155 is an oncogene which regulates several cancer cell process, including cell proliferation, migration, invasion, metastasis, and epithelial-mesenchymal transition (EMT). 35 We demonstrated that miR-127-3p, miR-133a-3p, miR-199b-5p, and miR-342-5p were down-regulated; however, miR-155-5p represented up-regulation in tumor samples in comparison with those of their matched non-tumor controls. The expression level of these miRNAs was associated with the extensiveness of the malignancy and HER-2 expression status. In addition, the results revealed that miR-127-3p, miR-199b-5p, miR-342-5p, miR-133a-3p, and F I G U R E 4 ROC curve analyses were performed to discriminating tumor from non-tumor breast tissue samples. ROC curve and its calculated area under curve (AUC) of miR-127 (78%), miR-199b (72%), miR-342-5p (67%), miR-133a (70%), and miR-155 (74%) suggest that all of these five miRNAs may be appropriate tumor biomarkers for breast cancer miR-155-5p could serve as valuable biomarkers for the diagnosis of BC with AUCs of 0.78, 0.72, 0.67, 0.70, and 0.74, respectively.
One of the most important points for an effective prognosis and diagnosis approach is to identify early-stage BC, and our study suggested that evaluating the expression levels of miR-127-3p, miR-133a-3p, miR-155-5p, miR-199b-5p, and miR-342-5p might be clinically useful in BC patients. Our results confirmed the previous findings which showed that aberrant expression of these five miRNAs can improve cancerous growth. 27,30,35,39,41 Altogether, dysregulation of this five-miRNA signature might be used as a promising biomarker in detecting and following the disease progression in BC patients.

ACK N OWLED G M ENTS
This work was supported by a research grant from Tarbiat Modares University, Tehran, Iran.