Altered Expression of miR-146a-5p and Bcl-2 in Oncocytic Carcinoma of the Breast: A Case Report

Background: Oncocytic carcinoma of the breast is extremely rare, and its molecular prole is poorly understood. The distinctive feature of oncocytic carcinoma of the breast is that the granular eosinophilic cytoplasm contains numerous mitochondria. Recently, microRNA-146a-5p has been identied as a contributor to carcinogenesis and as a mitochondria-related microRNA, which regulates the mitochondrial function affecting Bcl-2. Bcl-2 plays a role in mitochondrial apoptosis. Case presentation: We report the clinical features, histopathological features, and immunohistochemical and molecular ndings of oncocytic carcinoma of the breast in a 76-year-old woman. Immunohistochemistry studies revealed that the tumor cells were positive for antimitochondrial antibody but negative for gross cystic disease uid protein 15, which conrmed the diagnosis. For molecular proling, expression of microRNA-146a-5p and Bcl-2 messenger RNA (mRNA), which are mitochondria-related small molecules, were evaluated using real-time reverse transcription polymerase chain reaction. We found that the expression of microRNA-146a-5p was signicantly lower (p < 0.01) and that of Bcl-2 mRNA was signicantly higher (p < 0.01) compared to the control group (with no specic type of breast cancer). Conclusions: The signicant changes in the expression of microRNA-146a-5p and Bcl-2 are specic to oncocytic carcinoma of the breast. Therefore, we suggest that the use of microRNA-146a-5p to target Bcl-2 has a potential therapeutic effect on oncocytic carcinoma of the breast.


Background
Oncocytic carcinomas (OC) are rare malignant tumors composed of oncocytes, which are epithelial cells characterized by a granular eosinophilic cytoplasm containing numerous mitochondria [1]. OC of the breast is uncommon, accounting for less than 1% of all breast cancers [2]. Hence, little is known about its pathologic molecular features.
MicroRNAs (miRNAs) are small, non-coding RNAs that play an important role in post-transcriptional gene regulation [3,4]. They interact with their target messenger RNAs (mRNAs) to control translation through mRNA degradation or translational repression [5,6]. Research has revealed that miRNAs are involved in various biological processes, including cancer [7,8]. The miRNA expression pro les differ between diseases, suggesting that miRNAs have an important role in the diagnosis and treatment of cancers [9][10][11].
One of the miRNAs that has recently received much attention is miR-146a-5p because of its function as a tumor suppressor and its deregulation observed in various cancers [12]. It has also been identi ed as a mitochondrial miRNA (mito-miR) that targets the mitochondria and regulates mitochondrial protein expression and function [13,14]. A previous study suggested that miR-146a-5p affects mitochondrial function by targeting Bcl-2, which plays an apoptogenic role in mitochondria [15].
In view of the tumor-speci c expression of miRNAs and characteristic features of OC, we hypothesized that the expression of mitochondria-related miRNAs could be altered in OC. To test our hypothesis, we compared the expression of miR-146a-5p in tissues from specimens of OC with that in tissues from specimens of a more typical type of invasive ductal carcinoma (IDC) of the breast. We also assessed the mRNA and expression levels of Bcl-2, which is the target of miR-146a-5p. In this report, we describe a case of OC of the breast that exhibited alterations in the expression of miR-146a-5p and Bcl-2, in comparison with IDC. To the best of our knowledge, these alterations have not been previously reported. For molecular pro ling, we collected fresh tissue samples from the surgical specimens and stored them at 80°C until use.For comparison, we also collected fresh tissue samples of IDC from a 72-year-old patient, who had the same phenotype (positive estrogen and progesterone receptors and negative HER2).Reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate the expression levels of miR-146a-5p and Bcl-2 mRNA. miRNA was extracted using an miRNeasy Mini Kit (QIAGEN, Crawley, West Sussex, UK), in accordance with the manufacturer's protocol. RT-PCR was performed as previously described [16].U6 small nuclear RNA and glyceraldehyde 3-phosphate dehydrogenase were Page 4/10 used for normalization in RT-PCR with miRNA and mRNA, respectively. All experiments were conducted in triplicate. For RT-PCR, the results were calculated as the mean±standard deviation of triplicate data, and the expression level was illustrated as a fold difference of 1 for IDC. We used JMP® 12 software (SAS Institute Inc., Cary, NC, USA) to perform statistical analysis and compared the data of the two groups using the t-test. The signi cance threshold was set at P<0.05. In addition, we determined the Bcl-2 protein level by immunohistochemical staining using formalin-xed, para n-embedded samples.

