Abstract
Leydig cells are essential for male reproductive development throughout life. Production of androgens as well as intermediate steroids is tightly regulated. Although microRNAs (miRNAs) are suggested to play important roles in spermatogenesis, little is currently known regarding the regulation of steroidogenesis by miRNAs in Leydig cells. Here, we found that miR-150 was predominantly expressed in Leydig cells within mouse testis. Therefore, we determined steroidogenesis of the Leydig cells in which miR-150 was knocked down or overexpressed using miR-150 antagomir and agomir, respectively. Compared with negative control group, a significant increase of STAR expression was observed in miR-150 antagomir-treated Leydig cells. Conversely, STAR expression was significantly reduced in miR-150 agomir-transfected Leydig cells. Production of sex-steroid precursors and testosterone of Leydig cells was also negatively controlled by miR-150. We further identified Star as a target of miR-150 using luciferase reporter assay. Finally, we confirmed that miR-150 was necessary for steroidogenesis and spermatogenesis in vivo via intratesticular injection of miR-150 antagomir or agomir. Taken together, our studies suggest that miR-150 negatively regulates the expression of STAR and steroidogenesis of Leydig cells in mice.
Introduction
Leydig cells dominate the interstitial space after puberty, which transform from stem Leydig cells to adult Leydig cells (Mendis-Handagama & Ariyaratne 2001, Haider 2004). The main role of adult Leydig cells is to produce steroids, including testosterone. Testosterone from Leydig cells is essential to initiate, maintain and regulate spermatogenesis; thus, malfunctions of Leydig cells cause reduced androgen synthesis, leading to aspermatogenesis (O’Shaughnessy 2014, Smith & Walker 2014, Chen & Liu 2015). Enzyme-catalyzed reactions contribute to testosterone synthesis, in which steroidogenic acute regulatory (STAR) protein mediates the transportation of cholesterol into mitochondria (Clark & Stocco 1997). Cholesterol is converted to pregnenolone by cytochrome P450 SCC, and further converted to progesterone by 3b-hydroxysteroid dehydrogenase (3B-HSD) (Payne & Hales 2004). Cytochrome P450, family 17 (CYP17A1) subsequently activates the metabolism of progesterone to androstenedione. Finally, testosterone is metabolized from androstenedione by 17-hydroxysteroid dehydrogenase (HSD17B1, B3) (Grossmann et al. 2013). Spermatogenesis will be disrupted by the deficiencies of steroid hormone biosynthesis and steroidogenic enzyme expression in adult Leydig cells (Caron et al. 1997). However, the critical regulators of steroidogenic enzyme expression are poorly explored.
MicroRNAs (miRNAs) are a class of small non-coding RNA (~22 nt) to control pathophysiological processes such as cell proliferation, differentiation and apoptosis by regulating their mRNA targets (Bartel 2004, Plasterk 2006). In male reproductive system, miRNAs play essential roles in various processes of spermatogenesis by modulating the expression of their targets (Tang et al. 2007, Hayashi et al. 2008, Bouhallier et al. 2010, Wang & Xu 2015). However, the studies of miRNAs mainly focus on spermatogonial stem cells, spermatocyte meiosis, spermiogenesis and Sertoli cells (reviewed in Wang & Xu 2015, Pratt & Calcatera 2016). The studies of the role of miRNAs in Leydig cells are limited in comparison. A previous study suggests that miR-125a and miR-455, by targeting steroidogenic SR-BI, negatively regulate steroid hormone production (Hu et al. 2012). A significant increase of Leydig cell number is observed in miR-140-knockout embryos (Rakoczy et al. 2013). Moreover, stem Leydig cell commitment is determined by basic fibroblast growth factor (bFGF), which regulates several miRNA (miR-29a, -29c, -142, -451 and -335) expression (Liu et al. 2014).
In this study, we showed that steroidogenesis and STAR expression were negatively regulated by miR-150 in primary Leydig cells. In detail, miR-150 can bind directly to the 3′UTR of Star and negatively regulate STAR expression. This present study will be helpful for understanding the role of miRNAs in testosterone-producing Leydig cells.
