MicroRNA-based potential diagnostic, prognostic and therapeutic applications in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is a distinct subtype of breast cancer characterized by high recurrence rates and poor prognosis compared to other breast cancers. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of various post-transcriptional gene and silence a broad set of target genes. Many recent studies have demonstrated that miRNAs play an important role in the initiation, promotion, malignant conversion, progression, and metastasis of TNBC. Therefore, the aim of this review is to focus on recent advancements of microRNAs-based potential applications in diagnosis, treatment and prognosis of triple-negative breast cancer.


Introduction
Breast cancer (BC) is the most common malignant tumors affecting females worldwide, with an estimated 1,300,000 new cases and 465,000 deaths annually [1,2]. Triple-negative breast cancer (TNBC), which accounts for approximately 10-25% of all BC cases [3], usually show more rapid tumor growth, higher recurrence, poorer prognosis and more aggressive biological behavior compared to other breast cancer subtypes [4]. Based on the gene expression profiles, TNBC can be further classified into six different subtypes including immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL), basal-like 1, basal-like 2 and a luminal androgen receptor (LAR) subtype [5]. In addition, TNBC is characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor-2 (HER2), which are common therapeutic targets [6]. Herein, TNBC does not respond to endocrine therapy or other available targeted drugs [7,8].
Over the past several decades, the basic knowledge of TNBCs morphology, genetic and functional properties and its associated heterogeneity, invasive and metastatic phenotypes is increasingly understood by people [9,10]. Also, the therapeutic strategies including surgery, radiotherapy, chemotherapy, immunological therapy and targeted therapy are currently available for the patients with TNBC [4]. However, TNBC exhibits low survival due to its highly invasive and metastatic cap cells that are associated with higher recurrence behavior in local and distant lymph nodes and have a higher proliferation rate [11]. In addition, patients with TNBC have limited therapeutic options because they do not benefit from the traditional anti-hormonal or anti-HER2-based therapy. Therefore, new approaches for the diagnosis, treatment and prognosis of this breast cancer subtype are required.
MicroRNAs(miRNAs), a family of 21-25 nucleotide small non-protein-coding RNAs, are significantly involved in a variety of pathophysiological processes such as cell migration, invasion, proliferation and differentiation [12,13]. MicroRNAs can silence target genes efficiently and regulate a broad set of genes of interest simultaneously, which are involved in the initiation, progression and survival of TNBC [14][15][16]. Therefore, miRNAs have the potential to act as therapeutic targets and markers of TNBC [6,17]. However, only a limited number of literature reviews on the miRNAs in TNBC have been published to date [17,18]. Few literature reviews discussing the role of miRNAs in diagnosis, therapy and prognosis of TNBC have been recently published. Herein, this review aims to provide an analysis of miRNA involved in TNBC development as well as potential diagnostic, therapeutic and prognostic applications in triple-negative breast cancer.
The data given in Table 1 acquired through individual study has potential bias and limits, so expression patterns of some miRNAs associated with TNBC were retrieved from dbDEMC (a differentially expressed miRNA database in human cancer, http://www.picb.ac.cn/dbDEMC/) portal for analyses.
In this updated version of dbDEMC, a total of 209 newly released datasets were collected from Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA). The current version contains 2224 differentially expressed miRNAs out of 36 cancer types, planned by 436 experiments. As shown in Table 2, we utilized the dbDMEC database to screen a total of 122 miRNAs with up-regulation or downregulation in triple-negative breast cancer, of which 48 miRNAs were up-regulated and 74 miRNAs were down-regulated.
Some miRNAs in Table 1 have been shown to be up-regulated or down-regulated, which were coordinated with data in dbDMEC database, including miR-182, miR-210, miR-146a, miR-155, miR-10b, miR-145, miR-125b, miR-638, and the like. However, expression patterns of three miRNAs (miR-200a/b/c) between data in Table 1 and Table 2 are not matched. The three miRNAs acquired through individual study (in Table 1) that are down-regulated but up-regulated in high-throughput databases (dbDMEC, in Table 2), which may be data obtained by the individual study that is accidental and may require more experiment or other data for analysis. microRNA-based potential diagnostic and prognostic implications in TNBC microRNAs distinguish TNBC from other breast cancer subtypes Numerous reports suggested that aberrant expression miRNAs could clearly separate breast cancer specimens versus normal tissues [85]. Some studies confirmed that profiling of miRNAs can help to distinguish TNBC from other breast cancer subtypes [14,86,87]. Crippa et al. [26] demonstrated that miR-342 has negatively regulated the expression of BRCA1 in breast cancer, which may be a diagnostic marker for TNBC. A panel of 4 miRNAs(miR-155, miR-493, miR-30e and miR-27a) was found to act as a diagnostic and prognostic tool through sub-classifying TNBC into basal-like or core basal (CB) and five negative (5NP). This classification using miRNA could help in predicting the outcome of TNBC since BC subdivision tends to have a poor prognosis compared to 5NP [29]. Hu et al. assessed the expression of miR-93 by in situ hybridization in 119 cases of breast cancer. Also, the miR-93 expression level in TNBC tissues was significantly higher than that in non-triple-negative breast cancer tissues, suggesting that miR-93 may be a biomarker associated with the biological and clinical characteristics of TNBC [88]. Altogether, these findings indicate that the up-regulated miRNAs can be considered as a suitable candidate in the diagnosis of TNBC.
In addition, some miRNAs down-regulated in TNBC can also be used as biomarkers for diagnosis. The miR-199a-5p expression is significantly reduced in TNBC patients in comparison with other patients with non-TNBC breast cancer and a healthy group [42]. Also, other studies demonstrated that miR-199a-5p was associated with early stages of TNBC and miR-199a-5p may be a diagnostically valuable TNBC-specific marker in a large number of patients [43,89]. Savad et al. showed that miR-205 and miR-342 expressions were significantly down-regulated in the TNBC group compared to other BC groups in 59 patients with breast cancer. Their results suggested that miR-205and miR-342 may be used as potential biomarkers for diagnosis of TNBC [90]. Different miRNAbased diagnostic implications in TNBC are summarized in Table 3.

