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Effect of Silence Smad8 Gene on Proliferation of Granulosa Cells in Mice Yu
Daolun1#, Chen Fanghui2#, Zhang Li1, Wang Hejian1, Chen Jie1, Zhang Zongmeng1, Li Jie1, Xing Chaofeng1, Li Jun1*, Cai Yafei1,2*
1College of Life Sciences, Anhui Provincial Key Lab of the Conservation and Exploitation of Biological Resources, Anhui Normal University, Wuhu 241000, China; 2College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
Abstract: SMAD8 (also named SMAD9) protein is one of the important transcription factorsin TGF-β/ SMADs signaling pathway. In this study， RNAi was used to explore the effect of silencing Smad8 gene expression on proliferation of mouse granulosa cells (MGCs). Immunohistochemistry was used to locate SMAD8 expression in mouse follicle. Smad8 gene siRNA was designed and synthesized to transfect MGCs and Smad8 gene was detected by qPCR and Western blot after transfection with Smad8-siRNA. Cell proliferation capacity was analyzed by CCK-8 and the concentrations of E2 and P4 in supernatant were determined by ELISA. The qPCR was performed to measure FSHR， LHR， Cyclin D2， CDK4 mRNA levels in granulosa cells after transfection with Smad8-siRNA. The results showed that Smad8 gene expression was effectively inhibited by Smad8-siRNA (P<0.01)， cell proliferation capacity was significantly weakened. E2， LHR， Cyclin D2 and CDK4 mRNA level were significantly decreased， but P4 and FSHR mRNA remain unchanged. These results indicated that the silencing Smad8 gene decreased the proliferation ability of MGCs， and its mechanism may be related to the decline of E2 secretion and LHR， Cyclin D2， CDK4 expression decrease after Smad8 gene silence.
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