Materials and methods
Cell isolation and culture
Animal experiments were approved by the Institutional Committee on the Ethics of Animal Experiments of The Second Affiliated Hospital of Zhengzhou University (approval ID: ZZU-2015-43). C57BL/6 mice were purchased from Zhengzhou University. Germ cells, Sertoli cells and peritubular myoid cells were isolated using a second-step enzymatic digestion with collagenase 4, hyaluronidase, trypsin and DNase, as previously described (Chang et al. 2011, Chen et al. 2014, 2015). Leydig cells were isolated from 3-month-old C57BL/6 male mice by the combination of enzyme digestion and Percoll separation, as previously described (Akingbemi et al. 2001, Chang et al. 2011, Guang-Yu et al. 2016). Briefly, seminiferous tubules were placed into 10 mL of enzymatic solution consisting of 200 mg/mL DNase 1 and 0.5 mg/mL collagenase 1A. Tubules were digested for 10 min at 37°C in a shaking water bath, and then layered over 40 mL of 5% Percoll. After stewing for 20 min, the top 35 mL of Percoll was collected and centrifuged at 500 g for 10 min. The sediment was suspended in 55% Percoll, and centrifuged at 20,000 g for 30 min. Finally, the top 2 mL was discarded, and 5 mL fraction was collected from the top of the tube. Isolated Leydig cells were cultured in DMEM/F-12 with 5% FBS and 0.5% antibiotics. Immunofluorescence staining for P450 SCC (ABS236, Millipore) was used to determine the cell purity of isolated Leydig cells, as previously described (Guang-Yu et al. 2016). Leydig cell markers, such as 3b-Hsd, Star and Lhr, were highly expressed in isolated Leydig cells (Supplementary Fig. 2, see section on supplementary data given at the end of this article). Relative low expressions of Wt1 (a Sertoli cell marker), Vasa (a germ cell marker) and Myh11 (a myoid cell marker) were observed in isolated Leydig cell samples (Supplementary Fig. 2).
miR-150 antagomir and agomir
Primary Leydig cells were cultured in 6-well plates at a density of 1 × 106 cells. Complete medium without antibiotics was used to culture the cells at least 24 h prior to transfection. miR-150 antagomir (5′-CAC UGG UAC AAG GGU UGG GAG A-3′), miR-150 agomir (5′-GUG ACC AUG UUC CCA ACC CUC U-3′) and their negative controls (NC) were chemically synthesized by GenePharma (Shanghai, China). All of the bases were 2′-OMe modified. A final concentration of 50 nM miR-150 antagomir, miR-150 agomir or NC was transfected into Leydig cells by Lipofectamine 2000 (Invitrogen). 72 h after transfection, Leydig cells and their conditioned media were collected separately for further analysis.
Intratesticular injection was performed as previously described (Wu et al. 2015). Briefly, 4-week-old male mice were anesthetized and the testes were then exteriorized via abdominal incision. miR-150 antagomir or miR-150 agomir (10 μg) was mixed with Entranster in vivo transfection reagent (Engreen, Beijing, China), and then injected into rete testis. An equal amount of antagomir NC or agomir NC was injected into the other testis. qRT-PCR and Western blot were analyzed after 72 h. Morphology and hormone assays were determined after 1 week.
Star siRNA and Star adenovirus
In order to silence Star expression, a small interfering RNA (siRNA) against mouse Star was obtained (5′-GGG CUG UUA AAU AGA GAA AUU GCC CU-3′, Santa Cruz, CA, USA) and a scramble siRNA (5′-CGA GAA AUA AGA GUC ACA AUC CUG CU-3′) was employed as a control. Transfection was performed using 40 nM siRNA by Lipofectamine 2000 (Invitrogen). Star adenovirus was purchased from GenePharma (Shanghai, China). 72 h after transfection, Leydig cells and their conditioned media were collected separately for further analysis.
Steroid hormone measurements
Hormone level in conditioned media of Leydig cells were determined by liquid chromatography-tandem mass spectrometry (LC–MS/MS), as described previously (Keski-Rahkonen et al. 2011, Guang-Yu et al. 2016). Steroid separation and subsequent quantification in conditioned media of Leydig cells were detected using an Agilent 1200 Series HPLC system connected to a mass spectrometer (Agilent 6410 Triple Quadrupole LC/MS). The amounts of testosterone produced by cultured Leydig cells were measured with tritium-based radioimmunoassay (RIA), as previously described (Cochran et al. 1981). For serum hormone measurement, the blood was collected from mouse eyeballs and was left standing for 1 h, followed by centrifugation at 2000 g for 30 min. For testicular testosterone detection, tunica albuginea was removed and testicular homogenates were prepared with 0.9% saline. After centrifugation at 7200 g for 30 min, the supernatant was collected. The levels of serum testosterone, FSH, LH as well as the levels of testicular testosterone were detected by RIA kits (Neobioscience, Beijing, China), according to the manufacturer’s instructions.
miR-150 probe
miR-150 staining was performed in adult mouse testes by a specific probe. The horse radish peroxidase (HRP)-labeled probe to miR-150 was obtained from BioGenex (San Francisco, CA, USA). Hybridizations were operated overnight in a 60°C drying oven. Testicular sections were further stained with diaminobenzidine (DAB) and hematoxylin and the signal was visualized by light microscope (Shanghai Optical Instrument Factory, Shanghai, China).
