MicroRNAs in the prognosis of triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous group of tumors characterized by their aggressive nature and poor associated survival. MicroRNAs (miRs) have been found to play an important role in the occurrence and development of human cancers, but their role in the prognosis of TNBC patients remains unclear. We performed a meta-analysis to explore the prognostic value of miRs in TNBC. Methods: We systematically searched the PubMed, Embase, and Web of Science databases to identify eligible studies. A meta-analysis was performed to estimate the pooled hazard ratios (HRs) and their corresponding 95% confidence intervals (CIs) for the associations between levels of miR expression (predictive factors) and overall survival (OS) and disease-free survival (DFS) (outcomes) in patients with TNBC. Results: After performing the literature search and review, 21 relevant studies including 2510 subjects were identified. Six miRs (miR-155, miR-21, miR-27a/b, miR-374a/b, miR-210, and miR-454) were assessed in the meta-analysis. Decreased expression of miR-155 was associated with reduced OS (adjusted HR = 0.58, 95% CI: 0.34–0.99; crude HR = 0.67, 95% CI: 0.58–0.79). High miR-21 expression was also predictive of reduced OS (crude HR = 2.50, 95% CI: 1.56–4.01). We found that elevated levels of miR-27a/b, miR-210, and miR-454 expression were associated with shorter OS, while the levels of miR-454 and miR-374a/b expression were associated with DFS. Conclusions: Specific miRs could serve as potential prognostic biomarkers in TNBC. Due to the limited research available, the clinical application of these findings has yet to be verified.


Introduction
Breast cancer is the most commonly diagnosed malignancy and the leading cause of cancer-related mortality among women worldwide, with an estimated 1.7 million new cases and 521,900 deaths in 2012. [1] Of the breast cancer cases, approximately 10% to 20% have been reported to be triple-negative breast cancer (TNBC). [2] TNBC is a heterogeneous group of tumors characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2/ Neu (HER2), and this malignancy has been found to be often, but not always, a basal-like breast cancer. [3] Because it cannot be treated with current endocrine therapies and exhibits an aggressive nature, TNBC has been regarded as being associated with one of the worst prognoses of all breast cancer subtypes. [2,4] Advances in in-depth research on genetic biomarkers, such as miRs, in TNBC have promoted the utility of biomarkers in the diagnosis, treatment, and prognosis of the disease. MiRs are a class of small noncoding RNA molecules that are 19 to 25 nucleotides in length, can modulate gene expression, and are easily accessible and quantifiable. [5] A growing body of evidence indicates that aberrant expression of miRs may be linked with the development and progression of human cancers, [6] including renal cell carcinoma, [7] pancreatic ductal adenocarcinoma, [8] and Editor: Giovanni Tarantino.
LL and XM contributed equally to this work.
The present study was supported by the National Natural Science Foundation of China (81673249), Social Development Project in Jiangsu Province (BE2015694), Scientific Research Innovation Project for Graduate Student in Jiangsu Province (KYLX16_1125), and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAAD). The funding agencies had no role in the study design, data collection, data analysis, decision to publish, or preparation of the manuscript.
The authors have no conflicts of interest to disclose. brain tumors. [9] However, until now, no systematic review has been performed to explore the role of particular miRs in the survival of patients with TNBC.
In this study, we systematically reviewed relevant studies on the prognostic value of miRs in TNBC and pooled the effect estimates reported in these studies to provide a better understanding of associations between specific miRs and prognosis in TNBC and provide a rationale for miR-based therapeutics.

Search strategy
We followed the guidelines of the Meta-analysis of Observational Studies in Epidemiology (MOOSE) group and Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement. [10] We searched the PubMed, Embase, and Web of Science databases to identify relevant studies that assessed the utility of candidate miRs as prognostic factors in TNBC. The databases were searched to identify articles published from January 1990 to December 2016 using the following search strategy: (microRNA OR miRNA OR miR) AND (triple-negative breast cancer) AND (prognosis OR prognostic OR survival OR outcome OR mortality). The searches were limited to articles published in English. Two investigators (LL and XM) reviewed the titles and abstracts of studies identified in the initial search to determine the relevance of these publications. Then, the full texts of the remaining articles were obtained and carefully reviewed. We also manually screened the reference lists of retrieved articles to identify other potentially relevant studies.

