Mechanism of prolactin inhibition of miR-135b via methylation in goat mammary epithelial cells.
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.
Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A&F University, Yang ling, Shaanxi, PR China.
Zhi
Z
Chen
Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A&F University, Yang ling, Shaanxi, PR China.
Jun
J
Luo
Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A&F University, Yang ling, Shaanxi, PR China.
ChangHui
C
Zhang
Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A&F University, Yang ling, Shaanxi, PR China.
Yue
Y
Ma
Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A&F University, Yang ling, Shaanxi, PR China.
Shuang
S
Sun
Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A&F University, Yang ling, Shaanxi, PR China.
Tianyin
T
Zhang
Mammalian Nutrition Physiology Genomics, Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana, Illinois.
Juan J
http://orcid.org/0000-0003-1586-4365
JJ
Loor
D020413:3' Untranslated Regions; D000818:Animals; D001665:Binding Sites; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018899:CpG Islands; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic; D006041:Goats; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007774:Lactation / Q000187:drug effects*; D008321:Mammary Glands, Animal / Q000187:drug effects* / Q000378:metabolism; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D011388:Prolactin / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D020413:3' Untranslated Regions; D035683:MicroRNAs; C549289:Mirn135 microRNA, mouse; D007334:Insulin-Like Growth Factor I; D011388:Prolactin; D004248:DNA (Cytosine-5-)-Methyltransferases; D017346:Protein Serine-Threonine Kinases
LATS2; fat metabolism; methylation; miR-135b