MiRNA-143 mediates the proliferative signaling pathway of FSH and regulates estradiol production

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   Figure 1

   MiR-143 expression in the mouse ovary. (A) Real-time PCR analysis of miR-143 in the developing mouse ovary. (B) Relative miR-143 expression levels in isolated granulosa cells and oocytes by real-time PCR. GCs, granulosa cells; Oo, oocyte. Data are shown as mean ± s.e.m. of 3 samples for each group. (C) Localization of miR-143 in mouse ovary using LNA in situ hybridization. Primordial, primary, secondary and mature represent staged follicle. All the scale bars represent 100 μm (**P< 0.01).
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   Figure 2

   MiR-143 regulates estradiol and steroidogenesis-related genes expression. Periovulatory granulosa cells from large follicles stimulating by PMSG were cultured. (A) Overexpression and inhibition efficiency of miR-143 mimics or inhibitor, transfected into granulosa cells after 24 h. NC-in/NC-mi, miRNA inhibitor/mimics nonsense control, inhibitor: miR-143 inhibitor; mimics: miR-143 mimics. Results are mean ± s.e.m. of 3 independent experiments, ***P< 0.001 (t-test). (B) and (C) Granulosa cells were transfected with miR-143 mimics and mimics control, or miR-143 inhibitor and inhibitor control. The culture media were collected for measuring estradiol and progesterone levels at 24 h after transfection. Results are mean ± s.e.m. of 4 independent experiments, ***P< 0.001 (t-test). (D) and (E) mRNA levels of steroidogenesis-related enzymes expression in the granulosa cells were measured by real-time PCR after being transfected with an inhibitor and mimics for 24 h. Results are mean ± s.e.m. of 3 independent experiments done in triplicate and normalized to their respective control (*P< 0.05; **P< 0.01 and ***P< 0.001, by ANOVA. NS, not statistically significant). (F) Analysis of the 3B-HSD protein. Granulosa cells were transfected for 24 h and protein extracts were analyzed by Western blotting. (G) Quantification of 3B-HSD protein levels. Results are mean ± s.e.m. of 4 independent experiments; (*P< 0.05, by t-test). Granulosa cells transfected with miR-143 mimics and mimics control, or miR-143 inhibitor and inhibitor control for 24 h. (H) Western blotting analysis of CYP19A1 in granulosa cells transfected with miR-143 mimics and mimics control, or miR-143 inhibitor and inhibitor control for 24 h. (I) Quantification of CYP19A1 protein levels. Results are mean ± s.e.m. of 3 independent experiments; (*P< 0.05, by t-test). (J) Western blotting analysis of 17B-HSD in granulosa cells transfected with miR-143 mimics and mimics control, or miR-143 inhibitor and inhibitor control for 24 h. (K) Quantification of 17B-HSD protein levels. Results are mean ± s.e.m. of 4 independent experiments; (*P< 0.05, by t-test).
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   Figure 3

   FSH decreased miR-143 expression in vivo and in vitro. In vitro, Periovulatory granulosa cells from large follicles stimulating by PMSG were cultured. (A) Expression of miR-143 in estrous cycle ovaries by real-time PCR and normalized to the U6 transcript level. P, proestrus stage; E, estrous stage; M, metestrus; DI, diestrus. (B) Expression levels of miR-143 in the ovary from 0 to 48 h after injection of PMSG and HCG. Results are mean ± s.e.m. of 3 independent experiments, **P< 0.01 (t-test). (C) miR-143 levels at 0 (control), 6, 12, and 24 h after incubating the granulosa cells with 100ng/ml FSH. Results are mean ± s.e.m. of 4 independent experiments, **P< 0.01 (t-test). (D) Expression levels of miR-143 were measured by real-time PCR after incubating the granulosa cells with 0 (control), 1, 10 and 100 ng/mL FSH for 6 h. Results are mean ± s.e.m. of 4 independent experiments, **P< 0.01 (t-test). (E) After several signal pathway inhibitors including 20 μM of CH(PKC), H89 (PKA), PD98059 (ERK), SB203580 (p38 MAPK), and SP600125 (JNK) (final concentration 20 μM) and 100 ng/mL FSH treatment for 24 h, intracellular miR-143 levels in the granulosa cells were assayed by real-time PCR. Data are as shown as mean ± s.e.m. (n = 3). Significance differences are indicated by *P< 0.05, **P< 0.01, ***P< 0.001 and NS means not significant (by t-test). (F) MRNA levels of miR-143 at 0 (control), 6, 12, and 24 h after incubating the granulosa cells with 1 U/ml LH. Results are mean ± s.e.m. of 3 independent experiments done in triplicate and normalized to their respective control (*P< 0.05; **P< 0.01 and ***P< 0.001 by t-test).
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   Figure 4

