Long Non-Coding RNA MPNCR is a Negative Regulator of Bovine Mammary Epithelial Cell Proliferation

Abstract BackgroundNumerous studies have reported that many long non-coding RNAs (lncRNAs) may play multiple essential roles during mammary gland development; however, little is known about their functions and regulatory mechanism.ResultsIn this study, we confirmed a novel lncRNA, MPNCR, that was highly expressed during lactation compared to the dry period in dairy cattle. The potential biological and regulatory functions of MPNCR were explored in bovine mammary epithelial cells (BMECs). The MTT and EdU assays revealed that lncRNA MPNCR significantly suppressed cell viability and proliferation of BMECs. The subcellular localization indicated that MPNCR had a potential function as a competing endogenous RNA (ceRNA) to regulate the proliferation of BMECs. Bioinformatics analysis showed that lncRNA MPNCR had two binding sites for miR-31, further confirmed by the dual-luciferase report assay. The RT-qPCR result showed that the expression of miR-31 was downregulated following the overexpression of MPNCR in BMECs. Furthermore, the expression of MPNCR was significantly inhibited by miR-31 overexpression and upregulated by miR-31 inhibition. Cell viability and proliferation were significantly suppressed or promoted with the transfection of miR-31 inhibitor or miR-31 mimic into BMECs, respectively, whereas rescue with MPNCR reduced the proliferation of BMECs. The dual-luciferase report assay showed an interaction between miR-31 and the 3' UTR of the target gene CAMK2D. RT-qPCR and western blot further showed that the expression of CAMK2D was significantly increased with the overexpression of miR-31, while its expression was significantly suppressed with the inhibition of miR-31, indicating CAMK2D was a target of miR-31.ConclusionTaken together, these results demonstrate the function and regulatory mechanism of lncRNA MPNCR on the proliferation of BMECs, and that its regulation may contribute to bovine mammary cell population management.