Abstract
Prolactin (PRL) is a pituitary hormone that regulates multiple physiological processes. However, the mechanisms of PRL synthesis have not been fully elucidated. The aims of the present study were to study the functions and the related mechanisms of miR-375 regulating PRL synthesis. We initially found that miR-375 mainly expressed in the lactotrophs of mouse pituitary gland. To identify the function of miR-375 in the pituitary gland, the miR-375 knockout mice were generated by using Crispr/Cas9 technique. The results showed that miR-375 knockout resulted in the decline of pituitary PRL mRNA and protein levels by 75.7 and 60.4%, respectively, and the serum PRL level reduced about 46.1%, but had no significant effect on FSH, LH and TSH. Further, we identified that Estrogen receptor 1 (alpha) (Esr1) was a downstream molecule of miR-375. The real-time PCR and Western blot results showed that ESR1 mRNA and protein levels markedly decreased by 40.9 and 42.9% in the miR-375 knockout mouse pituitary, and these were subsequently confirmed by the in vitro study using transfections of miR-375 mimics and inhibitors in pituitary lactotroph GH4 cells. Further, Rasd1 was predicted by bioinformatic tools and proved to be the direct target of miR-375 in lactotrophs using the dual-luciferase reporter assay. Rasd1-siRNA transfection results revealed the negative effect of Rasd1 in regulating ESR1. Collectively, the results presented here demonstrate that miR-375 positively modulates PRL synthesis through Rasd1 and Esr1, which are crucial for understanding the regulating mechanisms of pituitary hormone synthesis.
Introduction
Prolactin (PRL) is a peptide hormone that is mainly synthesized and secreted from the anterior pituitary lactotrophs (Freeman et al. 2000, Cabrera-Reyes et al. 2017). With except the classical functions of PRL promoting the development of mammary gland and maintaining lactogenesis, PRL is involved in regulating other physiological processes, such as osmoregulation, angiogenesis, metabolism and immune response. And the disorder of PRL synthesis and secretion could lead to abnormal mammogenesis/lactogenesis, obesity, irregular humoral or cell-mediated immunity, defects in reproduction of both sexes and many other pathologic changes (Brisken et al. 1999, Freeman et al. 2000, McMurray 2001, Ben-Jonathan et al. 2006, Ignacak et al. 2012). It has been proved that PRL homeostasis in vivo is maintained and modulated by dopamine and estrogen signaling. Dopaminergic agonists are effective PRL secretion suppressors by their acting on D2 receptor (DRD2), which modulate Prl gene expression as well as the high intrinsic secretory activity of lactotrophs in vivo (Elsholtz et al. 1991, Ben-Jonathan & Hnasko 2001, Grattan 2015). Estrogen, conversely, stimulates PRL synthesis and secretion within the rodent lactotroph through the genomic regulation of Prl gene expression, the cell proliferation of lactotrophs and the modification of the lactotroph responsiveness to other factors mainly by binding to its nuclear receptor, Estrogen receptor 1 (alpha) (ESR1) (Chen & Meites 1970, Seo et al. 1979, West & Dannies 1980, Lieberman et al. 1981, Shull & Gorski 1985, Pelletier et al. 2003, Kansra et al. 2005, Nolan & Levy 2009, Grattan 2015). In addition, estrogen acts in the hypothalamus to inhibit the expression and the activity of dopamine synthetase tyrosine hydroxylase (TH), which subsequently modulates PRL homeostasis (Morel et al. 2009). Furthermore, it has been reported that EGF has the function to stimulate PRL release dependent upon ESR1 (Ben-Jonathan et al. 2009). Insulin and thyrotropin releasing hormone enhance the Prl gene expression primarily at the transcriptional level (Evans et al. 1978, Stanley 1988). Although numbers of factors modifying PRL synthesis and secretion have been identified, the related mechanisms and the molecular network in these signaling pathways have not been fully elucidated.
miRNA are evolutionarily conserved small noncoding RNAs which regulate gene expressions in various biological processes (Ambros 2004, Lewis et al. 2005). Up to date, numerous reports indicate that miRNA participate in the pituitary development as well as the hormone synthesis and secretion (Sivapragasam et al. 2011, Voglova et al. 2016, Cao et al. 2018). For example, it has been reported that the levels of miR-511 and miR-593 increase in the children with combined pituitary hormone deficiency (CPHD), which target the pituitary specific transcription factor prop paired-like homeobox 1 (PROP1) (Hu et al. 2015). miR-7a2 critically regulates FSH and LH synthesis and secretion through their effects on pituitary prostaglandin and bone morphogenetic protein 4 (BMP4) signaling (Ahmed et al. 2017). miR-26b targets lymphoid enhancer binding factor 1 (LEF-1), thus indirectly induces PRL, TSHβ and GH expressions (Zhang et al. 2010).
