AGO2 HITS-CLIP identifies functional miRNAs during mammary epithelial cells differentiation, reveals the role of Mmu-miR-122-5p in mammary cell development and differentiation

miRNAs are small non-coding RNAs of an average of 22 nucleotides in length, known for their post-transcriptional gene regulation, determining the cell fate of any organism. The role of miRNA has been shown to be involved from embryonic development to individual tissue-level differentiation. It plays an important role in modulating gene expression post-transcriptionally to execute mammary gland development and function. Recent studies highlighted many miRNAs and their tumorigenesis role in breast cancer. But a comprehensive study of miRNA on general mammary epithelial cell differentiation is missing. In this study, we performed global miRNA sequencing of the mammary epithelial stem-like cell (HC11), in vitro and high-throughput sequencing of crosslinking immunoprecipitation (HITS-CLIP) to characterize the miRNA:mRNA interactions. The integration analysis of miRNA sequencing and HITS-CLIP identified top regulatory miRNAs and their impact on suppressing cell cycle and cell proliferation pathways in course of mammary gland differentiation. Among them, one of the miRNAs, miR-122-5p which targeted the maximum number of genes post-transcriptionally in the undifferentiated state of mammary epithelial cells, was considered for functional study to illustrate the integrity of our HITS-CLIP dataset. The knockdown study of miR-122 showed increased cell proliferation through the Akt signaling pathway by targeting Gys1, which caused obstruction in the process of mammary cell differentiation and resulted in the downregulation of CASEIN.