Quantitative(q) RT-PCR
miRcute miRNA isolation kit was used to collect total RNA. Reverse transcription of purified miRNA was performed using miRcute miRNA first-strand cDNA synthesis kit. miRcute miRNA qPCR detection kit was used to conduct qRT-PCR. All kits were purchased from TIANGEN (Beijing, China). Relative expression of miR-150 was determined by the comparative Ct method (2ΔΔCt) after normalization to U6 (a housekeeping miRNA). Primers were all purchased from TIANGEN (Beijing, China). For mRNA analysis, qRT-PCR was performed as described everywhere (Chen et al. 2016). Primers of Star, Cyp11a1, Hsd3b1, Cyp17a1, Wt1, 3b-Hsd, Lhr, Vasa, Myh11 and Gapdh were designed using primerbank (http://pga.mgh.harvard.edu/primerbank/) and chemically synthesized by Sangon biotech (Beijing, China).
Western blot analysis
Western blot was carried out on Leydig cell extracts as described everywhere (Chen et al. 2016). 30 μg of proteins were electrophoresed on 10% SDS-PAGE and then transferred onto nitrocellulose (NC) membranes. Immunoblotting was performed using the following antibodies: anti-STAR (ab58013, Abcam) and anti-B-ACTIN (ab8226, Abcam).
Luciferase reporter assay
The full-length 3′UTR of Star was cloned by standard procedures into the pMIR-Report plasmid (Ambion), immediately downstream of stop codon of the luciferase gene (pMIR-Star-3′UTR luciferase reporter plasmid). Mutagenesis of the binding site of miR-150 of pMIR-Star-3′UTR plasmid was performed using a QuikChange site-directed mutagenesis kit (TIANGEN). MA-10 Leydig cells were maintained in 25-well plates and co-transfected with 200 ng wild-type pMIR-Star-3′UTR (or with mutated pMIR-Star-3′UTR), 20 pmol of either miR-150 agomir or agomir NC and 50 ng Renilla by Lipofectamine 2000 (Invitrogen). At 48 h after transfection, cells were lysed and luciferase activity was measured with the dual luciferase kit (Promega).
H&E staining and TUNEL assay
One week after intratesticular injection, testes were collected and fixed in 4% paraformaldehyde (PFA), embedded in paraffin and sectioned into 5 μm slices, which were stained with hematoxylin–eosin (H&E), as described everywhere. Apoptosis was analyzed by colorimetric TUNEL apoptosis assay kit (Beyotime, Shanghai, China), according to the manufacturer’s instructions.
Statistical analysis
Data are presented as mean ± s.e.m. for three independent experiments. Differences between means were examined using one-way ANOVA or t-test function of GraphPad Prism 5. Differences between means were considered significant at *P < 0.05 or **P < 0.01.
Results
miR-150 was predominantly expressed in mouse Leydig cells
Firstly, we found that miR-150 was expressed in various organs, including brain, stomach, lung, liver, kidney, spleen, muscle, small intestine, testis and ovary (Supplementary Fig. 1). Among them, miR-150 was expressed at a higher level in stomach, liver and testis. Then, we explored the expression of miR-150 within testis by qualitative and quantitative analysis. Enzyme digestion and Percoll-density gradient separation were used to isolate primary Leydig cells from 3-month-old mouse testicular tissues (Guang-Yu et al. 2016). Cell purity was assessed by immunofluorescence detection for Leydig cell-specific P450 SCC protein (Fig. 1A) and RT-PCR of more Leydig cell marker genes (Supplementary Fig. 2). Based on staining, the purity of Leydig cells was approximately 91% (Fig. 1B). Furthermore, we observed that, within the testis, miR-150 was predominantly expressed in Leydig cell population, while it exhibited a low expression in isolated Sertoli cells, germ cells and myoid cells (Fig. 1C). Using miR-150 probe staining, we detected that miR-150 was largely confined to Leydig cells, which are located in the interstitial region (Fig. 1D).
