MicroRNA-574 Impacts Granulosa Cell Estradiol Production via Targeting TIMP3 and ERK1/2 Signaling Pathway

Abstract

Estradiol represents a key steroid ovarian hormone that not only plays a vital role in ovarian follicular development but also is associated with many other reproductive functions. Our primary study revealed that miR-574 expression decreased in porcine granulosa cells during development from small to large follicles, and the increase of ERK1/2 phosphorylation accompanies this change. Since it has been well established that the ERK1/2 activity is tightly associated with granulosa cell functions, including ovarian hormone production, we thus further investigate if the miRNA is involved in the regulation of estradiol production in granulosa cells. We found that overexpression of miR-574 decreased phosphorylated ERK1/2 without affecting the level of ERK1/2 protein, and on the other hand, the inhibition of miR-574 increased phosphorylated ERK1/2 level (P<0.05); meanwhile, overexpression of miR-574 increased estradiol production but knockdown of miR-574 decreased estradiol level in granulosa cells. To further identify the potential mechanism involved in the miR-574 regulatory effect, in silico screening was performed and revealed a potential binding site on the 3'UTR region of the tissue inhibitor of metalloproteinase 3 (TIMP3). Our gain-, loss- of function experiments, and luciferase reporter assay confirmed that TIMP3 is indeed the target of miR-574 in granulosa cell. Furthermore, the siRNA TIMP3 knockdown resulted in decreased phosphorylated ERK1/2, and an increase in estradiol production. In contrast, the addition of recombinant TIMP3 increased phosphorylated ERK1/2 level and decreased estradiol production. In summary, our results suggest that the miR-574-TIMP3-pERK1/2 cascade may be one of the pathways by which microRNAs regulate granulosa cell estradiol production.

Keywords: ERK1/2 phosphorylation; Estradiol; Granulosa cell; Ovary; TIMP3; miR-574; microRNA.

Figure 1

The expression of miR-574, pERK1/2 and TIMP3, during the antral follicular maturation. (A) The quantitative real-time PCR analysis of mature miR-574 in granulosa cells (GC) isolated from small-size (1-3 mm) and large-size (4-6 mm) follicles. (B) The quantitative real-time PCR analysis of miR-574 precursor in LGCs and SGCs. (C) The quantitative real-time PCR analysis of miR-574 in the follicular fluid (FF) isolated from small (S-FF) and large (L-FF) follicles. (D) Western blot analysis image showing increase pERK1/2 in LGC compared to SGC. (E) The quantitative real-time PCR analysis of TIMP3 mRNA in granulosa cells (GC) isolated from small- and large follicles. (F) Western blot analysis image showing increase TIMP3 in LGC compared to SGC. ns, no significant difference. Error bars represent means ± SD of at least three biological replicates and Asterisks denote a significant difference between groups (P<0.05) as determined by Student’s t-test.

Figure 2

Gain- and loss-of-function of miR-574 change phosphorylated ERK1/2 level and Estradiol production in cultured granulosa cells. Cultured follicles granulosa cells were transduced with lentivirus that either expressed microRNA574 (miR574) or red fluorescent protein (RFP). (A) Relative miR-574 level after lentivirus transduction, snoU6 was used to normalize the mature miR-574 expression; (B) Representative western blot image of pERK1/2 and ERK1/2 protein in granulosa cells after overexpression of miR-574; (C) Estradiol production was measured by ELISA assay Overexpression of miR-574 promoted granulosa cells to produce more estradiol. (D) Relative levels of miR-574 after transfection with specific siRNA for miR-574 (miR-574 siRNA; 20 pmol/ml) and negative control siRNA (NC; 20 pmol/ml) in the pig granulosa cell. snoU6 was used to normalize the mature miR-574 expression data. (E) Representative western blot image of pERK1/2 and ERK1/2 protein in response to miR-574 downregulation. (F) Suppression of miR-574 resulted in granulosa cells to produce less estradiol. The data represents the mean ± SE of three independent experiments. Asterisks denote statistically significant differences between treatment and control groups (p <0.05).

Figure 3

Over-expression of miR-574 decreases the activity of pERK1/2 indicated by the downregulation of pFA2-Elk1 luciferase activity. in vivo (A) The PathDetect in vivo signal transduction pathway trans-reporting systems was used to monitor the activation of ERK1/2 pathways. The PathDetect vectors containing a synthetic promoter with five tandem repeats of the GAL4 binding site, and pFA2-Elk1 that express the activation domain of Elk1 transcription factor fused to the GAL4 DNA binding domain. The activation of ERK1/2 kinases will result in the activation of its Elk trans-activators, which in turn stimulate reporter expression. (B) Luciferase activity, indicative of transcription factor dependent activation, is expressed as relative light units compared to control. The data represents the mean ± SE of four independent experiments. Asterisks denote statistically significant differences between the RFP and miR-574 group (p <0.05).

