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. 2019 Mar 1;14:1-19.
doi: 10.1016/j.omtn.2018.10.012. Epub 2018 Oct 25.

miR-202-3p Regulates Sertoli Cell Proliferation, Synthesis Function, and Apoptosis by Targeting LRP6 and Cyclin D1 of Wnt/β-Catenin Signaling

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Free PMC article

miR-202-3p Regulates Sertoli Cell Proliferation, Synthesis Function, and Apoptosis by Targeting LRP6 and Cyclin D1 of Wnt/β-Catenin Signaling

Chao Yang et al. Mol Ther Nucleic Acids. .
Free PMC article

Abstract

MicroRNAs (miRNAs) play important roles in mammalian spermatogenesis, which is highly dependent on Sertoli cells. However, the functions and mechanisms of miRNAs in regulating human Sertoli cells remain largely unknown. Here, we report that hsa-miR-202-3p mediates the proliferation, apoptosis, and synthesis function of human Sertoli cells. miR-202-3p was upregulated in Sertoli cells of Sertoli cell-only syndrome (SCOS) patients compared with obstructive azoospermia (OA) patients with normal spermatogenesis. Overexpression of miR-202-3p induced Sertoli cell apoptosis and inhibited cell proliferation and synthesis, and the effects were opposite when miR-202-3p was knocked down. Lipoprotein receptor-related protein 6 (LRP6) and Cyclin D1 of the Wnt/β-catenin signaling pathway were identified as direct targets of miR-202-3p in Sertoli cells, which were validated by bioinformatics tools and dual-luciferase reporter assay. Differentially expressed LRP6 and Cyclin D1 between OA and SCOS Sertoli cells were also verified. LRP6 small interfering RNA (siRNA) interference not only mimicked the effects of miR-202-3p overexpression, but also antagonized the effects of miR-202-3p inhibition on Sertoli cells. Collectively, miR-202-3p controls the proliferation, apoptosis, and synthesis function of human Sertoli cells via targeting LRP6 and Cyclin D1 of the Wnt/β-catenin signaling pathway. This study thus provides a novel insight into fate determinations of human Sertoli cells and niche of human testis.

Keywords: Cyclin D1; LRP6; Wnt/β-catenin; human Sertoli cells; miR-202-3p; proliferation and apoptosis; synthesis.

