miR-142-3p Regulates Milk Synthesis and Structure of Murine

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Bioactive Constituents, Metabolites, and Functions

miR-142-3p Regulates Milk Synthesis and Structure of Murine Mammary Glands via PRLR-Mediated Multiple Signaling Pathways Lei Tian, Li Zhang, Yingjun Cui, Huiming Li, Xuejiao Xie, Ye Li, and Chunmei Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03734 • Publication Date (Web): 01 Aug 2019 Downloaded from pubs.acs.org on August 2, 2019

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Journal of Agricultural and Food Chemistry

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miR-142-3p Regulates Milk Synthesis and Structure of Murine Mammary

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Glands via PRLR-Mediated Multiple Signaling Pathways

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Lei Tian‡, Li Zhang†, Yingjun Cui†, Huiming Li†, Xuejiao Xie†, Ye Li*‡, Chunmei Wang*†

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†Key Laboratory of Dairy Science of Education Ministry, Northeast Agricultural University, Harbin, 150030,China

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‡Department of Life Sciences and Medicine, Kunming University of Science and Technology, Kunming,650500,

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China

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* Corresponding authors: Li Ye and Chunmei Wang, Tel: +86-18787464645 (Y.L.); +86-13936507139 (C.W.); Fax:

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+86-871-65916860 (Y.L.); +86-451-55191416 (C.W.); Email: [email protected] (Y.L.); [email protected]

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(C.W.).

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ABSTRACT

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Murine mammary gland is an ideal model for studying the development and milk synthesis of dairy animals.

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MicroRNAs play an important role in milk synthesis and mammary gland development，however, the molecular

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mechanism of miR-142-3p continue to be poorly understood. Here, we knocked down miR-142-3p expression in

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vitro and vivo increased prolactin receptor expression and activated many downstream cellular proteins, such as

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mammalian target of rapamycin, sterol regulatory element-binding transcription factor 1, cyclin D1 and signal

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transducer and activator of transcription 5. Additionally, miR-142-3p knockdown in mouse mammary gland epithelial

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cells increased proliferation but not viability, induced cell cycle progression, decreased apoptosis, and increased the

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expression of triglycerides and β-Casein. Moreover, knockdown miR-142-3p in murine mammary gland tissue in

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vivo affected the structure and function of the mammary gland, which showed an increased number of lobules and

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ducts and was more capable of producing milk. However, overexpression of miR-142-3p had the opposite effects. In

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summary, these data uncover that miR-142-3p regulates milk synthesis and structure of murine mammary glands via

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PRLR-mediated multiple signaling pathways.

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Keywords

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miR-142-3p, prolactin receptor, mammary gland, milk synthesis

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INTRODUCTION

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Proliferation and differentiation of mammalian mammary glands during pregnancy and synthesis of milk during

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lactation are tightly regulated by several factors, including hormones and signaling proteins and effector molecules.

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Prolactin (PRL) induces mammary gland development and lactogenesis. In pregnancy and lactation, the PRL receptor

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(PRLR) is one of the major receptors for lobuloalveolar maturation and synthesis of milk. The functions of PRL are

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mediated by PRLR, which is also an significant regulator of mammary gland growth and development. PRLR plays

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a major role in PRL signal transduction by initiating signaling cascades. It is a cytokine receptor, mainly using Janus

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kinase (JAK)/signal transducer and activator of transcription (STAT);1 signaling messengers in this pathway include

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mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B

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(AKT)/mammalian target of rapamycin (mTOR).2,3 At the molecular level, PRLR regulates the expression of genes

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necessary for growth and milk synthesis. PRLR pathway prompts mammary alveologenesis during pregnancy and

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induces β-casein synthesis during lactation. PRLR signaling also stimulates lipid biosynthesis by regulating sterol

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regulatory element-binding protein 1(SREBP1) and enhancing the key enzymes activity.4

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MicroRNAs (miRNAs) are endogenous, post-transcriptional regulating, non-coding RNAs that were critical

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regulators of various cellular processes, such as cell proliferation, differentiation, and death. Further, there are also

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some differentially expressed miRNAs during mammary gland development and milk synthesis, indicating that

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among the various regulatory mechanisms, the role of these differentially expressed miRNAs in post-transcriptional

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control is pivotal.5 In our previous work, we examined miRNA expression during different periods of mammary

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gland development and found that miR-142-3p is differentially expressed in virgin, pregnancy, lactation, and