   MiR-143 is involved in the FSH signaling pathway. Periovulatory granulosa cells from large follicles stimulating by PMSG were cultured. (A) and (B) Estradiol secreted by granulosa cells. The cells were transfected with either miR-143 inhibitors or miR-143 mimics. Twenty-four hours later, the cells were treated with FSH (100 ng/mL) for 12 h. Results are mean ± s.e.m. of 4 independent experiments, *P< 0.05 (t-test). (C) and (D) mRNA levels of CYP19A1. Results are mean ± s.e.m. of 4 independent experiments done in triplicate and normalized to their respective control. (*P< 0.05; **P< 0.01 by ANOVA).
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   Figure 5

   Effects of miR-143 on granulosa cell proliferation. Periovulatory granulosa cells from large follicles stimulating by PMSG were cultured then transfected with miR-143 mimics or inhibitors for 24 h. (A) Granulosa cell proliferation was measured by using BrdU analysis. All the scale bars represent 100 μm. (B) Percentages of BrdU-positive cells accounting for the total granulosa cells. (C) and (D) mRNA levels of cell cycle-related genes were measured by real-time PCR. NC-in or NC-mi were used as inhibitor or mimics controls. Results are mean ± s.e.m. of 3 independent experiments done in triplicate and normalized to their respective control (*P< 0.05, **P< 0.01 and ***P< 0.001 by t-test).
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   Figure 6

   MiR-143 binds to KRAS-3ʹUTR, which regulates its protein expression. (A) The predicted miR-143 binding site in CREB5-3ʹUTR and KRAS-3ʹUTR from www.miRNA.org. (B) Schematic of inserted CREB5-3ʹUTR and KRAS-3ʹUTRs sequences. (C) The psi-CHECKTM-2 reporter vector map. (D) and (E) Relative luminescence intensity detected by the Modulus TMII microplate multimode reader after miR-143 mimics and dual-luciferase vector were co-transfected into 293T cells for 24 h. Results are means ± s.e.m. of three independent experiments done in triplicate and normalized to their respective control. (**P< 0.01. NS, not statistically significant). (F) Quantification of KRAS mRNA levels. Periovulatory granulosa cells from large follicles stimulating by PMSG were cultured. Granulosa cells were transfected with an NC-inhibitor (NC-in), miR-143 inhibitor, NC-mimics (NC-mi), miR-143 mimics for 6 h. The data are mean ± s.e.m.. for multiple separate transfections (n = 3). (NS, not statistically significant by t-test). (G) Analysis of the KRAS protein. Granulosa cells were transfected an NC-inhibitor (NC-in), miR-143 inhibitor, NC-mimics (NC-mi), miR-143 mimics for 24 h and protein extracts were analyzed by Western blotting. (H) Quantification of KRAS protein levels. Results are mean ± s.e.m. of three independent experiments (*P< 0.05; NS, not significant). Data are presented as mean ± s.e.m. (n = 3) (P< 0.05, by t-test).
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   Figure 7

   KRAS functions in regulating estradiol stimulated by FSH. Periovulatory granulosa cells from large follicles stimulating by PMSG were cultured. (A) Expression levels of KRAS mRNA were measured by real-time PCR after incubating the GCs with 0 (control), 1, 10 and 100 ng/mL FSH for 6 h. Data are presented as mean ± s.e.m.. (n = 4) (*P< 0.05 by t-test). (B) KRAS protein expression levels were analyzed by Western blotting after incubating the granulosa cells with 0 (control), 1, 10 and 100 ng/mL FSH for 24 h. (C) Quantification of KRAS protein levels. Data are presented as mean ± s.e.m. (n = 3) (*P< 0.05, by ANOVA). (D) KRAS protein expression levels were analyzed by Western blotting after the granulosa cells transfected with the NC-inhibitor (NC-in), miR-143 inhibitor, NC-mimics (NC-mi), miR-143 mimics for 24 h. (E) Quantification of KRAS protein levels. Data are presented as mean ± s.e.m. (n = 3) (*P< 0.05 by ANOVA). (F) Quantification of intracellular KRAS mRNA levels after the granulosa cells were transfected with KRAS siRNA1-3 for 24 h. Data are presented as mean ± s.e.m. (n = 3) (*P< 0.05 by ANOVA). (G) Estradiol levels were detected after transfecting nc siRNA or KRAS siRNA1 and 24 h later, 100 ng/ml FSH was added to granulosa cells and were treated for 12 h (n = 4) (*P< 0.05 by t-test).

• © 2017 Society for Endocrinology