miR-375, which has been previously identified to be a key endocrine factor, participates in the regulation of multiple hormones. In pancreatic islets, miR-375 is one of the most abundant miRNA and affects insulin secretion by controlling β-cell mass and stimulating the exocytosis in β-cells (Poy et al. 2004, 2009, Eliasson 2017). In rat adrenal medulla, miR-375 negatively regulates the synthesis and secretion of catecholamines by targeting Sp1 (Gai et al. 2017). In addition, our previous study has shown that miR-375 is highly expressed in the intermediate lobe of mouse pituitary and regulates proopiomelanocortin (POMC) expression in corticotrophs through mediating the signaling pathway of corticotropin-releasing factor (CRF) and targeting mitogen-activated protein kinase 8 (MAPK8). In fact, the expression of miR-375 is almost five times higher in mouse pituitary than adrenal gland (Zhang et al. 2013). However, the cell type expressing miR-375 and the defined functions of miR-375 in the pituitary gland have not been well elucidated.
The present study was thus proposed to elucidate the physiological functions and the related mechanisms of miR-375 in anterior lobes of pituitary. We initially defined miR-375’s expression in mouse pituitary lactotrophs and subsequently determined its significance in modulating PRL synthesis by establishing miR-375 knockout mice. Upon further researches, Esr1 was proved to be regulated by miR-375 through Rasd1. These novel findings firstly demonstrate that miR-375 promotes PRL production through ESR1 signaling, and suggest that the miRNA are important factors regulating the synthesis of pituitary hormones.
Materials and methods
Mice maintenance and generation of miR-375 knockout mice
All mice were housed in a controlled temperature (25 ± 1°C), humidity (60 ± 5%) and photoperiod (12 h light: 12 h darkness cycle) environment with ad libitum access to regular water and food. MiR-375 knockout mouse line was generated by CRISPR-Cas9 genome editing technique. Briefly, the target sequences were designed by using an online CRISPR design tool (http://crispr.mit.edu) in which high scored sequences were selected and synthesized. The double-stranded target sequences were, respectively, inserted into a pGM-T3-sgRNA vector and subsequently transcribed to obtain specific sgRNAs for miR-375 gene editing. Meanwhile, the Cas9 mRNA was also transcribed. In this study, 20 ng/µL Cas9 mRNA and 40 ng/µL sgRNAs mixtures were injected into zygotes derived from crossed 6- to 8-week-old ICR female mice by using an Eppendorf microinjection system. The embryos were then cultured in 5% CO2, 37°C incubators for 24 h and put into the oviduct of pseudopregnant female ICR mice. Neonatal and adult miR-375 mutant mice and their respective littermate controls were used for analyses. All animal experiments were approved by the Chinese Association for Laboratory Animal Sciences.
Real-time PCR
Total RNAs were extracted from mouse tissues or cultured cells using the Trizol reagent (TaKaRa) according to the manufacturer’s instructions. Complementary DNAs were synthesized using M-MLV RT reagents (Promega). Gene expression levels were measured using SYBR Green master mix (TaKaRa) in the ABI PRISM 7500 Sequence Detection System (Applied Biosystems). The endogenous expressions of U6 and Gapdh were used for normalization of miR-375 and gene expression. The RT and real-time PCR primers for miR-375 and U6 were as previously described (Zhang et al. 2013). Other primer sequences were listed in Supplementary Table 1 (see section on supplementary materials given at the end of this article).
Western blots
Mouse tissues or cultured cells were lysed by radioimmunoprecipitation assay (RIPA) buffer (9806; Cell Signaling) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (8553S; Cell Signaling). The protein concentrations were determined using a bicinchoninic acid (BCA) assay reagent (Vigorous Biotechnology, Beijing, China) according to the manufacturer’s instructions. Samples containing equal amount proteins were electrophoresed on a 12% SDS-PAGE, and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories), which were subsequently blocked with 5% (w/v) non-fat dry milk for 1 h and incubated with either: anti-PRL antibody (1:1000; NHPP, Torrance, CA, USA), anti-ESR1 antibody (1:1000; Neomarkers, USA), anti-RASD1 antibody (1:2000; Santa Cruz Biotechnology)) and internal control anti-GAPDH antibody (1:10,000; Ambion) overnight at 4°C. The membranes were then washed three times in tris-buffered saline with tween-20 (TBST) and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10,000; Zymed, CA, USA) or HRP-conjugated goat anti-rabbit IgG (1:10,000; Zymed) for 1 h at room temperature. At last, the membranes were washed and treated with the SuperSignal West Pico Kit (Thermo Scientific) substrate at room temperature.