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involution.5 Some evidence suggests that miR-142 families may play crucial roles during various biological processes,

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such as apoptosis,6 inflammation,7 and cancer,8,9 especially in breast cancer.10 Blocking miR-142-3p promotes anti-

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apoptosis and inhibition by inducing KDM6A-mediated H3K27 demethylation in induced regulatory T cells.11 miR-

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142-3p promoted the IL-1β in neuroinflammation.12In breast cancer, miR-142-3p suppress tumor growth by

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knockdown estrogen receptor and Bach-1.13-15 However, there is yet no report about the function of miR-142-3p in

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mouse mammary gland development and lactation. The purpose of this work was to investigate the role of miR-142-

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3p in mammary gland development and milk synthesis through PRLR. Here, we show that miR-142-3p contributes

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to the remodeling and milk synthesis of murine mammary gland via PRLR-mediated multiple signaling pathways.

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MATERIALS AND METHODS

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Cell culture, tissue specimens, and treatment

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Murine mammary gland epithelial cells (MMGECs) were prepared from adult female BALB/c mice mammary glands

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as previously protocols16 and grown at 5% CO2 in Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12, Gibco,

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Carlsbad, CA, USA) + 10% fetal bovine serum (FBS) in 37 °C. Experimental assays were executed following 2-5

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passages when MMGECs reached 70-80% confluence. MMGECs were transfected with miR-142-3p precursor,

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inhibitor, and negative control using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to

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manufacturer’s protocol. The transfected RNA oligonucleotide sequences were synthesized (Genepharma, Shanghai,

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China) and depicted in Table 1.

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BALB/c mice were purchased from Harbin Veterinary Research Institute. Mammary tissues of female mice in

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different periods (virgin 4w,5w,7w; pregnant 5d,13d,18d; lactation 3d,7d,13d; and involution 2d,5d,10d) were

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collected, and six mice were used for each group. On the 1st day of lactation, miR-142-3p mimic (200ng)/inhibitor



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(200ng) were injected on the fourth mammary gland side of the mouse, and the other side was injected with mimic-

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NC (200ng)/inhibitor-NC (200ng). After 48 h (lactation 3d), the mice were sacrificed and breast samples were fixed

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in 10% paraformaldehyde or frozen in liquid nitrogen for further analysis. All animal experiments were performed

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in accordance with guidelines and regulations of Animal Ethics Committee.

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RNA immunoprecipitation assay

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RNA immunoprecipitation (RIP) assay was performed according to the manufacturer’s protocol of EZ-Magna RIP

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Kit (Millipore, Burlington, MA, USA).At the beginning,we lysed mammary gland tissue samples in RIP lysis buffer,

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and then, incubated tissue extract in RIP buffer with immunomagnetic beads conjugated to mouse anti-Ago2 antibody.

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Subsequently, these tissue extracts were treated with proteinase K, and isolated the immunoprecipitated RNA. After

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that, the concentration of RNAs was measured by a NanoDrop, and RNAs were subjected to the sequencing analysis.

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Cell viability assay

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CASY-TT Analyzer System (Schärfe System GmbH, Reutlingen, Germany) was used to detected the MMGECs

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viability according to the manufacturer’s protocols before.16 For determination of MMGECs activity suspension(100

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μL) were added into a CASY cup, which contained 10 mL CASY ton, and counted the cells in a CASY cell counter.

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The experiment was performed in triplicate.

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Quantitative RT-PCR analyses

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RNA was isolated from MMGECs and mammary gland tissues with the Trizol reagent (Thermo Fisher Scientific,

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Waltham, MA, USA) and miR VanaTM Kit (Applied Biosystems, Foster City, CA, USA). Total RNA was collected

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for cDNA synthesis with cDNA Synthesis Kit (Fermentas, Waltham, MA, USA) according to the manufacturer’s

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protocols. miRNA and mRNA levels were quantified by a SYBR Green Assay (Genepharma, Shanghai, China) and


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SYBR Green Master Mix (Takara Biotechnology, Kusatsu, Japan) following the manufacturers’ protocols. miRNA

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expression levels were normalized to 5s and U6, and gene expression levels were normalized to β-actin. The primer

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sequences used for PCR in Table 2. The samples were analyzed on an ABI 7500 System (ABI, Foster City, CA,

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USA).