Case Presentation
Interestingly, the expression level of miR-146a-5p was 0.18 times less in OC than in IDC (Fig. 3A), which was a signi cant difference. In addition, mRNA expression of Bcl-2 was 2.26-fold higher in OC than in IDC, which was also signi cant and could further explain the data from immunohistochemical staining, which showed a higher intensity of Bcl-2 protein (Figs. 3B and C).

Discussion And Conclusions
OC is an extremely rare type of breast cancer, characterized by mitochondrial accumulation. In our patient's tumor, we demonstrated signi cant changes in the expression of miR-146a-5p and Bcl-2 in OC compared to that of IDC, which suggests that miR-146a-5p, by targeting Bcl-2, has a potential therapeutic effect on OC.
The World Health Organization classi cation de nes OC of the breast as a tumor in which more than 70% of all cells have oncocytic features [2]. The distinctive feature of oncocytes is the enlargement of cells containing abundant eosinophilic granules in their cytoplasm due to an increase in altered mitochondria [17]. Morphologically, OC resembles apocrine carcinoma, which also has an abundant eosinophilic granular cytoplasm [18]. On hematoxylin and eosin staining, it is di cult to distinguish OC from apocrine carcinoma based on morphological features. Thus, accurate diagnosis of OC necessitates immunohistochemical staining for several markers, including mitochondrial markers. In addition, GCDFP-15, a marker for apocrine differentiation, must be absent for the diagnosis of OC. In our patient, the morphologic features and use of immunostaining for antimitochondrial markers and GCDFP-15 were consistent with OC.
The mechanism of oncocytic tumor formation remains to be fully elucidated. Mitochondrial DNA mutations or mitochondrial genome alterations have been found in OC, which lead to alterations in mitochondrial metabolism and tumorigenesis [19,20]. However, these observations have not been investigated in OC of the breast, and the unique miRNA expression in OC of the breast is poorly described because of its rarity. In other organs in which OC has been reported, such as the thyroid, kidneys, and salivary glands, miR-146a-5p expression has not been found. In relation to miR-146a and oncocytes, Fridman et al. demonstrated a speci c downregulation of miR-146a in renal oncocytoma in comparison with other histological types of renal tumors [21]. Other investigators have detected miRNAs in multiple subcellular compartments, including the mitochondria [22][23][24]. Regarding the accumulation of mitochondria as the hallmark of OC, we hypothesized that the expression of mito-miR would be altered in OC, in contrast to IDC. Indeed, our data showed that the expression of miR-146a-5p, a mito-miR, was signi cantly decreased in OC compared to that in IDC. We found no evidence that the altered expression of miR-146a-5p is associated with carcinogenesis of oncocytes; however, our results raise the possibility of a connection between miR-146a-5p dysregulation and mitochondrial abnormality in OC of the breast.
In breast cancer, miR-146a-5p has been shown to be upregulated, promoting proliferation and invasion and inducing epithelial-mesenchymal transition [25,26]. Furthermore, upregulation of miR-146a-5p plays an important role in regulating Bcl-2 proteins via the mitochondrial pathway of apoptosis in aging-related diseases, including cancer [15]. Bcl-2 proteins exist in the outer membrane of mitochondria and their overexpression in breast cancer has been shown to increase invasion and migration [27,28]. The higher expression of Bcl-2 and lower expression of miR-146a-5p in OC tissues compared to IDC tissues in our study indicate that miR-146a-5p may contribute to altered expression of Bcl-2 through mitochondria.
Our study has several limitations. First, we investigated only one sample of OC; we could not obtain multiple OC samples because of its rarity. We acknowledge that our results would be insu cient to generalize to all cases of OC; however, providing descriptions of molecular pro ling in a fresh sample is important and bene cial for patients with very rare tumors, from both clinical and translational standpoints [29]. In addition, although investigations of both tumors and noncancerous samples would be more desirable, we could not obtain noncancerous fresh samples for experimentation. Finally, we could not identify the mechanisms underlying the association between miR-146a-5p and Bcl-2 in OC in this study. Further studies are needed to con rm these ndings.
OC is a rare type of breast cancer. To the best of our knowledge, signi cant changes in the expression of miR-146a-5p and Bcl-2 in OC, in comparison with IDC, have not been previously reported. Therefore, our ndings could help improve the understanding of this rare tumor.