miR-150 was predominantly expressed in mouse Leydig cells. (A) Isolation of primary Leydig cells from 3-month-old mouse testes. Purity of Leydig cell cultures, as assessed by immunofluorescence staining for the Leydig cell marker P450 SCC (green). Cell nuclei were counterstained with DAPI (blue). Star (white) indicated P450 SCC-negative cells. Scale bars, 50 μm. (B) Proportion of P450 SCC-positive and -negative cells in the primary cultures. All quantitative data are mean ± s.e.m. of three different cultures. **P < 0.01 by Student’s t-test. (C) miR-150 relative level in different testicular cells, including germ cells, Sertoli cells, Leydig cells and peritubular myoid cells. Housekeeping U6 served as a loading control. (D) miR-150 probe was restricted to Leydig cells (L) in mouse adult testis section. Scale bars, 100 μm.
Citation: Reproduction 154, 3; 10.1530/REP-17-0234
Gain-of-function and loss-of-function strategies in primary Leydig cells
After obtaining primary Leydig cells, endogenous miRNAs regulating Leydig steroidogenesis were firstly studied in vitro. Thus, we investigated the role of miR-150 in the regulation of cultured Leydig cells in vitro using miRNA antagomir and agomir. qRT-PCR analysis showed that miR-150 level was downregulated by 65% in primary Leydig cells transfected by miR-150 antagomir, compared with Leydig cells treated with antagomir negative control (NC) (Fig. 2A). Conversely, miR-150 expression was significantly increased (more than 25-fold) in primary Leydig cells after miR-150 agomir treatment, compared with Leydig cells transfected by agomir NC (Fig. 2B).
Gain-of-function and loss-of-function strategies in primary Leydig cells. (A) Relative expression of miR-150 in mouse Leydig cells transfected with miR-150 antagomir or antagomir negative control (NC). (B) miR-150 abundance in miR-150 agomir and agomir NC-treated primary Leydig cells. Housekeeping U6 served as a loading control. All quantitative data are mean ± s.e.m. of three different cultures. **P < 0.01 by Student’s t-test.
Citation: Reproduction 154, 3; 10.1530/REP-17-0234
miR-150 negatively regulates the production of sex-steroid precursors
A series of steroidogenic enzymes were in charge of testosterone production from cholesterol in Leydig cells (Fig. 3A). We explored the effects of miR-150 in controlling the steroidogenesis of Leydig cells in vitro using above gain-of-function and loss-of-function strategies. Significant higher levels of pregnenolone (Fig. 3B), progesterone (Fig. 3C), androstenedione (Fig. 3D), androstenedione (Fig. 3E) and testosterone (Fig. 3F) were observed in the media from miR-150 antagomir-transfected Leydig cells, compared with antagomir NC-transfected cells. Conversely, miR-150 overexpression by the induction of miR-150 agomir significantly reduced the levels of these sex-steroid precursors and testosterone in Leydig conditioned media (Fig. 3B, C, D, E and F).
Steroid hormone measurements in the conditioned media sampled from Leydig cells after treatment. (A) A schematic drawing that illustrates the synthetic pathway from cholesterol to testosterone in mouse adult testes. The concentrations of (B) pregnenolone, (C) progesterone, (D) androstenedione, (E) androstanedione or (F) testosterone observed in conditioned media from primary Leydig cells treated with miR-150 antagomir, miR-150 agomir or NCs. All quantitative data are mean ± s.e.m. of three different cultures. *P < 0.05 by Student’s t-test.
Citation: Reproduction 154, 3; 10.1530/REP-17-0234
miR-150 negatively regulates the expression of Star, a steroidogenic gene, in Leydig cells
The deficient testosterone production in miR-150 agomir-treated Leydig cells implied that the transcription of steroidogenic factors was regulated by miR-150. Thus, we detected the expression of steroidogenic enzymes, such as Star, Cyp11a1, Hsd3b1 and Cyp17a1 in Leydig cell samples transfected by miR-150 antagomir/agomir or their NCs (Fig. 4). The mRNA level of Star was significantly upregulated in Leydig cells after miR-150 antagomir treatment, while a significant decrease in transcript abundance was observed for Star in miR-150 agomir-treated Leydig cells (Fig. 4A). However, the transcript expression of other testosterone synthesis-related enzymes, such as Cyp11a1 (Fig. 4B), Hsd3b1 (Fig. 4C) and Cyp17a1 (Fig. 4D), was not altered. Furthermore, miR-150 antagomir treatment elevated the protein level of STAR, while miR-150 agomir treatment attenuated STAR protein expression (Fig. 4E and F). Collectively, these results suggest that miR-150 negatively regulates the production of sex-steroid precursors likely by targeting STAR, a protein facilitating the transport of cholesterol to mitochondria for steroid synthesis.
