Smad7介导TGF-β信号通路对绵羊卵泡颗粒细胞增殖的影响

doi:10.3864/j.issn.0578-1752.2023.13.013

Abstract

Abstract:

【Background】Follicle development is a complex regulatory process, in which the proliferation and differentiation of ovine follicular granulosa cells (Granulosa Cells, GCs) will directly affect the development of follicles. TGF-β signaling pathway plays an important role in sheep ovarian development and follicle growth, and Smad7 also plays an important role as a key inhibitor of TGF-β signaling pathway. 【Objective】In this study, the effect of Smad7-mediated TGF-β signaling pathway on the proliferation and apoptosis of ovine follicular GCs was explored to provide a basis for further research on the regulatory role of Smad7 in the process of follicular GCs proliferation and apoptosis. 【Method】20 non-pregnant Jinzhong Duhu hybrid ewes aged 4-6 months were selected. After slaughtering, bilateral ovaries were collected, and GCs were isolated and cultured in vitro. And then, FSHR cell and expression localization of Smad7 cells was identified by immunofluorescence, the cell proliferation was measured by CCK8 method, and the growth curve was drawn. After the cells were passaged, different concentrations of Smad7 agonist Asiaticoside AS (0, 100, 200, 400, 600 ng·mL^-1) were added exogenously, and the optimal concentration of AS was selected to treat the second generation GCs. The expression levels of key genes as well as apoptosis and cycle-related genes in the TGF-β signaling pathway were detected using qRT-PCR and Western blotting. Three siSmad7s were synthesized and transfected into follicular GCs, and the one with the best interference effect was selected. The expression levels of key genes in the TGF-β signaling pathway, apoptosis and cycle-related genes were detected by qRT-PCR and Western blotting. 【Result】Smad7 was expressed in ovine follicular GCs; 200 ng·mL^-1 AS could significantly up-regulate the expression of Smad7 in GCs (P<0.01), siSmad7-1 could effectively transfected ovine follicle GCs (P<0.01); Up-regulation of Smad7 could significantly increase the expressions of Smad4, TFDP-1 and EP300 in the TGF-β signaling pathway (P<0.05), extremely significantly increase the expression of SP1 in the TGF-β signaling pathway (P<0.01), and significantly increase the expression of apoptosis-related gene Caspase8 (P<0.05), extremely significantly increased the expressions of apoptosis-related genes Caspase3 and Bim (P<0.01), significantly increased the expressions of cell cycle-related genes P21 and P27 (P<0.05), extremely significantly increased the expression of cell cycle-related gene CCND2 (P<0.01), while the expression of CCND1 was significantly decreased (P<0.05); Smad7 siRNA significantly decreased the expressions of Smad4, TFDP-1 and EP300 in the TGF-β signaling pathway (P<0.05), extremely significantly decreased the expression of SP1 in the TGF-β signaling pathway (P<0.01), and significantly decreased the apoptosis-related gene Caspase8 (P<0.05), extremely significantly decreased the expressions of apoptosis-related genes Caspase3 and Bim (P<0.01), extremely significantly decreased the expressions of cell cycle-related genes P21, P27 and CCND2 (P<0.01), extremely significantly increased the expression of CCND1 (P<0.01); CCK8 analysis demonstrated into that when Smad7 was up-regulated, the proliferation rate of GCs was significantly down-regulated at 24 h and 48 h (P<0.01). Smad7 siRNA significantly increased the proliferation rate of GCs at 24 h and 72 h (P<0.05), which also extremely significantly increased the proliferation rate of GCs (P<0.01).【Conclusion】In summary, it was suggested that Smad7 mediated TGF-β signaling pathway to inhibit the proliferation of ovine follicular GCs and to promote cell apoptosis.

Key words: asiaticoside (AS), siRNA, Smad7, TGF-β signaling pathway, sheep, granulosa cells

GUO ZeYuan, DU ZhangSheng, ZHANG YaQi, CHEN ChunLu, MA XiaoYan, CHENG Ying, WANG Kai, LÜ LiHua. Effects of Smad7-Mediated TGF-β Signaling Pathway on Proliferation of Sheep Granulosa Cells[J].Scientia Agricultura Sinica, 2023, 56(13): 2597-2608.

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Effects of Smad7-Mediated TGF-β Signaling Pathway on Proliferation of Sheep Granulosa Cells

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基因 Gene	引物序列 Primer sequence (5´→3´)	长度 Product size (bp)
Smad7	F:CCCATCACCTTAGCCGACTC	101
Smad7	R:CACAGCATCTGGACAGTCAGT	101
β-actin	F:GCAAAGACCTCTACGCCAAC	90
β-actin	R:GGGCAGTGATCTCTTTCTGC	90
TFDP1	F:GTGGTCGCGGGAAAACCA	175
TFDP1	R:GACAGCAACCAAGGACACCA	175
SP1	F:GCTACCATGAGCGACCAA	101
SP1	R:CTACCATTTCCATTGCCCCC	101
EP300	F:CCAGCCTCTTAAGATGGGAAT	110
EP300	R:TCGGAATCTGGAGACCAAGG	110
Smad4	F:GAGCAAGGTTGCACATAGGC	80
Smad4	R:GTCACTAAGGCACCTGACCC	80
CCND1	F:TGATCAGATGTGACCCGGACT	185
CCND1	R:CCCTCAAATGTTCACGTCGC	185
CCND2	F:AGACCTTCATCGCTCTGTGC	186
CCND2	R:TGAGGCAATCCACATCCGTG	186
P21	F:GCTTCTTGTACCAGGACCCAG	118
P21	R:CAGAGAGTTCGCTTCACCGAG	118
P27	F:ACCACCGTCAAACCTTACTCC	159
P27	R:TTCTCTGTAGGACCCCTCGT	159
Caspase3	F:CCCACTGCAGCAACATTAATCC	252
Caspase3	R:CGACAGGCCATGCCAGTATT	252
Caspase8	F:ACTGTGTGGAGCAGGTAACA	128
Caspase8	R:CCGGCTTAGGAACTTGAGGG	128
Bim	F:TGAGGCAGTCTCAGGCTGTA	153
Bim	R:GGAGGACCATTTGTGGGTGG	153

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