Synergistic Regulation of miR-29c and miR-106b in Buffalo Mammary Epithelial Cells Orchestrates Milk Protein Synthesis in a PTHLH-Mediated Fashion

Research has demonstrated that certain microRNAs (miRNAs) play a pivotal role in governing milk protein synthesis in mice and dairy cows. However, there remains a limited understanding of specific miRNAs that regulate milk protein synthesis in buffalo. While our previous transcriptome data identified miR-29c and miR-106b as potential regulators influencing buffalo milk protein synthesis, the precise mechanism by which they modulate this process remains elusive. To address this gap, we conducted overexpression and knockdown experiments in this study. Our findings reveal that miR-29c and miR-106b exhibit inhibitory effects on the mRNA levels of CSN2 and CSN3 genes, along with diminishing β-casein production, within buffalo mammary epithelial cells (BuMECs). Furthermore, these miRNAs exert regulatory effects on the transcription and phosphorylation states of pivotal components within the mammalian target of rapamycin (mTOR) and JAK2–STAT5 signaling pathways. Employing the luciferase reporter system and qPCR, we confirmed the PTHLH gene as a shared target of miR-29c and miR-106b. Notably, they collaboratively regulate PTHLH gene expression. Our previous investigation highlighted the role of the PTHLH gene in augmenting milk protein production within BuMECs, achieved via stimulation of the mTOR and JAK2–STAT5 signaling cascades. Consequently, the inhibitory effect of miR-29c and miR-106b on buffalo milk protein synthesis is attributed to their coregulation of PTHLH. These findings emphasize the significance of miR-29c and miR-106b as essential regulators of milk protein synthesis, shedding light on the underlying mechanism governing milk production in buffalo.