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Construction of luciferase plasmids and reporter assay

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The 3′UTR of Prlr including the predicted sites for miR-142-3p seizing was obtained by PCR. Then, the Prlr 3′

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UTR cloned into the pMIR-REPORT vector (Thermo Fisher Scientific, Waltham, MA, USA). The mutational site

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was got by site-directed mutagenesis. miR-142-3p or control together with these luciferase vectors were co-

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transfected into 293T cells by Lipofectamine 3000 in triplicate. Cells were lysed at 2 d post-transfection, then

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luciferase activity was assayed by Dual-Light Luciferase & β-Galactosidase Reporter Gene Assay System (Thermo

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Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocols.

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Western blot analysis

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Total protein was extracted from frozen tissue samples and cells. In brief, total protein samples were sonicated in

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homogenization buffer (1:100 protease inhibitor cocktail set III, 0.5 mM MgSO4, 1 mM of HEPES, 5 mM

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benzamidine, 2 mM of 2-mercaptoethanol, 3 mM ethylene diamine tetraacetic acid, 0.5‰ NaN3, 1:100 phosphatase

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inhibitor cocktail set II). Proteins (12 μg/lane) were loaded onto 10% SDS-PAGE gels, transferred onto PVDF

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membranes (Millipore, Burlington, USA), and incubated the PVDF membranes with skim milk or BSA for 2 h at

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25℃, after that incubated with the primary antibodies at room temperature 1.5h. The primary antibodies as follows:

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β-actin (sc-47778), PRLR (sc-30225), p-AKT (sc-33437), AKT (sc-8312), p-STAT5 (sc-81524), STAT5 (sc-74442),

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SREBP1 (sc-13551), GLUT1 (sc-1603), and CCND1 (sc-8396), Santa Cruz Biotechnology, CA, USA; mTOR

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(#2972), p-p38 MAPK (#4631) , p38 MAPK (#8690), p-mTOR (#5536), Cell Signaling Technology, MA, USA.



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Next, the membranes were incubated with secondary antibody with HRP (Santa Cruz Biotechnology, CA, USA),

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reacted with chemiluminescence western blotting substrate (Millipore, Burlington, MA, USA), and developed.

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Western blotting images were analyzed by ImageJ (NIH, MD, USA). β-actin was the control.

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Cell cycle and apoptosis analysis

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The transfected MMGECs were digested by 0.25% trypsin and centrifugated. MMGECs were washed with PBS three

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times, and then treated within 70% ethanol 12 h at 4 degrees temperatures. MMGECs were washed with PBS again,

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and treated with 2 mM Triton X-100 and 50 mg/mL PI for 30 min to detect the cell cycle in the dark. For apoptosis

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analysis, cells were stained by an Apoptosis Detection Annexin V-FITC/PI kit (Roche Diagnostics, Basel,

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Switzerland).MMGECs were prepared in 0.5 mL PBS and detected on the Cytomics FC500 flow cytometer

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(Beckman Coulter, Brea, CA, USA). The ratio of MMGECs within each cell cycle phase and apoptosis were analyzed

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by ModFit LT 3.2 (Verity Software House, Topsham, ME, USA).

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Immunofluorescence, hematoxylin and eosin staining, and in situ hybridization

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MMGECs on coverslips and mammary gland slices were incubated with PRLR primary antibody 12 h after fixation

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and blocking, after that treated with the fluorescent secondary antibody. Cell nuclei were labeled with PI, digital

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images were acquired with a laser scanning confocal microscope (TCS-SP2 AOBS, Leica, Heidelberg, Germany).

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The localization of PRLR staining were observed and analysed by the Leica LCS software. Others mammary gland

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tissue samples were collected for physiological structure observations after 10% paraformaldehyde fixation, trimming,

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embedding in paraffin, conventional sectioning, and staining with hematoxylin and eosin, as described previously,17

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and viewed under a light microscope.miR-142-3p was measured in tissue microarrays using In situ hybridization

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with digoxigenin-labeled probes. The slides were deparaffinized and rehydrated, and then treated with proteinase K


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5 min at 37 °C; the samples were washed with 0.1 M PBS/glycine 10 min three times. After treated with 5× saline

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sodium citrate solution at 25 °C for 15 min, the probes were added for hybridization 18 h at 55 °C. The samples were

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washed with graded-diluted saline sodium citrate solutions at 60 °C for 5 min, and treated at 37 °C with a digoxigenin

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antibody (Invitrogen, CA, USA) for 30 min. Finally, hybridization signal was visualized by NBT/BCIP (Sigma-

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Aldrich, MO, USA). Washing with H2O for 5 min to stop the reaction. The nuclear fast red was used to counterstain

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with, and the sections mounted using an aqueous solution. Images were captured at 200× magnification and analyzed

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with ImagePro 6.0 software.