In situ hybridization (ISH)
The expression of miR-375 in the pituitary gland was determined using digoxigenin-labeled locked nucleic acid probes (Exiqon, Woburn, MA). In situ hybridization was performed according to our previous report (He et al. 2018). Briefly, dried frozen sections of mouse pituitary glands were fixed in fresh 4% paraformaldehyde for 10 min at room temperature and washed in 1× PBS for 2× 3 min. The sections were then subjected to acetylation for 10 min and washed for 10 min in 1× PBS. After 8 h prehybridization at room temperature, the sections were hybridized at 54°C overnight. The sections were then washed with 5× SSC for 10 min and 0.2× SSC for 1 h at 60°C. After blocking for 1 h at room temperature, the sections were incubated with alkaline phosphatase-conjugated antibody to digoxigenin at 4°C overnight. The sections were then washed and incubated in Nitrotetrazolium blue chloride (NBT) and 5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) in the dark for staining.
Immunofluorescence staining
The pituitary glands were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 5 μm slices, and then dewaxed, rehydrated. After antigen retrieval by microwaving in 0.01 mol/L sodium citrate buffer (pH 6.0), the sections were blocked with 10% normal donkey serum at room temperature for 1 h and then incubated with anti-PRL antibody (1:150) at 4°C overnight. After washing, the sections were subsequently incubated with the secondary antibody cy2-conjuncted donkey anti-rabbit IgG (1:200; 711-225-152; Jackson ImmunoResearch) for 1 h at room temperature. The nuclei were stained by propidium iodide (PI) for 10 min. Finally, sections were mounted with Vectashield (H-1000; Vector Laboratories) and photographed under a fluorescence microscope photograph system (Leica Microsystems). All the positive cell counts were accomplished by the blinded observer.
ISH and immunofluorescence dual staining
ISH was completed according to the procedures above. The sections were then treated with 10% normal donkey serum in PBS and incubated with anti-PRL antibody (1:150), anti-FSHβ antibody (1:100; NHPP, Torrance, CA, USA), anti-LHβ antibody (1:100; NIDDK, Bethesda, MD, USA) or anti-TSHβ antibody (1:200; NIDDK) overnight at 4°C. After washing, the sections were subsequently incubated with the secondary antibody, Cy2-conjuncted donkey anti-rabbit IgG for 1 h at room temperature. Finally, sections were mounted and photographed as demonstrated above.
Radioimmunoassay (RIA)
Samples of serum and cell culture media were collected as indicated. PRL, FSH and LH concentrations were measured by using RIA reagents (Beijing North Institute Biological Technology, Beijing, China) according to the manufacturer’s procedures. The experiments were accomplished on a XH-6080 RIA system (CNNC Xi'an nuclear instrument factory, Xi'an, Shanxi, China).
Cell culture and transfections
GH4 cell line was grown in Dulbecco’s modified eagle medium (DMEM) (Invitrogen) containing 10% FBS (Invitrogen) and 1% penicillin–streptomycin at 37°C in a humidified atmosphere of 5% CO2. MiR-375 mimics (miR-375-mi), inhibitors (miR-375-in) and their respective negative controls were purchased from Gene Pharma (Shanghai, China). Lipofectamin2000 agent (Invitrogen Life Technologies) was used for transfections according to the manufacturer’s instructions. Rasd1 siRNA kit was purchased from RiboBio (Guangzhou, China). The cells and media were collected after 24 h and stored at −80°C before assays. For each assay, at least three independent experiments were conducted.
Bioinformatic analysis and dual-luciferase reporter assay
Potential miR-375 targets were searched and selected using miRNA.org and TargetScan (http://www.targetscan.org) databases. The psiCHECKTM-2 vector (Promega) was used for the dual-luciferase reporter assay. Initially, WT and mutant 3’-UTR sequences of Rasd1 mRNA were cloned by overlap PCR and were subsequently inserted into the vector to construct dual-luciferase reporter plasmids. 293T cells were transiently transfected with miR-375-mi (or nc-mi) and plasmids (WT or mutant) using Lipofectamine2000 reagent. The luciferase activities were measured by ModulusTM II microplate multimode reader. All transfection experiments were performed independently at least three times.
Statistical analysis
Data and statistical analyses were performed using GraphPad Prism 8.0 software. The results were expressed as means ± s.e.m. of at least three independent experiments. The differences among groups were determined using a Student’s t test or one-way ANOVA. P < 0.05 was considered to be statistically significant.
