Mechanism of Prolactin Inhibition of miR-135b via Methylation in Goat Mammary Epithelial Cells

ABSTRACT

Prolactin is an important endocrine activator of lactogenesis. This study investigated the function and mechanism of miR-135b in the enhancement of lactation by prolactin in goat mammary epithelial tissue. We utilized S-Poly (T) sequencing to evaluate changes in gene regulation in the goat mammary gland after incubation with 2.5 ug/ml prolactin and 2.5 ug/ml IGF-1 by examining highly expressed miRNAs during early-lactation and late-lactation. The results illustrated that miR-135b is highly expressed in the goat mammary gland during early-lactation and late-lactation, and also after treatment with 2.5 ug/ml prolactin and 2.5 ug/ml IGF-1. We used Q-RT PCR, Western Blot, immunofluorescence, and luciferase reporter assay analysis, and found that PRL was significantly down-regulated in response to the expression of miR-135b in a manner that was functionally related to TAG synthesis via the large tumor suppressor 2 gene (Lats2), an important regulator of adipocyte proliferation via Hippo Signaling. Furthermore, using bisulfite-sequencing PCR (BSP), Q-PCR and Western Blot we discovered an increase in expression of DNMT I (DNA methyl transferase I) in goat mammary epithelial cells with the 2.5 ug/ml PRL incubation, which led to DNA methylation of the CpG island upstream of miR-135b and inhibited the transcription and expression of miR-135b. This article is protected by copyright. All rights reserved

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