microRNAs act as potential prognostic markers in TNBC
Lack of highly sensitive and specific prognostic markers is a major obstacle for effective therapy against TNBC. Recently, several studies found that miRNA signature was correlated with the patients' overall survival (OS), outcome and recurrence of TNBC and acted as prognostic markers. Kleivi et al. have found that microRNA signature (miR-18b, miR-103, miR-107 and miR-652) was associated with tumor recurrence and reduced OS in TNBC patients as it was exclusively up-regulated in relapsing TNBC compared to non-relapsing TNBC, healthy subjects or ER þ patients [17]. Gasparini et al. [29] also found that upregulation of miR-493 and miR-155 was correlated with better patient outcome, whereas downregulation of miR-30e and miR-27a was correlated with a negative outcome. Liu et al. provided strong evidence that the expression levels of miR-374b-5p, miR-27b-3p, miR-126-3p, and miR-218-5p in tumor tissues were in association with disease-free survival (DFS) and OS of TNBC, which predict TNBC outcomes [91].
MiR-21, one of the most studied oncomiRs in cancer, is predominantly over-expressed in the majority of human tumors. It plays an important role in cancer formation and development, and it has been shown in multiple studies to serve as a potentially prognostic biomarker for TNBC [92][93][94]. A number of reports confirmed that other up-regulated microRNAs also have potential prognostic value for TNBC patients [38,[95][96][97][98]. High level of miR-9 expression showed significant association with poor disease-free survival and distant metastasis-free survival (DMFS) in TNBC, while a high level of miR-155 expression was associated with better DMFS, which suggests that expression levels of both miR-9 and miR-155 can serve as a candidate for prognostic biomarkers in TNBCs [38]. Shen et al. [95] confirmed that over-expression of miR-27b-3p can act as an independent predictor for distant metastasis of TNBC patients. High expression of miR-210, miR-454 and miR-34b were positively correlated with poor prognosis of TNBC patients [96][97][98]. Another study also found that miR-301a level was positively correlated with tumor size, depth of invasion, TNM stage and LNM by detecting the expression level of miR-301a in TNBC and adjacent non-cancerous tissues [99].
In addition, some down-regulated miRNAs in TNBC have a potential prognostic value for TNBC patients [100,101]. Deng et al. [101] selected five miRNAs that were differentially expressed in 125 patients with different prognosis for validation prognosis value of miRNAs and the results suggested that low expression of miR-221-3p may result in poor prognosis in patients with TNBC by regulating PARP1. The expression levels of miR-195 and miR-497 were lower in the MDA-MB-231 cell line than in cell line MCF10a. Also, miR-195/miR-497 regulate CD274 expression in TNBC, which may further influence tumor progression and may be able to predict the effect of immunotherapy on patients [102]. The miRNAs with potential prognostic implications in TNBC are listed in Table 4.  [84] microRNA-based potential therapeutic application in TNBC The therapeutic application of miRNAs involves two strategies. One strategy aims to inhibit oncomiRs by using miRNA antagonists. The other strategy, miRNA replacement, involves the reintroduction of tsmiRs mimic to restore a loss-of-function.