Results
miR-375 is expressed in the lactotrophs of mouse pituitary gland
We initially examined miR-375 in different tissues of adult mouse using real-time PCR. The results showed that miR-375 expression level in pituitary was 27 times higher than other tissues examined (Fig. 1A). In addition, we assayed miR-375 expression in the developmental pituitaries from 14.5 dpc (day post coitus) to the adult, and the results demonstrated that miR-375 kept rising in the duration examined and reached a maximum at adult (Fig. 1B). We then detected the locations of miR-375 in adult pituitary using ISH and found that miR-375 strongly expressed in the anterior and intermediate lobes whereas no miR-375 ISH staining was observed in the posterior lobe (Fig. 1C). The Further double staining of miR-375 with pituitary hormones revealed that about 52.2% PRL-positive cells expressed miR-375, but the dual staining of miR-375, respectively, with LHβ and FSHβ was not observed, although a few TSHβ positve cell expressed miR-375. These results infer that miR-375 involves in regulating pituitary PRL synthesis.
miR-375 is expressed in the mouse pituitary gland. miR-375 expressions in different tissues (A) and developmental stages (B) were analyzed by real-time PCR and normalized to U6. One-way ANOVA was used, F(A) = 946.5, F(B) = 24.29, P < 0.0001, n = 3. (C)ISH detection of miR-375 in the mouse putuitary gland. Bars: 100 μm. (D) ISH and immunofluorescence dual staining of miR-375 (Red) and PRL, FSHβ, LHβ or TSHβ (Green) in mouse pituitary glands. Bars: 60 μm. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 is expressed in the mouse pituitary gland. miR-375 expressions in different tissues (A) and developmental stages (B) were analyzed by real-time PCR and normalized to U6. One-way ANOVA was used, F(A) = 946.5, F(B) = 24.29, P < 0.0001, n = 3. (C)ISH detection of miR-375 in the mouse putuitary gland. Bars: 100 μm. (D) ISH and immunofluorescence dual staining of miR-375 (Red) and PRL, FSHβ, LHβ or TSHβ (Green) in mouse pituitary glands. Bars: 60 μm. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 is expressed in the mouse pituitary gland. miR-375 expressions in different tissues (A) and developmental stages (B) were analyzed by real-time PCR and normalized to U6. One-way ANOVA was used, F(A) = 946.5, F(B) = 24.29, P < 0.0001, n = 3. (C)ISH detection of miR-375 in the mouse putuitary gland. Bars: 100 μm. (D) ISH and immunofluorescence dual staining of miR-375 (Red) and PRL, FSHβ, LHβ or TSHβ (Green) in mouse pituitary glands. Bars: 60 μm. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 is essential for PRL synthesis
In order to identify the function of miR-375 in the pituitary gland, the miR-375 knockout mice were generated using Crispr/Cas9 technique (Fig. 2A and B). The ISH and real-time PCR results confirmed that miR-375 was deleted in homozygotes (Fig. 2C and D), although no morphological and histological differences in the pituitary gland were observed between the WT and miR-375 knockout mice.
miR-375 knockout mice were generated using Crispr/Cas9 technique. (A) Target sequences for miR-375 gene editing and miR-375 knockout sequence after editing. (B) Genotyping of miR-375 WT, knockout heterozygous and knockout homozygous mice. MiR-375 knockout efficiency of different genotypes were examined by real-time PCR (C) and ISH (D) Real-time PCR results were means ± s.e.m. of three independent experiments and normalized to U6. Bars: 100 μm. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 knockout mice were generated using Crispr/Cas9 technique. (A) Target sequences for miR-375 gene editing and miR-375 knockout sequence after editing. (B) Genotyping of miR-375 WT, knockout heterozygous and knockout homozygous mice. MiR-375 knockout efficiency of different genotypes were examined by real-time PCR (C) and ISH (D) Real-time PCR results were means ± s.e.m. of three independent experiments and normalized to U6. Bars: 100 μm. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 knockout mice were generated using Crispr/Cas9 technique. (A) Target sequences for miR-375 gene editing and miR-375 knockout sequence after editing. (B) Genotyping of miR-375 WT, knockout heterozygous and knockout homozygous mice. MiR-375 knockout efficiency of different genotypes were examined by real-time PCR (C) and ISH (D) Real-time PCR results were means ± s.e.m. of three independent experiments and normalized to U6. Bars: 100 μm. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
We then assayed the FSHβ, LHβ, TSHβ and PRL expressions in WT and miR-375 knockout mouse pituitaries. The immunofluorescence results showed that PRL-positive cell number decreased by 64.4% in miR-375 knockout pituitaries (Fig. 3A) but did not show significant changes in FSHβ, LHβ, TSHβ positive cell numbers (Supplementary Fig. 1). The real-time PCR and Western blot results showed that the PRL mRNA and protein levels in knockout mouse pituitaries decreased by 75.7 and 60.4%, respectively, compared with the controls (Fig. 3B and C). In addition, we assayed the serum PRL, FSH and LH concentrations by RIA, and the results showed that miR-375 knockout markedly decreased serum PRL concentrations by 46.1%, parallel to the mRNA and protein levels, but there was no significant effect either on serum FSH or LH concentration (Fig. 3D). These data demonstrate that miR-375 has function to enhance the PRL synthesis.
