In TNBC several miRNAs were observed with deregulated expression presenting with major roles in cancer progression [43, 44]. Most of the studies reporting on the miRNA expression in TNBC do not focus on minority groups or biological disparities based on the race of the patients. In fact, a search on PubMed database using the words “African American” OR “African women” OR “biological disparity” AND microRNA OR microRNAs OR miRNA OR miRNAs resulted in 83 studies related to AA women and microRNAs with only six of them with TNBC patients, which included the previous study of our group .
In this study, we report on the global miRNA profiling of genomically ancestral characterized AA patients with TNBC stratified in three clinical groups of patients based on tumor size, LN metastasis and breast cancer recurrence status.
Tumor size is one of the most important prognostic determinants for breast cancer . Larger tumors can be a result of late diagnosis, high proliferation rate, lack of treatment, or poor response to neoadjuvant treatment. In this study, the average tumor size in the non-treated TNBC patients was 5.86 ± 5.17cm; six of these patients were diagnosed with tumors larger than 5cm, being categorized as at least Stage IIB (no regional LN metastasis and no clinical or radiological evidence of distant metastasis), which as expected, presents lower 5-year survival rates when compared to patients with smaller tumors . Eight miRNAs were found differentially expressed between the groups of patients based on tumor size, among them, the miR-2117, miR-378c, miR519c-3p and miR-934 presented high power (AUC ≥ 0.8) to discriminate the patients in this group. Among these miRNAs, the expression of miR-2117 was observed in colorectal cancer inversely correlated with the expression levels of the target gene TGFBR1 . This gene is found overexpressed in breast cancer  and is involved in the MAPK-Signaling pathway . Downregulation of miR-378c was observed in head and neck squamous cell carcinoma (HNSCC) and appeared as one of the most important prognostic variables for HNSCC . However, in our study we observed lower levels of miR-519c-3p from patients diagnosed with larger tumors (> 5cm). Increased levels of miR-519c-3p were found to promote tumor growth and proliferation in hepatocellular carcinoma cells, by targeting the BTG3 gene . Increased expression of miR-934 was previously reported on TNBC samples compared to ER + tumors, however, its expression level was not associated with tumor size .
Lymph node (LN) status is still one of the strongest prognostic factors in breast cancer . In this group, 23 miRNAs were found differentially expressed between the LN + and LN- patients, among them, miR-1253, miR548l and mIR-873-5p, indicating that these miRNAs might be involved in conferring the invasion ability to the tumor cells. A combination of seven of these miRNAs (let-7f-5p, miR-1255b-5p, miR-1268b, miR-200c-3p and miR-520d-5p + miR-527 + miR-518a-5p), presented a more robust discriminatory power (AUC > 0.9). Interestingly, altered expression levels of miR-1253 and miR-548l were reported to interfere in the migration and invasion capacity of non-small cell lung cancer (NSCLC). Altered expression of miR-548l and miR-1253 were negatively associated with LN metastasis in NSCLC, by targeting the AKT1 and WNT5A genes, respectively [53, 54]. In our study, both of these miRNAs were observed with lower expression levels in the LN + group, indicating as in NSCLC, a similar tumor suppressor activity in breast cancer. The long non-coding RNA DCST1-AS1 was reported to act as a “sponge” by binding miR-873-5p, resulting in the upregulation of other target genes such as IGF2BP1, MYC, LEF1, and CDD4 and conferring the increase of cell proliferation and metastasis capacity in TNBC cells . The lower expression levels of this miRNA in the LN + group of our study could be among other mechanisms, a consequence of high levels of this lncRNA.
In relation to recurrence (REC), 27 miRNAs were observed differentially expressed between REC + and REC- groups, with 12 presenting an AUC value higher than 0.8; a combined analysis of three of these miRNAs (miR-1200, miR-1249-3p, and miR-1271-3p), presented a robust power (AUC > 0.9) in discriminating the patients based on REC status. Deregulated expression levels of miR-1249-3p in breast cancer cells were associated with interference of lncRNA MIF-AS1 . This lncRNA acts as a sponge resulting in lower expression levels of miR-1249-3p which impedes its interaction with the HOXB8 target gene. In vitro upregulation of miR-1249-3p resulted in suppression of proliferation and migration activity and reversion of the Epithelial Mesenchymal Transition (EMT) progress, indicating a tumor suppressor role of this miRNA in breast tumors, and corroborating with our findings that showed lower expression levels of miR-1249-3p in the REC + group. In gliomas and osteosarcomas, it was observed that the downregulation of miR-1200, led to the up-regulation of the HOXB2 gene, which resulted in the increase of proliferation and invasion capacity of the tumor cells [57, 58]. These data indicate a tumor suppressor activity of miR-1200, that as miR-1249-3p, presented lower expression levels in the REC + group. Finally, for miR-1271, to our knowledge, there is no reported deregulation of its expression in tumor cells.