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Measurement of triglyceride, lactose, β-casein,and milk composition

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After transfection of MMGECs and mammary gland tissues, the supernatants were collected, and β-casein,

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triglyceride (TG), and lactose were measured by ELISA with β-casein (CUSABIO, Houston, TX, USA), a TGGPO-

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POD Assay Kit (Applygen Technologies, Beijing, China), and a Lactose & D-Galactose (Rapid) Assay Kit

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(Megazyme, Wicklow, Ireland) following the protocols. Milk composition change was analyzed by a MilkScan

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FT120 Dairy Ingredients Fast Detector (Foss, Hillerød, Denmark), and the levels of milk fat, protein were recorded.

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The milk production was collected and measured by the change of bodyweight of offspring mice before and after

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suckling. Each female mouse feed 8 offspring mice, and every 4 hours as a lactation unit, isolation for 3h and lactation

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for 1h, and weighed the offspring mice before and after suckling, recorded the data. In 48 hours, we detected 12

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times, and added the results of 12 times, got the total lactation of female mice for 48h.

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Statistical analysis

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All experiments were performed in three times. The blank control groups were not given any treatment, and the

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negative control groups were treated with negative control oligonucleotides. All results were compared and analyzed



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by Student’s t tests, p < 0.05 or p < 0.01 was considered statistically significant. Statistical analyses were performed

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by SPSS 18.0 software (SPSS Inc., IL, USA).

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RESULTS

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miR-142-3p expression in murine mammary gland of different periods

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miRNA microarray was used to compare the miRNA expression profiles in mouse mammary gland of four

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developmental stages: virgin(V), pregnancy(P), lactation(L), and involution(I), and we found the expression of miR-

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142-3p was down-regulated during pregnancy and lactation (Figure 1A, p < 0.01) compared with virgin. The results

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of qRT-PCR showed that the expression of miR-142-3p was also decreased in pregnancy and lactation (Figure 1B,

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p < 0.01, p < 0.05). Using ISH, we observed the levels of miR-142-3p in pregnancy and lactation were lower than

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other developmental periods (Figure 1C, D, p < 0.05).

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Prlr is a target gene of miR-142-3p in MMGECs

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RIP-Seq was adopted to analyze which proteins were regulated directly by miR-142-3p in mammary gland tissue.

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We injected mmu-miR-142-3p mimic into one side of the fourth mammary gland, and the other side was injected

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with control. We identified some miR-142-3p targets which were relevant to the development and milk synthesis of

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mammary (Table 3), such as PRLR. TargetScan, PicTar, and miRanda predicted that Prlr 3′UTR was a potential

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target of miR-142-3p. To validate this in silico prediction, the plasmids were constructed contained the Prlr 3′UTR

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predicted binding sites, or nucleotide substitutions were made in the mutant 3′UTR (Figure 2A). The luciferase assay

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showed that miR-142-3p overexpression inhibited the activity of the Prlr 3′UTR luciferase reporter (p < 0.01), but no

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obvious effect on the mutant Prlr 3′UTR luciferase vector (Figure 2B). Additionally, Prlr mRNA expression was up-

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regulated (p < 0.05) by miR-142-3p inhibitor (Figure 2C); and miR-142-3p downregulated also increased PRLR (p


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< 0.01) (Figure 2D) in MMGECs. Taken together, these data reveal that Prlr is a target gene of miR-142-3p in

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MMGECs.

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miR-142-3p affects Prlr downstream signaling gene expression in MMGECs

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The Prlr-mediated PRL signaling pathway has been verified.18 To study how miR-142-3p modulated Prlr and its

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downstream pathway in MMGECs, we investigated the expression and activation of AKT, mTOR, STAT5, MAPK,

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SREBP1, GLUT1, and CCND1. Prlr mRNA and protein level were up-regulated by miR-142-3p inhibitor and

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decreased by miR-142-3p mimic (both p < 0.05) (Figures 3A , 3B, 3C). In MMGECs knockdown miR-142-3p

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increased(p < 0.05) AKT, mTOR , STAT5, MAPK, SREBP1, CCND1 and significant increased (p < 0.01) GLUT1