miR-375 knockout results in reduce of adult mouse pituitary PRL synthesis. (A) Immunofluorescence staining of PRL (Green) and nucleus (Red) of WT and knockout mouse pituitaries and relative statistical data. Bar: 100 μm. (B) Relative mRNA levels of different hormones in WT and knockout pituitaries were assayed by real-time PCR and normalized to Gapdh. (C) PRL protein levels in WT and knockout pituitaries were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. (D) Hormone concentrations in WT and knockout mouse serums were assayed by RIA, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 knockout results in reduce of adult mouse pituitary PRL synthesis. (A) Immunofluorescence staining of PRL (Green) and nucleus (Red) of WT and knockout mouse pituitaries and relative statistical data. Bar: 100 μm. (B) Relative mRNA levels of different hormones in WT and knockout pituitaries were assayed by real-time PCR and normalized to Gapdh. (C) PRL protein levels in WT and knockout pituitaries were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. (D) Hormone concentrations in WT and knockout mouse serums were assayed by RIA, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 knockout results in reduce of adult mouse pituitary PRL synthesis. (A) Immunofluorescence staining of PRL (Green) and nucleus (Red) of WT and knockout mouse pituitaries and relative statistical data. Bar: 100 μm. (B) Relative mRNA levels of different hormones in WT and knockout pituitaries were assayed by real-time PCR and normalized to Gapdh. (C) PRL protein levels in WT and knockout pituitaries were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. (D) Hormone concentrations in WT and knockout mouse serums were assayed by RIA, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 has no significant effect on pituitary PRL expression in the duration of pituitary development
In order to identify whether miR-375 modulate PRL levels through affecting pituitary lactotroph development, we assayed the effects of miR-375 knockout on the PRL-positive cell number and Prl mRNA expression using immunofluorenscence and real-time PCR at PND1 (postnatal day), PND7, PND14 and PND21. The results showed that neither PRL immunofluorescence positive cell number nor Prl mRNA levels had a significant difference between the miR-375 knockout mice and controls at PND1, PND7 and PND14, but the PRL-positive cell and Prl mRNA decreased by 64.5 and 75.4%, respectively, in miR-375 knockout mice compared with the WT mice at PND21 (Fig. 4A and B). These confirm that miR-375 has the function to enhance pituitary PRL synthesis without affecting pituitary lactotroph development.
miR-375 knockout affects Esr1 expression in adult mouse pituitaries. (A) Immunofluorescence staining of PRL (Green) and nucleus (Red) of WT and knockout mouse pituitaries in developing stages. Bar: 100 μm. (B) Relative Prl mRNA levels of WT and knockout pituitaries in developing stages were assayed by real-time PCR and normalized to Gapdh. (C) Relative mRNA levels of PRL regulation related genes in WT and knockout mouse pituitaries were assayed by real-time PCR and normalized to Gapdh. (D) PRL protein levels in WT and knockout pituitaries were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 knockout affects Esr1 expression in adult mouse pituitaries. (A) Immunofluorescence staining of PRL (Green) and nucleus (Red) of WT and knockout mouse pituitaries in developing stages. Bar: 100 μm. (B) Relative Prl mRNA levels of WT and knockout pituitaries in developing stages were assayed by real-time PCR and normalized to Gapdh. (C) Relative mRNA levels of PRL regulation related genes in WT and knockout mouse pituitaries were assayed by real-time PCR and normalized to Gapdh. (D) PRL protein levels in WT and knockout pituitaries were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 knockout affects Esr1 expression in adult mouse pituitaries. (A) Immunofluorescence staining of PRL (Green) and nucleus (Red) of WT and knockout mouse pituitaries in developing stages. Bar: 100 μm. (B) Relative Prl mRNA levels of WT and knockout pituitaries in developing stages were assayed by real-time PCR and normalized to Gapdh. (C) Relative mRNA levels of PRL regulation related genes in WT and knockout mouse pituitaries were assayed by real-time PCR and normalized to Gapdh. (D) PRL protein levels in WT and knockout pituitaries were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH, n = 4. *P < 0.05, **P < 0.01, ***P < 0.001. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 directly targets Rasd1 in mouse pituitary
In order to investigate the mechanism of miR-375 affecting PRL synthesis, we detected the gene expressions, including Pit-1, Ser1, Drd2, Ets-1, Igf1, Cdk4, Hes1, Notch2, Tgfb and Esr2, in the miR-375 knockout and control pituitary glands by real-time PCR. These genes are acknowledged to be pivotal to influence the PRL synthesis. We found that Esr1 and Drd2 mRNAs were significantly lower than WT mice (Fig. 4C). The further Western blot results showed that ESR1 protein level decreased by 42.9% in the miR-375 knockout mice, compared to WT mice (Fig. 4D). These results suggest that miR-375 probably affects Prl gene expression and PRL synthesis through ESR1.
