抑制BMP/Smad信号通路对水牛卵巢颗粒细胞生长和类固醇激素合成的影响

doi:10.16431/j.cnki.1671-7236.2023.06.019

Abstract

Abstract: 【Objective】 The aim of this study was to investigate the effect of BMP/Smad signaling pathway on the growth and steroid hormone synthesis of buffalo ovarian granulosa cells.【Method】 Three pairs siRNAs of Smad4 gene (Smad4-siRNA1, Smad4-siRNA2 and Smad4-siRNA3) and NC-siRNA (control group) were transfected into buffalo granulosa cells by lipofection.The expression level of Smad4 gene was detected by Real-time quantitative PCR to compare the interference efficiency of different siRNAs.Then the siRNA with the highest interference efficiency was transfected into buffalo ovarian granulosa cells.The cell proliferation in the control group and interference group was detected by CCK-8, and the expression of the apoptosis-related genes Bax and Bcl2, cell cycle-related regulation genes CyclinD2 and CDK4 and the steroid hormone synthesis related genes Cyp19A1 and Cyp11A1 were also detected by Real-time quantitative PCR, and the contents of estradiol (E₂) and progesterone (P₄) were detected by ELISA.【Result】 The results of gene interference test showed that the three pairs of siRNAs had extremely significant interference effects on the expression of Smad4 gene (P<0.01), and the interference efficiency of Smad4-siRNA1 was the highest, reaching 64%.The CCK-8 test results showed that compared with control group, the cell proliferation rate of the interference group was significantly decreased (P<0.05).The results of Real-time quantitative PCR showed that compared with the control group, the expression of Bcl2, CyclinD2, CDK4 and Cyp19A1 genes in the interference group were significantly or extremely significantly down-regulated (P<0.05 or P<0.01), while the expression of Cyp11A1 gene was extremely significantly up-regulated (P<0.01), and the expression of Bax gene was not affected.The results of ELISA showed that compared with the control group, the estradiol secretion in the interference group decreased by 10.2%, and the progesterone secretion increased by 24.7%, and the difference was significant (P<0.05).【Conclusion】 The disruption of endogenous BMP/Smad signaling pathway by RNAi-mediated Smad4 gene silencing could not only significantly inhibit the growth of buffalo granulosa cells, but also induce the apoptosis of buffalo granulosa cells.Furthermore, inhibiting BMP/Smad signal would change the production of steroid hormones. BMP/Smad signal pathway played an important role in regulating the growth of buffalo granulosa cells and the production of steroids.

Key words: buffalo; BMP/Smad signaling pathway; Smad4 gene; granulosa cells; RNAi; steroidogenesis

CLC Number:

S823.8⁺3

XU Yuanyuan, ZHENG Haiying, YANG Chunyan, DENG Tingxian, HUANG Chenqian, FENG Chao, LU Xingrong, DUAN Anqin, MO Xia, MA Xiaoya, SHANG Jianghua. Effects of Inhibition of BMP/Smad Signaling Pathway on the Growth and Steroid Hormone Synthesis of Buffalo Ovarian Granulosa Cells[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2354-2362.

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Effects of Inhibition of BMP/Smad Signaling Pathway on the Growth and Steroid Hormone Synthesis of Buffalo Ovarian Granulosa Cells

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