miRNA-based inhibition therapeutic applications
A large number of studies have demonstrated that dysregulation of specific miRNAs can promote tumor cell proliferation, differentiation, migration, invasion and lead to drug resistance (as shown in Table 1, Figure 1), which act as promising therapeutic targets for TNBC. The inactivation of oncomiRs was accomplished successfully through knockdown target oncomiRs or the administration of a synthetic anti-miRNA oligonucleotide. MicroRNA-21, well-known over-expressed oncomiRs, can promote proliferation and migration of MDA-MB-231cells. Knockdown of miR-21 significantly prevented the proliferation of TNBC cell, in vitro migration and lung metastasis [28]. Some antisense oligonucleotides (antagomiR) can block the function of endogenous micro-RNA and  miR-204 miR-498 miR-636 "miRNA Ã " is a piece of RNA on a pre-miRNA that is located just as opposed to a mature miRNA.
normalize the gene regulatory network and signaling pathways and sensitize cancerous cells to chemotherapy. For example, antagomiR-21 could restore trastuzumab sensitivity in resistant breast cancer by inducing PTEN expression [103]. However, miRNAs and antagomiR are easily degraded, some scientists deliver miRNAs into TNBC cells to improve stability through some nanomaterials. Shu et al. [103] developed efficient delivery systems that can specifically deliver antagomiR-21and improve therapeutic effect in TNBC cells and living animal models. Similarly, Devulapally et al. [104] proved that antisense miR-21-PS and miR-10b can effectively inhibit the proliferation and metastasis of triple-negative breast cancer, which also indicates that multi-target antagonism of endogenous miRNA may be an effective strategy for the treatment of metastatic cancer targeting metastasis and antiapoptosis. Zhou et al. [105] also reported that a novel calcium phosphate-polymer hybrid nanoparticle system can codeliver paclitaxel and miRi-221/222 (inhibitors for miRNA-221 and miRNA-222) to their intracellular targets, thereby inhibiting the proliferation mechanism of miR-221/222, thereby significantly improving the therapeutic efficacy of paclitaxel.

miRNA-based replacement therapeutic applications Suppress metastasis
Metastasis is a process in which tumor cell breaks away from the original site and travels through the blood or lymphatic system to the other parts of the body and form new tumors. Some miRNAs such as miR-146a-5p, miR-26a, miR-136 and miR-136 have been proved to inhibit the expression of numerous cancer-related genes that subsequently suppress metastasis in TNBC [43][44][45].