Further, to find out the direct target of miR-375 in lactotrophs, we searched miRNA.org and TargetScan databases, predicted the potential miR-375 target genes, in which the genes related to PRL synthesis were selected, including Rasd1, Map3k8, Igfbp, Pax6 and Tcf4. Meanwhile, Prl and Esr1 were predicted not to be the potential miR-375 direct target. Thus the mRNAs of the above genes in miR-375 knockout and WT mouse pituitary glands were assayed. The real-time PCR results showed that the pituitary Rasd1 mRNA level in the miR-375 knockout mice was 2.2 times higher than WT mice (Fig. 5A), and the RASD1 protein level was also significantly upregulated (Fig. 5B).
Rasd1 is a direct target of miR-375. (A) Relative mRNA levels of Gh in WT and knockout pituitaries was assayed by real-time PCR and normalized to Gapdh. (B) RASD1 protein levels in WT and knockout pituitaries were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. (C) The predicted binding sites of miR-375 in the 3’UTR of mouse Rasd1 gene and strategies for vector constructions were shown. (D) Effects of miR-375 mimics and inhibitors on the transiently transfected Rasd1 3’UTR fused to luciferase reporter vectors were assayed, n = 4. *P < 0.05. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
Rasd1 is a direct target of miR-375. (A) Relative mRNA levels of Gh in WT and knockout pituitaries was assayed by real-time PCR and normalized to Gapdh. (B) RASD1 protein levels in WT and knockout pituitaries were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. (C) The predicted binding sites of miR-375 in the 3’UTR of mouse Rasd1 gene and strategies for vector constructions were shown. (D) Effects of miR-375 mimics and inhibitors on the transiently transfected Rasd1 3’UTR fused to luciferase reporter vectors were assayed, n = 4. *P < 0.05. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
Rasd1 is a direct target of miR-375. (A) Relative mRNA levels of Gh in WT and knockout pituitaries was assayed by real-time PCR and normalized to Gapdh. (B) RASD1 protein levels in WT and knockout pituitaries were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. (C) The predicted binding sites of miR-375 in the 3’UTR of mouse Rasd1 gene and strategies for vector constructions were shown. (D) Effects of miR-375 mimics and inhibitors on the transiently transfected Rasd1 3’UTR fused to luciferase reporter vectors were assayed, n = 4. *P < 0.05. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0001.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
To verify whether Rasd1 was a direct target of miR-375, we conducted dual-luciferase reporter assay. The Rasd1 putative 3’UTR sequences of WT and mutant were cloned and inserted into the multiple cloning sites of a psiCHECKTM-2 vector, respectively (Fig. 5C). These WT or mutant vectors, together with miR-375-mi or nc-mi were co-transfected to the 293T cells. The results showed that miR-375 downregulated the activities of the WT 3’UTR about 36.1% but no effect was detected for the mutant (Fig. 5D). These indicate that miR-375 directly binds to Rasd1 3’UTR to regulate its expression.
miR-375 modulates Esr1 expression through Rasd1
To further investigate the mechanisms of miR-375 in regulating PRL synthesis, we transfected pituitary lactotroph GH4 cells with miR-375-mi and miR-375-in, respectively. Real-time PCR results showed that miR-375-mi upregulated miR-375 level by 12.2-fold while miR-375-in downregulated miR-375 level by 80.6% (Fig. 6A), which, respectively, resulted in the elevation or reduction of Prl mRNA levels in the cultured GH4 cells and the PRL concentrations in the culture media (Fig. 6B and C). In addition, Western blot analyses revealed that the ESR1 protein level increased 1.3-fold, whereas RASD1 protein was downregulated about 38.3% after 24 h miR-375-mi transfection. Conversely, miR-375-in transfection had contrary effects on ESR1 and RASD1 protein levels, which, respectively, decreased by 34.8% and increased by 1.3-fold (Fig. 6D). These results confirmed that miR-375 regulated PRL synthesis through Esr1 and Rasd1, consistent with the in vivo results.
