MicroRNAs in the development and neoplasia of the mammary gland

Introduction

MicroRNAs (miRs) are small, endogenous noncoding RNAs that regulate gene expression post-transcription, by degrading target mRNA or repressing translation. They are hardy in nature, being resistant to RNAse degradation, acidic pH, and repeated freezing and thawing, and are stable at room temperature¹. In addition to their involvement in many other physiological processes, they play crucial roles in mammary gland development and lactation, and their deregulation can lead to breast cancer². This review provides an overview on the recent findings concerning the role of miRs in the developmental stages of the mammary gland and breast cancer progression.

miRs in mammary glands

The study of miRs in mammary gland development is essential, as miRs in milk are derived from the mammary gland and can be a biomarker of a healthy lactating gland, as well as protecting infants and promoting their development³. The role of miRs in milk is still being debated. For example, whether they are only simple nutrients or if they have some regulatory functions in milk recipients after entering systemic circulation is still disputed⁴.

Comparative analysis of the miRNome of bovine milk, mammary tissue, milk fat, and whey revealed 188 miRs in common, with some novel miRs discovered⁵. The miR signature of milk fat was similar to that of mammary gland tissue miRs. Functional studies of highly expressed miRs in those fractions indicated their role as regulators of mammary gland functions contributing to healthy milk production⁵.

miRs controlling lactation

It was observed that miR-126 plays a role in mammary epithelial cells (MECs) by modulating lipid synthesis⁶. FASN (fatty acid synthase) gene expression is increased when miR-126-3p is inhibited, suggesting its involvement in lipid metabolism in the mammary gland. In addition, estradiol and progesterone enhanced lipid synthesis by downregulating the levels of miR-126-3p⁶. Similarly, miR-150 hampers lipogenesis in MECs and reduces secretory activity⁷, while miR-145 facilitates milk fat synthesis in lactating goats by targeting the gene INSIG1 (insulin induced gene 1)⁸.

The role that miRs play as regulators of lipogenesis was confirmed by overexpression studies, which showed that there was more synthesis of fat droplets, accumulating triacylglycerols, and a higher proportion of unsaturated fatty acids in lactating MECs. For instance, miR-24 was found to be expressed at a much higher level during peak lactation in goats and affects triacylglycerol content, unsaturated fatty acid concentration, and expression of target genes, such as FASN, SREBF1 (sterol regulatory element binding transcription factor1), SCD (stearoyl-CoA desaturase), GPAM (glycerol-3-phosphate acyltransferase; mitochondrial), and ACACA (acetyl-CoA carboxylase)⁹.

miR expression in MECs is regulated by lactogenic hormones (dexamethasone, insulin, and prolactin), as evidenced by an increased miR-148a level in bovine MECs, which is probably associated with increased milk production during lactation in cows¹⁰. The miR-29 family affects lactation by regulating the DNA methylation of target genes DNMT3A and DNMT3B (DNA (cytosine-5)-methyltransferase 3A and 3B) in MECs of dairy cows¹¹. Moreover, inhibition of the miR-29 family resulted in hypermethylation of promoters of lactation–related genes, leading to decreased secretion of triglycerides, proteins, and lactose by the epithelial cells. miR-486 facilitates lactation by downregulating PTEN (phosphatase and tensin homolog) target gene. Downregulation of this gene affects the expression of downstream genes, such as AKT, mTOR and β-casein, which have crucial roles in mammary gland development and lactation¹². Additionally, diet restriction has been shown to affect the miRNome of lactating mammary glands, indicating the role of miRs in regulating milk composition¹³.

miRs controlling involution and breast cancer

Identification and characterization of miRs involved in breast cancer will facilitate targeting miRs for possible therapy. Improper involution possibly contributes towards tumour development¹⁴. miR-424(322)/503 regulates mammary involution in humans by targeting BCL-2 (B-cell lymphoma 2; anti-apoptotic) and IGF1R (insulin like growth factor-1-receptor) genes. The loss of this miR leads to malignancy and nonresponse to chemotherapy, demonstrating its role as a tumour suppressor¹⁴. By contrast, some miRs, such as miR-660-5p, promote tumour development and metastasis, and the level of miR-660-5p was found to be increased in MCF7 breast cancer cell lines¹⁵. This miR’s tumour promoting activity was confirmed by observation of reduced invasion of MCF7 cells at reduced miR-660-5p levels.

One of the major causes of breast cancer is disturbed estrogen signaling, where the altered expression of estrogen receptor α (ERα) and its cross-talk with the related miR culminates in neoplasia¹⁶. Studies on the effect of E2 on the expression pattern of miRs in MCF7 and ZR75 cell lines revealed 172 miRs that were up or downregulated. Notable miRs are miR-206, miR-34a, miR-17-5p, and miR-125 a/b, which act as tumour suppressors, and miR-21, miR-10B, and miR-155, which act as oncogenes¹⁷. Another study using an ACI rat model for the effect of E2 on the miR signature showed 33 dysregulated miRs¹⁸. Additionally, the use of ellagic acid (natural phenol antioxidant) reversed the dysregulation of miR-206, miR-182, miR-375, miR-127, miR-183, and miR-122, subsequently modulating the target proteins ERα, RASD1, cyclin D1, FoxO1, FoxO3a, Bcl-w, Bcl-2 and cyclin G118¹⁸. Furthermore, overexpression of the tumour suppressing miR-133a in MCF-7 and MDA-MB-231 cells suppressed phosphorylated Akt (p-Akt) protein and inhibited p-Akt nuclear translocation, and this miR also regulates the cell cycle of cancerous cells by targeting the EGFR (epidermal growth factor receptor) gene¹⁹.

miR-206 is found to play a crucial role in BRCA1 (Breast CAncer susceptibility gene; tumour suppressor) depleted mouse mammary gland²⁰. Overexpression of miR-206 showed no effect on lactation, but did have a role in tissue remodeling through increasing fat tissue and reducing branching morphogenesis. There may be a possibility of increased miR-206 levels due to BRCA1 loss, culminating in mammary gland remodeling and tumour development²⁰. miR-184 is found to be a tumour suppressor by regulating the number of genes in the PI3K/AKT/mTOR pathway, as observed by miR profiling of the pubertal mouse mammary gland²¹. This pathway is important in mammary gland development and lactation²². miR-184 is only expressed in epithelial cells, and the level increases during differentiation of cells from terminal end bud into ductal epithelial cells²¹.

Conclusions

miRs have been shown to be one of the major regulators of mammary gland development and neoplasia. miRNome studies of mammary gland in different developmental stages and cancerous tissues will elucidate biomarkers for early cancer diagnosis, and may be used as therapeutic targets. Future studies focusing on the cross-talk between miRs and target genes with the signaling pathways involved in development and neoplasia of mammary gland will open the door to understand mammary gland biology and oncogenesis in molecular detail.