MiR-21-3p Centric Regulatory Network in Dairy Cow Mammary Epithelial Cell Proliferation

MicroRNA-mediated gene regulation is important for the development of the mammary gland and the lactating process. A previous study has shown that the expression of microRNA-21 (miR-21) is different in the dry and early lactation period of the dairy cow mammary gland, but the molecular mechanisms underlying the lactation cycle are not fully understood. Here, the function of miR-21-3p on bovine mammary gland epithelial cells (BMECs) was detected by MTT assay and flow cytometry analysis, which showed that miR-21-3p significantly promoted the cell viability and proliferation. Then, the regulating mechanism of miR-21-3p on cell viability and proliferation was elucidated. Dual luciferase assay, RT-qPCR, and Western blot results revealed that IGFBP5 was a target gene of miR-21-3p. It was known that lncRNA could act as a competing endogenous RNA to sequester miRNAs and reduce the regulatory effect of miRNA-targeted genes. Based on our previous lncRNA-seq data and bioinformatics analysis, lncRNA NONBTAT017009.2 was potentially associated with miR-21-3p, and its expression was specifically inhibited with the transfection of miR-21-3p mimic into BMECs. Inversely, the overexpression of NONBTAT017009.2 significantly decreased the expression level of miR-21-3p in BMECs, while the expression of IGFBP5, the target gene of miR-21-3p, was significantly upregulated. In addition, the promoter region of miR-21 contained two STAT3 binding sites, and the dual luciferase reporter assays revealed that the overexpression of STAT3 significantly reduced the promoter activity of miR-21, implying that the transcription factor STAT3 may act as an upstream regulator affecting the regulation process of miR-21-3p. The overexpression of STAT3 significantly inhibited the expression of miR-21-3p, while the mRNA expression of IGFBP5 was significantly increased compared with the control group. Besides, there are no STAT3 binding sites in the promoter region of IGFBP5 as we predicted by gene-regulation and JASPAR software. Therefore, it could infer that STAT3 might regulate the expression of IGFBP5 by miR-21-3p. Taken together, these results established a regulatory network of miR-21-3p to illustrate the regulating mechanism on promoting cow mammary epithelial cell proliferation.