miR-375 modulates Esr1 expression through Rasd1. Cultured GH4 cells were transfected with miR-375 mimics, inhibitors or their respective negative controls for 24 h. (A) Relative miR-375 expression in cells were assayed by real-time PCR and normalized to Gapdh. (B) Relative Prl mRNA expression in cells were assayed by real-time PCR and normalized to Gapdh. (C) PRL concentrations in culture media were assayed by RIA. (D) Protein levels of ESR1 and RASD1 in cells were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. Cultured GH4 cells were transfected with Rasd1 siRNA or its negative control for 24 h. (E) Protein levels of ESR1 and RASD1 in cells were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. (F) PRL concentrations in culture media were assayed by RIA, n = 4. *P < 0.05, **P < 0.01.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 modulates Esr1 expression through Rasd1. Cultured GH4 cells were transfected with miR-375 mimics, inhibitors or their respective negative controls for 24 h. (A) Relative miR-375 expression in cells were assayed by real-time PCR and normalized to Gapdh. (B) Relative Prl mRNA expression in cells were assayed by real-time PCR and normalized to Gapdh. (C) PRL concentrations in culture media were assayed by RIA. (D) Protein levels of ESR1 and RASD1 in cells were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. Cultured GH4 cells were transfected with Rasd1 siRNA or its negative control for 24 h. (E) Protein levels of ESR1 and RASD1 in cells were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. (F) PRL concentrations in culture media were assayed by RIA, n = 4. *P < 0.05, **P < 0.01.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
miR-375 modulates Esr1 expression through Rasd1. Cultured GH4 cells were transfected with miR-375 mimics, inhibitors or their respective negative controls for 24 h. (A) Relative miR-375 expression in cells were assayed by real-time PCR and normalized to Gapdh. (B) Relative Prl mRNA expression in cells were assayed by real-time PCR and normalized to Gapdh. (C) PRL concentrations in culture media were assayed by RIA. (D) Protein levels of ESR1 and RASD1 in cells were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. Cultured GH4 cells were transfected with Rasd1 siRNA or its negative control for 24 h. (E) Protein levels of ESR1 and RASD1 in cells were assayed by Western blot. Relative protein levels were analyzed by gray scanning and normalized to GAPDH. (F) PRL concentrations in culture media were assayed by RIA, n = 4. *P < 0.05, **P < 0.01.
Citation: Journal of Endocrinology 250, 1; 10.1530/JOE-20-0001
A further attempt with Rasd1-siRNA transfection to knockdown Rasd1 was conducted to verify whether Rasd1 was involved in the pathway of miR-375 regulating Esr1. Real-time PCR results showed that the RASD1 protein level in transfected GH4 cells decreased about 43.7%, whereas ESR1 protein level and PRL concentration were markedly elevated (Fig. 6E and F). These collected data suggest that miR-375 regulates Esr1 expression by targeting Rasd1, and finally regulates PRL synthesis.
Discussion
Our previous study has shown that miR-375 is highly expressed both in the anterior and intermediate lobes (Zhang et al. 2013). The in vivo and in vitro results of the present study firstly demonstrate that miR-375 co-localizes with PRL and acts as a positive factor of PRL synthesis in mouse pituitary gland but has no significant effects on the development of the pituitary gland and PRL cell differentiation.
It has been proved that miR-375 has multiple physiological functions, including its regulating effects on hormone synthesis and secretion, such as insulin, catecholamines and POMC. In pancreatic islets, miR-375 negatively regulates insulin secretion through a direct effect on insulin exocytosis (Poy et al. 2004), and miR-375 knockout results in a decrease of β-cell mass due to impaired proliferation, whereas insulin secretion per cell is enhanced (Poy et al. 2009). Negative effects of miR-375 are also discovered in the regulation of catecholamines in the adrenal medulla and POMC in the pituitary gland, through the modulation of expression and synthesis of both hormones (Zhang et al. 2013, Gai et al. 2017). However, the results presented here showed that miR-375 behaves a positive effect on the synthesis of PRL. In support, we have shown that the pituitary PRL expression and global PRL concentration are markedly decreased in miR-375 knockout mice, which have been confirmed by the in vitro overexpression and knockdown of miR-375 in GH4 cells, whereas there are no significant effects on the cell proliferation, apoptosis (Supplementary Fig. 2) and pituitary development. In addition, we examined other hormones produced in anterior pituitary lobes, and did not observe any changes in the pituitary Fshβ, Lhβ, Tshβ and Gh mRNAs and the relative global hormone levels, although somatotroph shared a common progenitor with lactotroph and showed co-localizations of GH with miR-375 (Supplementary Fig. 3). These data highlights the importance of miR-375 in regulating pituitary PRL synthesis.