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mRNA expression (Figure 3A) ,meanwhile increased(p < 0.05) AKT, SREBP1, GLUT1, CCND1 and significant

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increased (p < 0.01) mTOR, STAT5, MAPK protein expression. But AKT, mTOR , STAT5, and MAPK protein

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phosphorylation levels were much higher than the negative control (Figure 3B). Interestingly, miR-142-3p

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overexpression reduced (p < 0.05) the protein expression of AKT, mTOR, STAT5, MAPK, SREBP1, GLUT1,

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CCND1, and significant reduced (p < 0.01) the protein expression of PRLR, at the same time reduced (p < 0.05) the

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phosphorylation levels of AKT, mTOR, STAT5, and MAPK (Figure 3C).

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Effect of miR-142-3p on MMGEC proliferation, apoptosis, activity, and synthesis of milk composition

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The MMGECs were harvested after transfection with inhibitor or mimic at 48 h. Cell count was increased in the

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inhibitor group (p < 0.05), but not significantly changed in the mimic group, similarly cell viability of the mimic

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group was not significantly different to negative control (Figure 4A).Knockdown miR-142-3p could increased PRLR

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expression and overexpression could suppress it in MMGECs (p < 0.05) (Figure 4B, C). Flow cytometry revealed

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that miR-142-3p inhibitor could contribute to MMGECs proliferation and cell cycle progression, and suppressed



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apoptosis (p < 0.05, Figure 4D), conversely, miR-142-3p mimic increased MMGEC apoptosis and suppressed

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proliferation (p < 0.05, respectively, Figure 4E).β-Casein and TG secretion was increased (both p < 0.05) by miR-

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142-3p inhibitor and reduced(p < 0.01, p < 0.05, respectively) by miR-142-3p overexpression (Figure 4F, G).

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Differently, lactose secretion was not changed by knockdown or overexpression of miR-142-3p (p > 0.05) (Figure

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4H). This result showed that miR-142-3p suppressed the secretion of β-casein and TG, but had little effect on lactose

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secretion in MMGECs.

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miR-142-3p inhibits Prlr and its downstream signaling pathway in murine mammary glands in vivo

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In vivo, we injected miR-142-3p mimic or inhibitor into one side of the fourth mammary gland and the other side

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with negative control. PRLR mRNA was increased (p < 0.05) and protein was significantly increased(p < 0.01) in

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murine mammary glands by miR-142-3p knockdown according to qRT-PCR and western blotting (Figure 5A, B),

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whereas miR-142-3p overexpression had the opposed effects (Figure 5C, D). Western blotting analysis detected that

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silencing miR-142-3p improved the levels of Prlr downstream proteins including p-AKT, p-mTOR, p-eIF2α, p-

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STAT5, p-MAPK, cyclin D1, SREBP1, and β-casein (all p < 0.05) in murine mammary glands (Figure 5C). By

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contrast, up-regulating miR-142-3p repressed the levels of multiple PRL signaling pathway proteins in murine

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mammary glands (Figure 5D). Thus, these data demonstrated that miR-142-3p also inversely regulates PRL signaling

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via Prlr in vivo.

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Effect of miR-142-3p on structural and functional changes in murine mammary gland

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miR-142-3p could influence the synthesis and secretion of mammary gland proteins, lipids, and lactose. miR-142-3p

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inhibition increased PRLR expression (Figure 6A, p < 0.05), the development of the mammary acinus (Fig. 6C, p
0.05, compared with

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the NC group.

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Figure 7. Diagram summarizing our findings.

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miR-142-3p regulates milk synthesis and structure of murine mammary glands via PRLR-mediated multiple signaling

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pathways.

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Table 1. The transfected RNA oligonucleotide sequences Table 1. The transfected RNA oligonucleotide sequences

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RNA oligonucleotide

Sequences

mmu-miR-142-3p mimic sense

5'UGUAGUGUUUCCUACUUUAUGGAU3'

mmu-miR-142-3p mimic antisense

5'CCAUAAAGUAGGAAACACUACAAA3'

mmu-miR-142-3p mimic-NC sense

5'AGUACUGCUUACGAUACGGTT3'

mmu-miR-142-3p mimic-NC antisense

5'CCGUAUCGUAAGCAGUACUTT3'

mmu-miR-142-3p inhibitor sense

5'AUCCAUAAAGUAGGAAACACUACATT3'

mmu-miR-142-3p inhibitor-NC sense

5'UUCUCCGAACGUGUCACGUTT3'

