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 μg/ml prolactin and 2.5 μg/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 μg/ml prolactin and 2.5 μg/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 μg/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. K E Y W O R D Sfat metabolism, LATS2, methylation, miR-135b | INTRODUCTIONMilk provides optimal nutrition for growth and development of humans. Compared with cow milk, goat milk (the milk of Capra hircus) has a higher level of unsaturated fatty acids, total fat, calcium, vitamins, carbohydrates, and proteins (Han et al., 2004;Hinrichs, 2004;Luna, Bach, Juarez, & de la Fuente, 2008;Qu, Chen, Fan, Sun, & Zeng, 2016).Prolactin (PRL) plays a key role in the process of lactation and is secreted by the hypothalamus. PRL is an important hormone for the initiation of lactation in ruminants, can stimulate mammary epithelial cell metabolism, and stimulate milk protein synthesis (Inaba et al., 2016). In non-ruminants, PRL promotes mammary development during early pregnancy by stimulation of the ovaries to produce estrogen and progesterone (Campo Verde Arbocco et al., 2016).During pregnancy, PRL concentrations in plasma are significantly elevated and it can promote the secretion of IGF 1 and enhance the activity of the PKB/AKt (protein kinase) pathway (Roman, Seres, Herichova, Zeman, & Jurcovicova, 2003; Milewicz et al., 2011;Xiong et al., 2016). In addition, PRL-PRLR complexes (PRL combined with PRLR) can enter cells and elicit physiological effects through the JAK/ STAT and PKB/AKt pathway (Akers, 2006). Numerous studies have revealed that PRL plays an indispensable role in the process of lactation. However, how PRL works at the molecular level and the related molecular mechanism remain a "black box" that requires careful research (Akers, 2006;Erb et al., 1976;Hart, Bines, Morant, & Ridley, 1978;Kazmer & Oyler, 1991 Wang et al., 2014). MiRNA regulation is precise and its expression has stri...

PRL/microRNA-183/IRS1 Pathway Regulates Milk Fat Metabolism in Cow Mammary Epithelial Cells

“…Milk traits may be regulated by using DNA methylation and histone modification to interfere with the expression of galactolipid-related genes [41]. Chen et al 2018 [20] reported that PRL could regulate the expression of DNMT1 and affected the body function through the apparent modification, which is consistent with our results. PRL has a certain regulatory effect on genomic DNA methylation, but information on the regulation effect of PRL on breast fatty acids is still limited.…”

“…Genomic DNA modified with bisulphite was used as the template, and BSP was used as a primer to amplify the methylated fragment of the miR-183 5 promoter. The PCR product was cloned into the pmd-19t vector, and the Top10 competent cells were transformed [20]. The mono-clones were collected and sent to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing (Yingjun, Shanghai, China).…”

MicroRNA‐145 regulates immune cytokines via targeting FSCN1 in Staphylococcus aureus‐induced mastitis in dairy cows

“…The RT‐qPCR assays were performed according to the manufacturer's instructions (SYBR Premix TaqMan®, Perfect Real Time) with the specific primers reported previously. Expression was normalized to that of GAPDH (Chen et al, ; Chen, Luo, et al, ). The primer sequences are listed in Table .…”

Screening candidate microR-15a- IRAK2 regulatory pairs for predicting the response to Staphylococcus aureus-induced mastitis in dairy cows

“…RNA samples complying with standardized criteria were used for subsequent experimentation. The full procedure is described in Chen et al (2018aChen et al ( , 2018b.…”