A variety of factors have been described to affect PRL synthesis and secretion in rodents, in which dopamine and estrogen signaling have been clearly defined. Dopamine, synthesized and released from hypothalamus, is a major hormone suppressing pituitary PRL synthesis and secretion (Elsholtz et al. 1991, Ben-Jonathan & Hnasko 2001). In our initial results, it has been shown that the miR-375 expression in the hypothalamus is much lower than the pituitary gland, and as expected, miR-375 knockout did not have a significant influence on the mRNA levels of the dopamine synthetases tyrosine hydroxylase (TH) and its transporter (DAT), compared with the WT mouse (Supplementary Fig. 4). We observed; however, the mRNA level of its receptor DRD2 significantly decreased in miR-375 knockout. But since dopamine signaling negatively regulates PRL synthesis and secretion, this change should not lead to the decrease of PRL levels. These suggest that the lack of PRL in miR-375 knockout mouse is not due to the change of dopamine signaling.
In addition, the results of the present study demonstrate that miR-375 regulates pituitary PRL synthesis by targeting Rasd1 andsubsequently influences Esr1 expression. Initially, the real-time PCR results revealed a reduction of Esr1 mRNA in miR-375 knockout mouse pituitary gland and subsequent in vitro transfections of miR-375-mi/in confirmed its positive role in regulating Esr1 expression. Estrogen receptor was shown to be involved in the Prl gene transcription by binding to a response element of Prl gene (Day et al. 1990, Anderson & Gorski 2000). Besides, ESR1 plays a dominant role in estrogen signaling regulating PRL production, rather than ESR2 (Scully et al. 1997, Pelletier et al. 2003, Kansra et al. 2005). However, using bioinformatics technique, we have confirmed neither Prl nor Esr1 gene contains potential binding sites for miR-375 in gene sequences. Instead, in a list of genes, we further predicted and selected, Rasd1 was proved to be a direct target of miR-375, which has been verified both in vivo and in vitro. These findings are consistent with the results in breast cancer cells in which miR-375 targets Rasd1 and subsequently regulates Esr1 expression (de Souza Rocha Simonini et al. 2010), which infer that miR-375 – Rasd1 – Esr1 pathway involves in regulating multiple physiological processes in different tissue or/and cell types. In fact, the repressive interaction between miR-375 and Rasd1 is partially conserved across evolution as predicted by TargetScan database. The conserved binding site can be observed in 80% species analyzed, such as human, rhesus, squirrel, mouse, rat, cat, dog and even non-mammalian vertebrates like alligator, parrot, sparrow and chicken. Combining with above-mentioned miR-375 – Rasd1 – Esr1 pathway in different species and tissues, this would suggest a pathophysiological role to this miRNA in the context of pathologies in which the decrease or increase in PRL levels could be associated with alterations in miR-375 levels.
It is known that adaptations of global PRL level are facilitated during pregnancy and lactation, mostly through the alteration of hypothalamic and ovarian feedbacks (Grattan 2015, Grattan et al. 2008). Our results have provided that miR-375 is a novel factor influencing basal PRL synthesis in pituitary lactotrophs during non-pregnant stage, which acts independently of both dopamine and estrogen. We could hypothesize that miR-375 might also functions during pregnancy and lactation, but it still needs further verifications. And meanwhile, as mentioned above, miR-375 has multiple physiological functions. Thus the global miR-375 knockout would definitely result in other effects despite PRL, for instance, the alteration of catecholamines and POMC levels. The study of these potential effects will lead to a better understanding of miR-375’s biological functions and will provide a basis for the clinical application of miR-375.
In the present study, we demonstrate a novel mechanism modulating PRL synthesis in mouse pituitary lactotrophs. In brief, we addressed that miR-375 highly expresses in the mouse anterior pituitary and locates in lactotrophs. MiR-375 knockout mouse model helps to establish miR-375’s positive role on PRL synthesis, which is mediated by the modulation of Esr1 expression. Moreover, Rasd1 is proved to be the direct target of miR-375 in lactotrophs. Together, we have highlighted the importance of this miR-375 mediated pathway in the maintenance of PRL homeostasis through regulating PRL synthesis.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-20-0001.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the National Key Research and Development Program of China (grant number 2018YFC1003500); the National Natural Science Foundation of China (grant numbers 31430083, 31772692); the Natural Science Foundation of Jiangsu Province (grant number BK20190879); and the Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Author contribution statement
Sheng Cui conceived and designed the experiments; Jinglin Zhang, Di Zhang, and Jie Gao carried out the experiments; Jinglin Zhang, Jie Gao and Kemian Gou analyzed the data; Hui Liu provided the reagents/materials/analysis tools; Sheng Cui and Jinglin Zhang wrote the manuscript.
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