Figure 4

Identification of putative miR-574 binding sites within the TIMP3 3-UTR. (A) Putative miR-574 binding sites at the 3’UTR of TIMP3 were computationally identified by the online target detection program (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid); Top lines: miR-574 sequence; middle line: wild-type 3-UTR sequences; bottom line: mutated 3’UTR sequences. asterisks indicate the mutated nucleotides; (B) Granulosa cells were first transduced with lenti-miR-574 and then transfected with the luciferase-TIMP3 3-UTR vector or a control vector (a construct in which the 3-UTR of TIMP3 was replaced with a random sequence without any miR-574 target sites), and luciferase activity was measured. Mut represents luciferase expression constructs in which the miR-574 binding sites in the luciferase-TIMP3 3-UTR vector were mutated as indicated in (A). The mean ± SEM of the relative luciferase expression ratio (Firefly luciferase/Rinella luciferase, Luc/R-luc) was calculated based on three biological replicates, and compared with the negative control (NC). Data represent the mean ± SE of three independent experiments. Asterisks denote statistically significant differences when compared to RFP (control; p <0.05).

Figure 5

miR-574 regulates TIMP3 in the cultured granulosa cell. (A) Relative levels of TIMP3 mRNA in the granulosa cell after overexpression of miR-574. (B) Representative western blot of TIMP3 protein in the granulosa cell after lentivirus transduction. (C) Densitometric quantitation depicting decreased expression of TIMP3 protein after over-expression of miR-574. GAPDH was used for western blot and mRNA normalization. The data represents the mean ± SE of three or four independent experiments. Asterisks denote statistically significant differences between the negative control (RFP) and miR-574 group (p <0.05). (D) Relative levels of TIMP3 mRNA after transfection with specific siRNA for miR-574 (miR-574 siRNA) and negative control siRNA (NC) in the granulosa cells. (E) Representative western blot image of TIMP3 protein after siRNA of miR-574 transfection. (F) Densitometric quantitation depicting increased expression of TIMP3 protein. (G) Relative levels of Aromatase mRNA in the granulosa cell after overexpression or knock-down of miR-574. (H) Relative levels of Cyp450scc mRNA in the granulosa cell after overexpression and knock-down of miR-574. GAPDH mRNA and protein were used for western blot and real-time qPCR normalization, respectively. The data represents the mean ± SE of three independent experiments. Asterisks denote statistically significant differences between NC and miR-574-siRNA groups (p <0.05). NS, not significant.

Figure 6

Inhibition of TIMP3 decreased phosphorylated ERK1/2 but increased estradiol production in cultured granulosa cells. (A) Relative levels of TIMP3 mRNA after transfection with siRNA for TIMP3 (si-TIMP3) and negative control siRNA (NC) in granulosa cells. (B) Representative western blot of TIMP3 in granulosa cells after transfection with siRNA for TIMP3 (si-TIMP3) and negative control siRNA (NC). (C) Representative western blot of pERK1/2 and ERK1/2 protein after siRNA transfection. (D) Densitometric quantitation depicting decreased level of pERK1/2. (E) Suppression of TIMP3 promoted granulosa cells to produce more estradiol. GAPDH protein and mRNA were used for western blot data and mRNA normalization. Estradiol production was measured by ELISA assay. Data represent the mean ± SE of three independent experiments. Asterisks denote statistically significant differences between NC and si-TIMP3 group (p <0.05).

Figure 7

Recombinant TIMP3 increased phosphorylated ERK1/2 level and decreased estradiol production. (A) The PathDetect in vivo signal transduction pathway trans-reporting systems (Promega) was used to measure transcription activator and ERK1/2 signal transduction pathway after the addition of Recombinant TIMP3. First, the cultured granulosa cells were co-transfected with a pFR-luciferase reporter with a pFA2-Elk1 plasmid. 24h after the transfection, the recombinant TIMP3(rTIMP3) was added into the culture plate well. Granulosa cell sample was collected after 6h treatment then used for luciferase assay. Luciferase activity is indicative of transcription factor-dependent activation, is expressed as relative light units compared to control. (B) Estradiol production was measured by ELISA assay. The addition of TIMP3 suppresses estradiol production in the cultured granulosa cells. The data represents the mean ± SE of three or four independent experiments. Asterisks denote statistically significant differences between the blank control and the rTIMP3 group (p <0.05).