Block tumor cell proliferation, invasion and migration
The tsmiRs can block tumor cell proliferation, invasion, migration and lead to cancer cell death  and function of these down-regulated tsmiRs may be replaced by introducing synthetic miRNA mimics. Moreover, miRNA mimics cannot only provide obvious benefits to those cancer cells with low tumor suppressor miRNA expression levels but also show therapeutic benefits in cancer with normal miRNA expression levels. Therefore, miRNA mimics could be a promising treatment for TNBC [106]. For example, miR-542-3p, a potent tumor suppressor, can control cancer aggressiveness through inhibiting cell proliferation, inducing cell cycle arrest and apoptosis and suppressing tumor angiogenesis [107]. It can also directly downregulate the expression of the anti-apoptotic protein surviving. Herein, Wang et al. developed a nanocarrier system of HA-coated PEI-PLGA NPs for simultaneously delivering chemotherapeutic drug DOX and a tumor suppressor miR-542-3p mimics into TNBC cells. The results indicated that intracellular restoration of miR-542-3p promoted breast cancer cell apoptosis via activating p53 and inhibiting surviving [107]. miR-34a is a miRNA that is regulated by the p53 network at the transcriptional level and has been shown to be significantly down-regulated in TNBC. Deng et al. [108] co-wrapped miR-34a with doxorubicin (DOX) in hyaluronic acidchitosan nanoparticles and into breast cancer cells.
The data obtained indicate that intracellular recovery of miR-34a inhibits breast cancer cell migration by targeting Notch-1 signaling, and in addition, co-delivery of DOX and miR-34a can achieve a synergistic effect on tumor suppression. Goyal et al. developed LbL-NS to deliver tumor suppressor miR-34a to TNBC cells. These constructs were shown to safely and efficiently regulate the expression of SIRT1 and Bcl-2 (two known miR-34a targets) to reduce cell proliferation [109].

Regulate radiosensitivity and chemosensitivity
Radiotherapy is an effective and well-established cancer treatment. However, radiation resistance poses a major clinical challenge in cancer treatment. Experimental evidence demonstrates that some specific miRNAs play critical roles in modulating radiosensitivity of TNBC [75,110,111]. Liang et al. [112] suggested that miRNA-302 sensitized resistant triplenegative breast cancer cells to irradiation in vitro and in vivo and miRNA-302 could act as a potential sensitizer to radiotherapy. Multidrug resistance (MDR) is generally considered to be a major factor in the failure of many forms of chemotherapy. Recently, growing body of reports have suggested that several miRNAs are closely associated with MDR and they can modulate drug sensitivity in TNBC [78][79][80][81][82][83][84]. Tan et al. [79] found that miR-638 can affect DNA repair and sensitivity to UV and cisplatin. Bockhorn et al. [113] reported that increased miRNA-30c levels did not affect TNBC cell growth but sensitized the drug response of TNBC cell lines to paclitaxel and doxorubicin. They found that miRNA-30c played a pivotal role in chemoresistance via direct targeting of the actin-binding protein Twinfilin 1 which is responsible for the promotion of epithelial-to-mesenchymal transition.
An increasing body of evidence suggests that there has been a tremendous improvement in understanding the mechanisms of miRNAs  and the development of creative strategies for using miRNA in TNBC therapy [103][104][105][107][108][109]. However, there is no currently available miRNA based clinical therapy. There are a few basic reasons for the aforementioned situation. First, naked miRNA antagonists and miRNA mimics are quickly degraded and cleared in the blood circulation [107,108,114]. Second, miRNA probably cause unexpected toxicities and significant undesirable side effects [115,116]. For example, tumor-secreted miR-21and miR-29a can bind to toll-like receptor(TLR) family as agonists, leading to NF-jB signaling activation and secretion of IL-6 and TNF-a, which ultimately may lead to tumor growth and metastasis and may cause systemic immune toxicity. MiRNA let-7b can bind and activate TLR 7 signaling in neurons and cause neurodegeneration [116]. Third, there are probably many off-target effects for miRNAs therapy [117].

Conclusion and future perspectives
In summary, deregulation of miRNAs is involved in the development of triple-negative breast cancer and these changes have important roles in modulating gene expression and cancer-relevant pathways, which qualify these miRNAs as promising candidate cancer biomarkers for TNBC diagnosis, prognosis and therapy prediction. However, miRNAs have not yet been clinically utilized as disease-specific markers due to the need for an optimized detection strategy. With the development of the times and continued progress with a better understanding of functional involvement of miRNAs, more and more evidence strongly supports miRNAs involved in tumorigenesis and offer the promise of developing a novel approach in the clinical care of TNBC patients.

Acknowledgement
Financial assistance received from Innovation platform Open Foundation of education department of Hunan province is acknowledged with thanks.

Disclosure statement
The authors declare that they have no conflict of interest.