Figures

Figure 1
Figure 1
Isolation and Identification of Human Sertoli Cells from OA and SCOS Patients (A) RT-PCR showed the transcripts of GDNF, GATA4, SOX9, WT1, BMP4, and SCF in the isolated cells. PCR with PBS but without cDNA served as a negative control. (B) Western blots showed the protein levels of BMP4, SCF, and GDNF in OA and SCOS Sertoli cells. (C–L) Immunofluorescence demonstrated the expression of SOX9 (C), GATA4 (D), WT1 (E), VIM (F), GDNF (G), OCLN (H), SCF (I), VASA (J), α-SMA (K), and CYP11A1 (L) in the isolated cells. Replacement of primary antibodies with PBS was used as a negative control (M). The cell nuclei were stained with DAPI. Scale bars, 5 μm (C–M).
Figure 2
Figure 2
Differentially Expressed miR-202-3p Inhibits the Proliferation of Human Sertoli Cells (A) Real-time qPCR revealed the expression of miR-202-3p in both OA and SCOS Sertoli cells (n = 20). (B and C) CCK-8 assay showed the growth curve of human Sertoli cells for 5 days in the pre-miR group (B) and the pre-miR inhibitor group (C) after virus infection and puromycin screening. (D) EDU incorporation assay showed the EDU-positive cells in human Sertoli cells after virus infection and puromycin screening. Cell nuclei were counterstained with Hoechst 33342. The percentages of EDU-positive cells were counted out of 500 total cells from three independent experiments. (E) Immunofluorescence revealed the ki-67-positive cells in the four cell strains. Cell nuclei were counterstained with DAPI. The percentages of ki-67-positive cells were counted out of 500 total cells from three independent experiments. (F) Western blots demonstrated the expression of PCNA and cell-cycle proteins in human Sertoli cells at 72 hr after virus infection and puromycin screening. β-actin served as a loading control of proteins. Results of the pre-miR group were normalized to the Normal ctrl group, and results of the pre-miR inhibitor group were normalized to the inhibitor ctrl group. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars, 10 μm (D and E).
Figure 3
Figure 3
miR-202-3p Inhibits the Synthesis Function of Human Sertoli Cells The mRNA levels of GDNF (A), SCF (B), BMP4 (C), FGF2 (D), CXCL12 (E), and EGF (F) at 72 hr after virus infection and puromycin screening. (G) Western blots demonstrated GDNF, SCF, BMP4, FGF2, and CXCL12 proteins in human Sertoli cells at 72 hr after virus infection and puromycin screening. Results of three independent assays were concluded in (H). β-actin served as loading control of proteins. (I and J) CCK-8 assay showed the growth curve of a human spermatogonial stem cell line that was cultured with culture medium collected daily from the four Sertoli cell strains with upregulation of miR-202-3p (I) and downregulation of miR-202-3p (J). (K) Western blot demonstrated the expression of PCNA in human spermatogonial stem cell line after 72 hr of culture with culture medium collected daily from the four Sertoli cell strains. Results of the pre-miR group were normalized to the normal ctrl group, and results of the pre-miR inhibitor group were normalized to the inhibitor ctrl group. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4
Figure 4
miR-202-3p Promotes the Apoptosis of Human Sertoli Cells (A–D) Annexin V-APC/PI and flow cytometry analysis revealed apoptosis in human Sertoli cells at 72 hr in the normal ctrl group (A), pre-miR group (B), inhibitor ctrl group (C), and pre-miR inhibitor group (D) after virus infection and puromycin screening. Results of three independent assays were concluded in (E). A total of 10,000 cells were analyzed. (F–I) TUNEL assay revealed the percentages of TUNEL+ cells in the normal ctrl group (F), pre-miR group (G), inhibitor ctrl group (H), and pre-miR inhibitor group (I) in human Sertoli cells after virus infection and puromycin screening. Results of three independent assays were concluded in (J). (K) Western blots demonstrated cPARP, Bcl2, and Bax proteins in human Sertoli cells at 72 hr after virus infection and puromycin screening. Results of three independent assays were concluded in (L). β-actin served as loading control of proteins. Results of the pre-miR group were normalized to the normal ctrl group, and results of the pre-miR inhibitor group were normalized to the inhibitor ctrl group. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars, 10 μm (F–I).
Figure 5
Figure 5