Table 2. The primer sequences used for qRT-PCR. Table 2. The primer sequences used for qRT-PCR Primer

Sequences

mmu-miR-142-3p RT-prime

5'GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATAC GACTCCATAA3'

mmu-miR-142-3p forward

5'ACGGGCTGTAGTGTTTCCTACT3'

mmu-miR-142-3p reverse

5'CAGTGCAGGGTCCGAGGTAT3'

5s forward

5'TCGTCTGATCTCGGAAGCACCCGGTT3'

5s reverse

5'AAGCCTACAGCACCCGGTAT3'

Prlr forward

5'CCCAAGCTTTGAGGAATCAGCCAAGAA3'



Prlr reverse

5'GGACTAGTGTGGAGGAGTGAAGTAGTGG3'

Akt1 forward

5'CAACCAGGACCACGAGAA3'

Akt1 reverse

5′ACACGATGTTGGCAAAGAA3′

mTOR forward

5′GCTGACCGAAATGAGGGC3′

mTOR reverse

5′AGAATCAGACAGGCACGAA3′

Stat5 forward

5′GGCCCTTCTCTTCTCTGGATTG3′

Stat5 reverse

5′ACCTCGGTCCTGGGGATTAT3′

MAPK forward

5′TCACAGGGACCTAAAGCCCA3′

MAPK reverse

5′CTGGGGTTCCAACGAGTCTT3′

SREBP1

5′CTTCTGGAGACATCGCAAAC3′

forward

SREBP1 reverse

5′GGTAGACAACAGCCGCATC3′

GLUT1 forward

5′GTATCGTCAACACGGCCTTC3′

GLUT1 reverse

5′GCCAGCCACAGCAATACGA3′

CCND1 forward

5′TCAAGTGTGACCCGGACTG3′

CCND1 reverse

5′ATGTCCACATCTCGCACGTC3′

β-actin forward

5′CTGTCCCTGTATGCCTCTG3′

β-actin reverse

5′ATGTCACGCACGATTTCC3′

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qRT-PCR data were analyzed using the formula: R = 2-(ΔCt sample - ΔCt control).

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Table 3. Molecules identified by RIP. Fold change in miR-142-3p expression in mammary gland tissue

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compared with control tissue Table 3. Molecules identified by RIP Gene symbol

Gene description

Fold change

phosphatase and tensin homolog

9.7

Tgfbr3

transforming growth factor, beta receptor III

6.1

Tgfbr1

transforming growth factor, beta receptor I

3.5

fatty acid binding protein 3, muscle and heart, pseudogene 1

3.1

Fas

fatty acid synthase

0.8

Prlr

prolactin receptor

0.8

Pgr

progesterone receptor

0.3

insulin-like growth factor I receptor

2.8E-05

Pten

Fabp3-ps1

Igf1r

438

TOC Graphic


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Figure 1. miR-142-3p expression in murine mammary gland tissues from different developmental periods. (A) RNA microarray analysis of miR-142-3p. V: virgin; P: pregnancy; L: lactation; and I: involution (n = 3 mice per group). (B) miR-142-3p expression determined by qRT-PCR. The expression of 5s was used as an endogenous control. V: virgin; P: pregnancy; L: lactation; and I: involution (n = 6 mice per group). Results are shown as relative expression *P < 0.05; **P < 0.01, significant difference versus virgin. (C) (D) miR142-3p expression by using ISH of different developmental periods. miR-142-3p was found to be downregulated during pregnancy and lactation compared with virgin and involution tissues (P < 0.05). 193x125mm (300 x 300 DPI)


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Figure 2.Prlr is a direct target of miR-142-3p. (A) Bioinformatics prediction showing that the seed sequence of mmu-miR-142-3p has a high level of complementarity to Prlr 3′UTR. Complementary sequences are conserved between humans, mice, and rats (highlighted in yellow). Three mutant nucleotide substitutions were made in their 3′UTR (highlighted in red). (B) miR-142-3p mimic inhibited the activity of a luciferase reporter gene linked to the 3′UTR of mouse Prlr. The activity of mutant Prlr 3′UTR luciferase reporter showed no obvious effect. (C) miR-142-3p knockdown in MMGECs led to an increase in Prlr mRNA levels. MMGECs treated with negative control served as a control. Prlr and miR-142-3p expression levels were determined by qRT-PCR. (D) miR-142-3p knockdown by transfecting MMGECs with inhibitor increased endogenous PRLR protein levels. β-Actin was used as a loading control. The results are shown as relative expression. n = 3, *P < 0.05, **P < 0.01, compared with the NC group. 163x143mm (300 x 300 DPI)



