TGFβR1介导TGF-β/Smad信号通路对绵羊颗粒细胞功能的影响

doi:10.11843/j.issn.0366-6964.2023.08.019

Abstract

Abstract: The study aimed to explore the mechanism of the TGF-β/Smad signaling pathway mediated by TGFβR1 regulating the function of sheep granulosa cells. In this study, sheep ovarian granulosa cells were collected, and pcDNA3.1(+)-TGFβR1 and pcDNA3.1(+)-SMAD4 overexpressed plasmid vectors and shRNA-TGFβR1 and shRNA-SMAD4 interfering plasmid vectors were constructed to overexpress and interfere with TGFβR1 and SMAD4 genes in sheep granulosa cells, respectively. The proliferation, cell cycle and apoptosis of sheep granulosa cells were detected by CCK-8 cell proliferation assay, flow cytometry and Annexin-V FITC/PI double staining. The mRNA levels of TGFβR1 and SMAD4, the protein expression levels of apoptosis-related proteins BAX, BCL2, and Caspase3 and the phosphorylation levels of TGFβR1, TGFβR2 and SMAD2/3 in the TGF-β/Smad signaling pathway were detected by Real-Time PCR and Western blot. The results showed that overexpression of TGFβR1 and SMAD4 extremely significantly promoted the ability of granulosa cells to enter the S phase from the G0/G1 phase (P<0.01), the proliferation of granulosa cells and the expression of anti-apoptotic protein BCL2 (P<0.01), and inhibited the expression of apoptotic protein BAX and Caspase3 (P<0.01). Interference with TGFβR1 and SMAD4 both promoted granulosa cell apoptosis and the expressions of apoptotic protein BAX and Caspase3 (P<0.01) and inhibited the expression of anti-apoptotic protein BCL2 (P<0.01). Further studies showed that overexpression of TGFβR1 could promote the phosphorylation levels of TGFβR1, TGFβR2 and SMAD2/3 in the TGF-β/Smad signaling pathway (P<0.01), and extremely significantly promote the protein expression level of SMAD4 (P<0.01). Interference with TGFβR1 significantly inhibited the phosphorylation levels of TGFβR1, TGFβR2 (P<0.01) and SMAD2/3 (P<0.05), and inhibited the protein expression of SMAD4 (P<0.01). Overexpression of SMAD4 could significantly promote the expression of TGFβR1 protein (P<0.01). The results showed that TGFβR1 promoted the proliferation and cycle of sheep granulosa cells, inhibited granulosa cell apoptosis, positively regulated the proliferation and cycle of sheep granulosa cells, and negatively regulated the apoptosis of sheep granulosa cells by mediating the expression of SMAD2/3 and SMAD4 in TGF-β/Smad signaling pathway.

Key words: TGFβR1, sheep, granulosa cell, TGF-β/Smad signaling pathway

CLC Number:

S826.3

LI Yuexin, LIU Aiju, MA Xiaofei, ZHENG Zhong, HU Boxin, ZHI Yunxia, TIAN Shujun. Effect of TGFβR1 on the Function of Sheep Granulosa Cells Mediated by TGF-β/Smad Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3335-3347.

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Effect of TGFβR1 on the Function of Sheep Granulosa Cells Mediated by TGF-β/Smad Signaling Pathway

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