Identification of Direct Targets of miR-202-3p in Human Sertoli Cells (A) There were two potential binding sites of miR-02-3p at the 3′ UTR region of LRP6 and Cyclin D1 mRNA based on the bioinformatic analysis. 3′ UTR of LRP6 and Cyclin D1 mRNA containing wild-type (WT), mutant-1 (Mut1), and mutant-2 (Mut2) was cloned into dual-luciferase plasmids. (B) The mRNA levels of LRP6 and Cyclin D1 were detected using real-time qPCR in 20 paired OA and SCOS Sertoli cells. (C) Western blots revealed the different expression patterns of LRP6 and Cyclin D1 between OA and SCOS Sertoli cells (n = 8). β-actin served as a loading control. (D) Empty plasmids (psiCHECK2 control) and dual-luciferase plasmids containing WT, Mut1, or Mut2 of LRP6 or Cyclin D1 3′ UTR were transfected into HEK293T cells and OA Sertoli cells with miR-202-3p overexpression plasmids or control plasmids, respectively. Firefly and Renilla luciferase signals were performed for luciferase activity after 36 hr of transfection. (E) mRNA levels of LRP6, c-Myc, Cyclin D1, and β-catenin, as measured by real-time qPCR in the four cell strains. (F) Western blots showed the LRP6, c-Myc, Cyclin D1, β-catenin, phospho-β-catenin, and non-phospho-β-catenin protein levels at 72 hr after virus infection and puromycin screening. β-actin served as a loading control. Results of the pre-miR group were normalized to the normal ctrl group, and results of the pre-miR inhibitor group were normalized to the inhibitor ctrl group. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 6
Figure 6
LRP6 Knockdown Inhibits the Proliferation and Synthesis Function of Human Sertoli Cells (A) CCK-8 assay showed the growth curve of human Sertoli cells after transfection of control siRNA or LRP6 siRNA-3 for 1–5 days. (B) Western blots demonstrated the expression of PCNA after 48 hr of transfection of control siRNA or LRP6 siRNA-3. Results of three independent assays were concluded in (C). β-actin served as a loading control of proteins. (D) EDU incorporation assay showed the EDU-positive cells in human Sertoli cells at 48 hr after transfection of control siRNA or LRP6 siRNA-3. Results of three independent assays were concluded in (E). Cell nuclei were counterstained with Hoechst 33342. The percentages of EDU-positive cells were counted out of 500 total cells from three independent experiments. (F) Western blots demonstrated GDNF, SCF, BMP4, FGF2, and CXCL12 proteins in human Sertoli cells at 48 hr after transfection of control siRNA or LRP6 siRNA-3. Results of three independent assays were concluded in (G). β-actin served as a loading control of proteins. (H) CCK-8 assay showed the growth curve of human spermatogonial stem cell line that cultured with culture medium collected daily from Sertoli cells transfected with control siRNA or LRP6 siRNA-3. (I) Western blots demonstrated PCNA protein in human spermatogonial stem cell line after 72 hr of culture with culture medium collected daily from Sertoli cells transfected with control siRNA or LRP6 siRNA-3. Results of three independent assays were concluded in (J). β-actin served as a loading control of proteins. *p < 0.05; **p < 0.01. Scale bars, 20 μm (D).
Figure 7
Figure 7
LRP6 Knockdown Promoted Apoptosis of Human Sertoli Cells (A) Annexin-V and propidium iodide (PI) staining and flow cytometry showed the percentage of apoptosis in human Sertoli cells after LRP6 knockdown. Results of three independent assays were concluded in (B). A total of 10,000 cells were analyzed. (C) TUNEL assay revealed the percentages of TUNEL+ cells in human Sertoli cells transfected with control siRNA or LRP6 siRNA-3. Results of three independent assays were concluded in (D). (E) Western blots demonstrated cPARP, Bcl2, and Bax proteins in human Sertoli cells at 48 hr after transfection with control siRNA or LRP6 siRNA-3. Results of three independent assays were concluded in (F). β-actin served as loading control of proteins. **p < 0.01; ***p < 0.001. Scale bars, 10 μm (C).
Figure 8
Figure 8
LRP6 Knockdown Attenuated the Effects of miR-202-3p Inhibition on Sertoli Cells (A) CCK-8 assay displayed the proliferation of human Sertoli cells treated with miRNA inhibitor control, miR-202-3p inhibitor, and miR-202-3p inhibitor+LRP6 siRNA-3 for 5 days. (B) Western blot showed the expression of PCNA in Sertoli cells treated with miRNA inhibitor control, miR-202-3p inhibitor, and miR-202-3p inhibitor+LRP6 siRNA-3 at 48 hr. Results of three independent assays were concluded in (C). (D) Western blot showed the expression of GDNF, SCF, BMP4, FGF2, and CXCL12 in Sertoli cells treated with miRNA inhibitor control, miR-202-3p inhibitor, and miR-202-3p inhibitor+LRP6 siRNA-3 at 48 hr. Results of three independent assays were concluded in (E). (F) Annexin V and propidium iodide (PI) staining and flow cytometry showed the percentage of apoptosis in human Sertoli cells treated with miRNA inhibitor control, miR-202-3p inhibitor, and miR-202-3p inhibitor+LRP6 siRNA-3 at 48 hr. Results of three independent assays were concluded in (G). A total of 15,000 cells were analyzed. (H) Western blots demonstrated cPARP, Bcl2, and Bax proteins in human Sertoli cells treated with miRNA inhibitor control, miR-202-3p inhibitor, and miR-202-3p inhibitor+LRP6 siRNA-3 at 48 hr. Results of three independent assays were concluded in (I). *p < 0.05; **p < 0.01.

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