Figure 3. miR-142-3p affects Prlr downstream signaling gene expression in MMGECs (A) miR-142-3p inhibitor significantly increased AKT, mTOR, STAT5, MAPK, SREBP1, GLUT1, and CCND1 mRNA levels in MMGECs. (B) miR-142-3p inhibitor significantly increased AKT, mTOR, STAT5, MAPK, SREBP1, GLUT1, and CCND1 protein expression, while AKT, mTOR, STAT5, and MAPK protein phosphorylation levels were increased. (C) Overexpression of miR-142-3p reduced the protein expression of PRLR, AKT, mTOR, STAT5, MAPK, SREBP1, GLUT1 and CCND1, and reduced the phosphorylation levels of AKT, mTOR, STAT5, and MAPK. n = 3, * p < 0.05, ** p < 0.01, compared with the NC group. 440x479mm (300 x 300 DPI)


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Figure 4.miR-142-3p affects MMGEC proliferation, apoptosis, activity, and synthesis of β-casein, TG, and lactose. (A) Cell count was increased in the miR-142-3p inhibitor group and not significantly changed in the mimic group, and cell viability did not change significantly. (B) PRLR expression was significantly upregulated by miR-142-3p inhibitor in MMGECs. (C) PRLR expression was decreased by miR-142-3p overexpression in MMGECs. (D) miR-142-3p knockdown increased MMGEC proliferation and cell cycle progression and reduced apoptosis. The x-axis indicates FITC-A, and the y-axis indicates cell count. (E) miR142-3p overexpression increased MMGEC apoptosis and suppressed proliferation. The x-axis indicates annexin V-FITC, and the y-axis indicates PI. (F) β-Casein was increased by miR-142-3p inhibitor and reduced by miR-142-3p mimic. (G) TG was increased by miR-142-3p inhibitor and reduced by miR-142-3p mimic. (H) miR-142-3p inhibitor and mimic had no significant effect on lactose. n = 3, *P < 0.05, ** P < 0.01, n.s. P > 0.05, compared with the NC group. 361x333mm (300 x 300 DPI)



Figure 5.miR-142-3p inhibits Prlr and its downstream signaling pathway in murine mammary glands in vivo. (A) miR-142-3p knockdown enhanced Prlr mRNA expression. (B) miR-142-3p overexpression reduced Prlr mRNA expression. (C) miR-142-3p knockdown increased PRLR, AKT, eIF2α, MAPK, mTOR, STAT5, cyclin D1, β-casein (CNS2), and SREBP1, while it increased AKT, eIF2α, MAPK, mTOR, STAT5, and MAPK protein phosphorylation in murine mammary glands. PTEN did not change significantly. (D) miR-142-3p upregulation reduced the expression levels of PRLR, AKT, eIF2α, MAPK, mTOR, STAT5, cyclin D1, β-casein (CNS2), and SREBP1, and decreased AKT, eIF2α, MAPK, mTOR, STAT5, and MAPK protein phosphorylation in murine mammary glands. PTEN did not change significantly. n = 6, *P < 0.05, **P < 0.01, n.s. P > 0.05, compared with the NC group. 224x190mm (300 x 300 DPI)


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Figure 6. Effect of miR-142-3p on structural and functional changes in the mammary gland (A) miR-142-3p inhibition increased PRLR expression. (B) miR-142-3p overexpression reduced PRLR expression. (C) miR-142-3p inhibition improved mammary acinus development, and miR-142-3p overexpression suppressed mammary acinus development. (D) Milk production was increased by miR-1423p inhibitor and reduced by miR-142-3p mimic. (E) TG concentrations were increased by miR-142-3p inhibitor and reduced by miR-142-3p mimic. (F) Lactose concentrations did not change significantly. n = 6, * p < 0.05, ** p < 0.01, n.s. p > 0.05, compared with the NC group. 366x443mm (300 x 300 DPI)



Figure 7. Diagram summarizing our findings. miR-142-3p regulates milk synthesis and structure of murine mammary glands via PRLR-mediated multiple signaling pathways 355x260mm (300 x 300 DPI)


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miR-142-3p Regulates Milk Synthesis and Structure of Murine

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