Interference RNA-based silencing of endogenous SMAD4 in porcine granulosa cells resulted in decreased FSH-mediated granulosa cells proliferation and steroidogenesis

Abstract

FSH plays a critical role in granulosa cell (GC) proliferation and steroidogenesis through modulation by factors including bone morphogenetic proteins family, which belongs to transforming growth factor b (TGFB) superfamily. TGFBs are the key factors in maintaining cell growth and differentiation in ovaries. However, the interaction of FSH and TGFB on the GCs' proliferation and steroidogenesis remains to be elucidated. In this study, we have investigated the role of SMAD4, a core molecule mediating the intracellular TGFB/SMAD signal transduction pathway, in FSH-mediated proliferation and steroidogenesis of porcine GCs. In this study, SMAD4 was knocked down using interference RNA in porcine GCs. Our results showed that SMAD4-siRNA causes specific inhibition of SMAD4 mRNA and protein expression after transfection. Knockdown of SMAD4 significantly inhibited FSH-induced porcine GC proliferation and estradiol production and changed the expression of cyclin D2, CDK2, CDK4, CYP19a1, and CYP11a1. Thus, these observations establish an important role of SMAD4 in the regulation of the response of porcine GCs to FSH.

A comprehensive transcriptomic view on the role of SMAD4 gene by RNAi-mediated knockdown in porcine follicular granulosa cells

“…Basically, as a co-SMAD, SMAD4 has a critical function in mediating TGF-β/SMAD signaling pathway, but its role on other SMADs is unclear. A previous study showed that silencing of SMAD4 did not affect the expression of SMAD1, SMAD3 and SMAD5 in porcine GCs (Wang et al 2011). In this study, SMAD1 and SMAD5 were not detected, and SMAD2 and SMAD3 expressions were not significant changes under SMAD4-silencing (Supplementary Tables S3 and S4), suggesting that these genes might not play important roles in SMAD4-mediated pathway.…”

“…In fact, the role of SMAD4 gene in steroidogenesis is still controversial. In ovarian-specific Smad4-knockout mouse GCs, Smad4 loss increased the expression levels of STAR, Cyp11a1, Hsd3b1, and Hsd17b7 and resulted in disrupting regulation of steroidogenesis (Pangas et al 2006), but SMAD4 knockdown in porcine GCs was shown to downregulate the expression levels of CYP19A1 and HSD3B, without any significant changes in expressions of CYP11A1 and STAR genes (Wang et al 2010c(Wang et al , 2011. In the present study, upregulated expressions of HSD3B1 and STAR under SMAD4-silencing were consistent with the results discovered by Pangas et al (2006), indicating SMAD4-silencing might enhance the synthesis of steroid.…”

“…Western blot was carried out to measure protein expression levels of SMAD4 as previously described by Wang et al (2011). In brief, GCs were collected after 48-h transfection and were extracted via cell lysis buffer.…”

Transactivation of MicroRNA-320 by MicroRNA-383 Regulates Granulosa Cell Functions by Targeting E2F1 and SF-1 Proteins

“…FSH Regulates the Expression of miR-320, E2F1, and SF-1-FSH is the major regulator of folliculogenesis, particularly in driving proliferation and steroidogenesis of GCs (44). These physiological functions of FSH are achieved by the activation of different target genes in GCs (45).…”

Non-coding RNA in Ovarian Development and Disease

“…These cellular changes were associated with increased (miR-224 inhibitor) and decreased (miR-224 mimic) SMAD4 levels, respectively. Reconciling these results with the observations of Wang et al (2011) is however difficult, as they show that direct loss of SMAD4 decreases cell proliferation and oestrogen synthesis, the opposite effect shown when miR-224 is used to modulate SMAD4 levels. It is possible that miR-224 may be mediating its effects on cell proliferation and oestradiol synthesis through the regulation of other genes.…”