Relative expressions of steroidogenic synthetases following miR-150 antagomir or miR-150 agomir treatment. Relative mRNA abundance of (A) Star, (B) Cyp11a1, (C) Hsd3b1 and (D) Cyp17a1 in miR-150 antagomir-, miR-150 agomir- or NC-treated primary Leydig cells. The mRNA quantities were normalized to Gapdh. (E, F) Protein level of STAR in miR-150 antagomir-, miR-150 agomir- or NC-treated primary Leydig cells. All quantitative data are mean ± s.e.m. of three different cultures. *P < 0.05 by Student’s t-test.
Citation: Reproduction 154, 3; 10.1530/REP-17-0234
miR-150 targets Star in Leydig cells
We further explore the mechanism of miR-150 in regulation of STAR and steroidogenesis by luciferase reporter assay. Using TargetScan (http://www.targetscan.org) prediction, we observed that miR-150 can bind to the 3′UTR region of Star mRNA through its conserved binding site (Fig. 5A). Then, the luciferase reporters (wild-type or mutated 3′UTR of Star) and pre-miR-150 were transfected into MA-10 Leydig cells. We observed that the luciferase activity of Star wild-type 3′UTR was significantly repressed by pre-miR-150 (Fig. 5B), while the reporter activity of Star mutated 3′UTR was not altered by pre-miR-150 (Fig. 5C). Thus, luciferase reporter assay suggests that miR-150 targets Star in Leydig cells. Moreover, we found that downregulation of miR-150 could reverse the decreased concentration of testosterone by Star silencing (Fig. 5D); conversely, overexpression of miR-150 rescued the increased concentration of testosterone after Star adenovirus treatment (Fig. 5E). Collectively, we conclude that miR-150 negatively regulates the expression of STAR in the steroidogenesis pathway.
Identification of Star as a target of miR-150. (A) The miRNA target pairing sequence within the 3′UTR of the Star gene. (B, C) Luciferase assays using reporter vectors containing the wild-type Star 3′UTR seed sequence (WT) or the mutant construct in which mutations were introduced into the seed sequence (MUT). MA-10 Leydig cells were transfected with the pre-miR-150 or NC in combination with one of the reporter constructs. (D, E) The concentration of testosterone in conditioned media from primary Leydig cells treated with Star siRNA or Star siRNA in combination with miR-150 antagomir, Star adenovirus (Ad) or Star Ad in combination with miR-150 agomir. All quantitative data are mean ± s.e.m. of three different cultures. *P < 0.05 by Student’s t-test.
Citation: Reproduction 154, 3; 10.1530/REP-17-0234
miR-150 is necessary for the steroidogenesis in vivo
Finally, we tested whether miR-150 is necessary for steroidogenesis to regulate spermatogenesis in the animal model. Gain of function and loss of function of miR-150 in testes were performed by intratesticular injection of miR-150 agomir or miR-150 antagomir, respectively (Fig. 6A). We observed that the protein level of STAR in testes was significantly upregulated by miR-150 antagomir intratesticular injection. By contrast, miR-150 agomir intratesticular injection caused significant downregulation of STAR protein in testes (Fig. 6B). In correlation with an increase of STAR expression in testes, serum testosterone (T) level was significantly elevated in miR-150 antagomir intratesticular-injected mice (Fig. 6C). In correlation with a decrease of STAR expression in testes, serum T level was significantly declined in miR-150 agomir intratesticular-injected mice (Fig. 6C). Similarly, the level of testicular T was significantly increased in miR-150 antagomir intratesticular-injected mice, while it was significantly decreased in miR-150 agomir intratesticular-injected mice (Fig. 6D). However, we found that the levels of serum FSH and LH were not significantly altered after miR-150 antagomir or agomir intratesticular treatment (Fig. 6E and F). H&E staining further indicated that spermatogenesis was severely disrupted in miR-150 antagomir intratesticular-injected testes, compared with antagomir NC group. However, thickness of seminiferous epithelium was observed after miR-150 agomir intratesticular injection (Fig. 6G). Furthermore, we found that miR-150 agomir injection induced massive apoptosis of germ cells, which might be the cause of the disrupted spermatogenesis in miR-150 antagomir intratesticular-injected testes (Fig. 6H). In combination with in vitro studies, we suggest that ‘miR-150-STAR’ pathway plays an essential role in supporting steroidogenesis and spermatogenesis.
