MicroRNA-21 Promotes the Viability, Proliferation and Milk Fat Synthesis of Ovine Mammary Epithelial Cells by Targeting PDCD4

TL;DR

This study shows that miR-21 boosts sheep mammary cell survival, growth, and milk fat production by targeting the PDCD4 gene.

Contribution

The novel finding is that miR-21 promotes ovine mammary epithelial cell functions by downregulating PDCD4.

Findings

miR-21 overexpression increases OMEC viability, proliferation, and triglyceride content.

PDCD4 is confirmed as a direct target of miR-21 through luciferase and RT-qPCR assays.

Reduced PDCD4 expression leads to enhanced milk fat synthesis and cell proliferation in OMECs.

Abstract

MicroRNAs (miRNAs) are short endogenous non-coding RNAs and play important roles in regulating mammary development and activities of ovine mammary epithelial cells (OMECs), which affect the milk yield and milk ingredient contents of ewes. We previously found that miR-21 was highly expressed in ovine mammary tissue, while the regulatory mechanisms of miR-21 underlying mammary development and lactation performance are still unclear. Accordingly, in this study, we investigated the functions of miR-21 in the activities of OMECs, and validated the target relationship of miR-21 with a predicted target gene programmed cell death 4 (PDCD4) by a dual-luciferase reporter assay. Finally, we investigated the regulatory effect of PDCD4 on the viability, proliferation and milk fat synthesis of OMECs. The overexpression of miR-21 significantly increased the viability of OMECs, the number and proportion of Edu-labeled positive OMECs, as well as the contents of triglyceride in OMECs. In fact, miR-21 inhibitor obtained opposite results with miR-21 mimics. The results obtained from the dual luciferase report and RT-qPCR assays confirmed that the seed sequence of miR-21 can complementarily combine with the 3′-untranslated regions (3′-UTR) of PDCD4, and miR-21 decreased the luciferase activity of PDCD4. Meanwhile, miR-21 also reduced the expression of PDCD4. These results indicate that PDCD4 is a target gene of miR-21. It was further found that PDCD4 decreased the viability and triglyceride content of OMECs, and the number and proportion of Edu-labeled positive OMECs. These findings suggest that miR-21 promotes the viability, proliferation and milk fat synthesis of OMECs by down-regulating the expression of PDCD4. The results revealed the regulatory mechanism by which miR-21 affected the activities and milk fat synthesis of OMECs in sheep.

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