Eligibility criteria
Articles were considered eligible if they met all the following initial inclusion criteria: focused on patients undergoing treatment for TNBC; measured miR expression levels in tumor or blood samples; clearly defined the utilized miR cut-off points; clearly described the utilized miR detection methods; analyzed the correlations between survival outcomes and miR expression; and clearly described the follow-up duration. Articles were excluded if they were case reports, letters, commentaries, conference records or reviews; had a sample size less than 30 cases; calculated HRs based on a combination of multiple miRs; lacked sufficient data for estimating HRs and 95% CIs; or used survival data that originated from the TCGA, PROGmiR, METABRIC, or BreastMark dataset. Data were extracted from articles fulfilling all the aforementioned selection criteria. Two individual investigators (LL and XM) independently assessed the eligibility of the retrieved articles. Discrepancies were resolved by consensus or consultation with a third investigator (PS).

Quality assessment
The quality of the included studies was assessed according to the following checklist, which was developed based on the criteria proposed by the MOOSE group: [10] clearly defined study design; clearly described study population (country); sufficiently large sample size (N > 30); clearly described the outcomes (OS or DFS); clearly defined the method of miR measurement (quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridization (ISH), etc.); clear defined the utilized cut-off values; measured miR expression in tumor or blood samples; and had a sufficiently long follow-up duration (>60 months). To assure the quality of this meta-analysis, studies were excluded if they did not meet these criteria.

Data extraction
Data were extracted independently by 2 investigators (LL and XM), who used a predefined sheet to retrieve information from all studies qualifying for final inclusion. The data sheet was designed based on previous studies focusing on similar topics and the PRISMA guidelines. [11] The following data were extracted: title; first author; publication year; study design; study population; participant number; sample types; miRs; miR expression assessment methods; cut-off values; follow-up duration; and HRs for OS or DFS and their corresponding 95% CIs and P values. If HRs (95% CIs) and P values could not be extracted from the original article, we estimated these values using the available data or the Kaplan-Meier curves presented in the articles using the methods described by Parmar et al [12] and Tierney et al. [13] 2.5. Statistical analysis OS was defined as the interval from the date of primary surgery to the date of all-cause mortality. DFS was defined as the interval from the date of primary surgery to the date of disease relapse or all-cause mortality. [14] We pooled the HRs (95% CIs) extracted from the studies using the Stata 13.0 software (StatCorp, College Station, TX). Heterogeneity was assessed using the Cochran Q test and Higgins I-squared statistic. P values less than .1 for the Q test and I 2 value >40% indicated the presence of significant heterogeneity across studies. The fixed-effects model was applied in the absence of between-study heterogeneity, while the randomeffects model was applied when heterogeneity was observed. An observed HR > 1 indicated worse prognosis in the group with elevated miR expression. Conversely, an observed HR < 1 indicated worse prognosis in the group with a decreased miR expression. [15] Egger test was used to assess publication bias.

Ethical consideration
Ethical approval was not required for this study.

Selection of studies
A flow diagram of the study selection process is shown in Fig. 1. A total of 370 publications were identified in the initial search. After reviewing the titles and abstracts of these articles, we identified 51 articles evaluating the use of prognostic miR biomarkers in TNBC. We then carefully reviewed the full texts of these articles and excluded an additional 32 articles. Two articles described independent cohorts that were analyzed separately. [16,17] Thus, we regarded these 2 articles as 4 studies. In total, 19 articles (21 studies) were eligible for inclusion in this meta-analysis.

The miR-374a/b and prognosis of TNBC
Two studies evaluated the association between miR-374a/b expression and the prognosis of TNBC patients (n = 589), of which 1 reported data on DFS [19] and 1 reported data on both OS and DFS. [21] All of these studies provided adjusted HR data for DFS, and no significant heterogeneity was observed (I 2 = 48.3%, P = .164). The fixed-effects model revealed that downregulation of miR-374 was associated with shorter DFS (combined adjusted HR: 0.77; 95% CI: 0.65-0.90) (Fig. 5).

Discussion
We conducted a comprehensive systematic literature review to explore the utility of miR biomarkers that can be easily and Table 2 Descriptive characteristics and related data from included studies.

