Investigating the Role of Mir-4725 and Its Novel Snp in Triple Negative Breast Cancer

Abstract

Breast cancer is the primary cause of female cancer-related deaths across the world. One specific subtype of breast cancer, known as triple-negative breast cancer (TNBC), responsible for ~45% of breast cancer frequency in West Africa and is distinguished by the null expression of estrogen, progesterone, and human epidermal growth factor 2 receptors. It is the most aggressive subtype and has a poor prognosis, especially in women of African ancestry. TNBC is a complex and heterogeneous disease with diverse molecular subtypes and its incidence is rapidly increasing in Africa. Genetic and epigenetic factors, including microRNAs (miRs), play a significant role in TNBC pathogenesis. miRs are short non-coding RNA sequences that are involved post-transcriptional regulation and their dysregulation is a key epigenetic factor in TNBC. Mutations (such as SNPs) in miRNA genes impact their processing and expression, and have been linked to TNBC pathophysiology. A novel SNP, rs73991220 was discovered in miR-4725, and has been associated with the risk of estrogen receptor-negative breast cancer in women of African ancestry. In a preliminary study of a Ghanaian breast cancer cohort, the SNP rs73991220 was identified in two out of six TNBC tumour samples. However, the function of miR-4725 and the impact of the SNP rs73991220 on miR-4725 expression have not been established in breast cancer. This study sought to identify the role of miR-4725 in TNBC and how the SNP affects miR-4725 expression. Twenty-four breast tumour samples and their matched normal adjacent tissues were screened for SNP rs73991220 (G) using PCR-RFLP. Two of the twenty-four (8%) tumour samples were found to have the heterozygous AG genotype and were both TNBC subtypes. The effect of the SNP on the stability of the secondary structure of the primary miR-4725 transcript was determined using the RNAfold online tool. Subsequently, the expression of miR-4725 was determined in the 2 AG and 2 AA TNBC tumour samples. It was observed that the SNP (G) conferred more stability to pri-miR-4725 compared to A; there was however, no significant difference in expression between the AA and AG genotypes of the TNBC tumour tissues. Furthermore, the putative targets of the mature miR (miR-4725-5p) were determined using online bioinformatics tools, and the target validation was performed by overexpressing miR 4725-5p in MDA-MB-468, a basal-like 1 subtype of the TNBC cell line obtained from an African-American woman. KIF2C was found to be significantly repressed with exogenous expression of miR-4725 and hence predicted to be the most likely target of miR-4725-5p in the MDA-MB-468 cell line out of five validated putative targets. Additionally, other tumour promoting markers (WISP1, SNAIL, MYC, and VEGFA) were upregulated following miR 4725-5p overexpression. These findings suggest that miR-4725-5p may have a tumour promoting function in MDA-MB468 cells, which may be independent of KIF2C suppression and the SNP rs73991220 may favour miR-4725 expression thus contributing to the increased risk of TNBC progression.