miR-150 is necessary for STAR expression and spermatogenesis in vivo. (A) miR-150 antagomir, miR-150 agomir, or NCs was injected into adult mouse testes. Three days after intratesticular injection, the relative level of miR-150 was determined in antagomir or agomir intratesticular-injected testes. (B) Protein level of STAR in miR-150 antagomir, miR-150 agomir, and NC intratesticular-injected injected testes. Serum T (C), FSH (E), and LH (F) levels, as well as testicular T level (D) in miR-150 agomir-, antagomir-, and NC intratesticular-injected mice. All quantitative data are mean ± s.e.m. of three different cultures. *P < 0.05 by Student’s t-test. (G) Histological sections of miR-150 antagomir, miR-150 agomir and NC intratesticular-injected testes analyzed by H&E staining. (H) TUNEL analysis. Scale bars, 100 μm.
Citation: Reproduction 154, 3; 10.1530/REP-17-0234
Discussion
miRNAs have been shown to play a role in almost all developmental processes (Pauli et al. 2011, Suh & Blelloch 2011). In reproductive system, miRNAs play essential roles in various processes of spermatogenesis via modulating the expression of the targets (Tang et al. 2007, Hayashi et al. 2008, Bouhallier et al. 2010, Wang & Xu 2015). However, their function in Leydig steroidogenesis has not been studied in detail. Here, we focused our analysis on miR-150 and showed that miR-150 was predominantly expressed in Leydig cells and played an essential role in steroidogenesis by targeting STAR.
Antagomir and agomir were utilized to knockdown and overexpress miR-150 in Leydig cells, respectively. In this study, we found that overexpression of miR-150 in primary Leydig cells by agomir significantly downregulated STAR expression and the production of pregnenolone, progesterone, androstenedione and testosterone. Silencing of miR-150 in Leydig cells by antagomir exhibited perfectly opposite effects. These in vitro studies were further confirmed by intratesticular injection of either miR-150 agomir or antagomir. Downregulation of STAR expression and failure of spermatogenesis in miR-150 agomir intratesticular-injected testes proved that ‘miR-150-STAR’ pathway plays an essential role in supporting both steroidogenesis and spermatogenesis.
In the steroid biosynthesis pathway, STAR is an essential protein that mediates the transfer of cholesterol into mitochondria where it is cleaved to pregnenolone (Chen et al. 1995). The miRNAs inhibit the expression of specific genes by either degrading the target mRNA or direct translational inhibition (Carrington & Ambros 2003, Meister & Tuschl 2004). A previous study suggests that Star may be a target gene of miR-376b, miR-150, miR-330 and miR-138 in mouse Leydig tumor cell line, MLTC-1 (Hu et al. 2013). Here, we provided evidence that miR-150 functions as a direct negative regulator of steroidogenic STAR. miR-150 can directly repress STAR protein expression through its binding to specific binding site (GGUAUAA–AUUGGGAG) in the 3′UTR of the mouse Star gene, thereby negatively regulating steroidogenesis of adult Leydig cells. Importantly, miR-150 can rescue the effect of STAR on the production of testosterone.
The role of most studied miRNAs is based on the in vitro studies of knockdown or overexpression (Bjork et al. 2010, He et al. 2013, Yang et al. 2013). Few miRNAs were examined for their in vivo function by knockout mouse models (Bao et al. 2012, Tong et al. 2012, Chen et al. 2017). In the future, it is essential to generate miR-150-knockout mice and is plausible to postulate that Leydig cell deficiency in expression of miR-150 will lead to the deficiency of steroidogenesis and could be an underlying cause of male infertility.
In conclusion, the present study provides novel data that miR-150 negatively regulates the expression of STAR in Leydig cells. These results may be valuable for understanding the regulatory mechanism of steroidogenesis in Leydig cells and diseases associated with the dysfunction of Leydig cells.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-17-0234.
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
The research was funded by a grant from the Key Programs of the Zhengzhou University (No. 315508).
Author contribution statement
Li Tan conceived and designed the experiments. Xu-Jing Geng and Dong-Mei Zhao performed the experiments. Gen-Hong Mao analyzed the data. Li Tan wrote the paper.
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