OS DFS
Author-year MiRNAs HR (95% CI) P HR (95% CI) P robustly evaluated in predicting prognosis in TNBC. To our knowledge, this is the first extensive meta-analysis to describe the role of miRs in TNBC prognosis. Although various miRs were found to be associated with prognosis in TNBC, most of these miRs were assessed in only a single study. Six miRs (miR-155, miR-21, miR-27a/b, miR-374a/ b, miR-210, and miR-454) were evaluated in at least 2 studies. We, therefore, performed a meta-analysis of the effect of these 6 miRs on the survival of TNBC patients. The results of this study showed that lower expression of miR-155 predicted worse OS in TNBC patients, while elevated levels of miR-21, miR-27a/b, miR-210, and miR-454 expression were associated with shorter overall survival times. Similarly, lower expression of miR-374a/b and higher expression of miR-454 were associated with shorter DFS.
The miR-155 locus is located within a region known as B-cell integration cluster, [34] and miR-155 is overexpressed in various solid tumors, including breast, lung, colon, pancreatic and thyroid cancers. [35,36] Some studies have reported the pro-oncogenic  properties of miR-155 in lung cancer [36] and T-cell leukemia. [37] However, we identified this miR to exhibit opposite behavior, finding that overexpression of miR-155 tended to have a protective effect on survival in TNBC patients. There are a number of molecular mechanisms that could explain this relationship. In TNBC, miR-155 may play a crucial role in DNA damage pathways. [15] miR-155 may regulate DNA repair activity and sensitivity to ionizing radiation by repressing RAD51 recombinase (RAD51), [27] while RAD51 has been identified as a central protein in homologous recombination. MiR-21 is one of the most extensively studied cancer-related miRs and might play an ever-expanding role in most cancers. [38] miR-21 may serve as a key regulator of oncogenic processes, including tumor growth, migration, and invasion. [39] Elevated miR-21 expression levels have been found to be associated with poor outcomes in cancer patients. [40] miR-21 may target the proapoptotic phosphatase and tensin homolog (PTEN) and promote tumor cell proliferation, which, in turn, may inhibit the apoptosis of tumor cells in TNBC cell lines in vitro. [22] miR-27a/b has been linked to the peroxisome proliferatoractivated receptor (PPAR) and PTEN signaling in TNBC cells, acting as a tumor suppressor by regulating the cell division cycle (CDC27) gene. [19] CDC27 has been identified as a core component of the anaphase-promoting complex (APC) and found to be involved in regulating mitotic checkpoints to ensure chromosomal integrity. [41] The results of a pathway analysis showed that miR-374b may regulate critical pathways involved in TNBC tumor development and progression, including the    fibroblast growth factor and transforming growth factor pathways. [19] miR-210, a known hypoxia-regulated miR, has been found to be upregulated in many cancers. This miR may serve as a key player in cell response to hypoxia and has been linked to a number of hypoxia-dependent diseases involved in mitochondrial metabolism, angiogenesis, DNA repair, and cell survival. [42] miR-454 has dual functionality, acting as either an oncogenic miR or a tumor suppressor. Previous studies have reported this miR to be downregulated in esophageal cancer [43] and upregulated in colorectal cancer and breast cancer. [44,45] miR-454 has been reported to function as an oncogenic miR by targeting PTEN. Patients with TNBC tumors that lose PTEN expression have poorer survival, as PTEN negatively regulates the PI3K-AKT signaling pathway. [46,47] Further studies are needed to understand the molecular mechanism underlying the effect of miRs in TNBC. Some limitations must be considered when interpreting the results of the current study. First, the number of studies available was limited. More studies are needed to further assess these associations in the future. Second, marked heterogeneity was observed in some of the analyses, findings that were likely identified due to differences in patient characteristics (ethnicity, nationality, gender, age, tumor stage, and grade) and the use of different assay methods, cut-off values for miR expression levels, sample preparation methods (i.e., paraffin-fixed, formalin-fixed, freshly frozen tumors or serum), follow-up durations, and HR extraction methods. Third, circulating biomarkers are more valuable than tissue biomarkers because they can be assayed before surgery and monitored throughout the lifespan. More studies should be conducted in the future to evaluate the prognostic value of specific miRs in serum in TNBC.

Conclusions
Specific miRs may serve as potential prognostic biomarkers in TNBC. Due to the limited research available, the clinical application of these findings has yet to be verified.