Smad3 promotes the autophagy of granulosa cells in rats

doi:10.3969/j.issn.0529-1356.2014.01.021

Abstract

Abstract:

Objective To study the effect of Smad3 on the autophagy of the ovarian granulosa cells (GCs) in the rat. Methods Ovarian granulosa cells were cultured and treated in the following conditions: control group in which GCs were cultured without any treatment; knockdown group in which GCs were transfected by specific Smad3 siRNA with Lipo RNAiMAX; overexpression group in which GCs were transfected by p3×FLAG-CMV-Smad3 plasmid with Lipo 2000. Immunocytochemistry was used to identify cell purity. To determine the efficiency of transfection, the expression of Smad3 protein was evaluated by Western blotting; Western blotting analysis was also used to detect the expression of autophagy associated gene 8(LC3), B cell lymphoma2(Bcl-2) proteins of ovarian GCs. Results Western blotting results showed that: in knockdown group, the ratio of LC3B II/I had no significant difference and the expression intensity of Bcl-2 protein was significantly decreased, compared to the control group. In overexpression group, the ratio of LC3BII/I was significantly increased and the expression intensity of Bcl-2 protein was significantly increased, compared to the control group. Conclusion Both Smad 3 SiRNA and P3×FLA-CMV-Smad 3 can be effectively transfected to GCs. The mechanism, by which Smad 3 promotes the autophagy of GCs, may be related to the expression of Bcl-2 protein.

Key words: Smad3, Ovarian granulosa cell, Autophagy, Immunohistochemistry, Western blotting, Rat

SHEN Chun-yan DONG Jing-xia XU Jian*. Smad3 promotes the autophagy of granulosa cells in rats[J]. AAS, 2014, 45(1): 109-113.

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