TNBC patients can show satisfactory response to chemotherapy, especially in the neo-adjuvant setting [59–61]. However early recurrence is more frequent in this breast cancer subytpe when compared to others, which usually occurs within the first 3 years after diagnosis [5, 62]. Chemotherapy adherence and uptake have also been shown to differ among patients' racial/ethnic groups [63–66]. In TNBC, a previous study conducted by our group , showed however that although a substantial number of TNBC patients failed to receive and/or complete chemotherapy, AA patients presented a higher chemotherapy uptake than White patients. Interestingly, and aware of the limitation of the sample size of this present study, seven patients presented recurrence after treatment while fifteen patients were disease-free. Considering both LN and REC status, 22 miRNAs were found differentially expressed among the LN+/REC+, LN+/REC-, LN-/REC+, and LN-/REC- groups, eight of them also presented in the comparison of LN and REC clinical groups comparisons: miR-10a-5p, miR-1253, miR-1271-3p, miR-184, miR-18a-5p, miR-411-5p, miR-542-3p, and again miR-595. Finally, a query of the KM Plot database of the TCGA and METABRIC TNBC cases, showed that several of the miRNAs observed with deregulated expression in the patients with larger tumor size, positive LN and REC were significantly associated with lower survival rates.
Interestingly, the involvement of 12 of the miRNAs observed in the TNBC cases of our study and that were differentially expressed in the above clinical groups was among the ones observed with differential expression in our previous study in TNBC cases of AA and non-Hispanic White (NHW) patients . Among these common miRNAs, 12 of them presented the same level of expression of this study, two of which associated with tumor size (miR-2117 and miR-617), eight with LN status (miR-1253, miR-1268a, miR-200c-3p, miR-520d-5p, miR-518a-5p, miR-528, miR-580 and miR-873-5p), and two with REC status (miR-1200 and miR-449b-5p). These associations could indicate that these 12 miRNAs are intrinsically regulated in the TNBC of AA patients, and could account for the observed more aggressive phenotype of their tumors when compared to the NHW patients. This suggestion can be supported by the analysis of the KEGG pathway of the differentially expressed miRNAs observed in each clinical group of this study, which revealed their involvement in signaling pathways often associated with tumor aggressiveness. In the LN group, for example, considering only the panel of seven miRNAs that presented the best capability to discriminate LN + and LN- cases, among the top ten KEGG pathways observed, three were affected by six of these miRNAs: signaling pathways regulating pluripotency of stem cells, TGF-beta and Hippo signaling pathways. In the REC group, among the top ten KEGG pathways, the thyroid hormone signaling pathway was potentially affected by eight of the 12 miRNA in the highest discriminatory panel (miR-1200, miR-1271-3p, miR-449b-5p, miR-4536-3p, miR-542-3p, miR-548n, miR-593-3p and miR-595). Considering these miRNAs in the above clinical groups, miRNA/mRNA pairings that were experimentally validated showed a number of targets that play relevant roles in breast cancer progression, including genes that are part of the subclassification of the TNBC into the six molecular subtypes  among them, genes involved in the epithelial-to-mesenchymal transition (EMT) process, which is essential to confer cell migration and invasion and stem cell tumor capabilities [69, 70], and which miRNAs are important regulators [71, 72]. Of note, one of the most prevalent TNBC subtypes in AA patients, the mesenchymal-like (ML) subtype [73–75], is enriched in genes involved in the regulation of EMT and in the biology of cancer stem cells, both markers that confer clinical aggressiveness [76, 77]. In addition, some of the differentially expressed miRNAs observed in our study are highly involved in breast cancer progression as shown by their predicted interactions with relevant genes of the Integrated Breast Cancer Pathway. Some of these interactions were already experimentally validated, including miR-184 and AKT1K, BCL2 and MYC, and miR-200c, miR-130a-3p, miR-18a-5p, miR-519a-3p and PTEN. These interactions indicate an important role for these miRNAs in the tumorigenesis of TNBC in AA patients, which should be further explored in other independent well-characterized and large AA populations.