SET蛋白对精母细胞株(GC-2 spd)增殖和组蛋白乙酰化的影响

Up-regulation of SET Protein in the Proliferation of GC-2 spd Cells and Histone Acetylation

目的:观察SET蛋白对GC-2 spd精母细胞株增殖和组蛋白乙酰化的影响,探讨SET蛋白调节精子形成的作用。方法:体外培养GC-2 spd细胞,通过精母细胞标志物乳酸脱氢酶C(LDHC)和睾丸特异激酶1(TESK1)鉴定。琼脂糖珠免疫共沉淀试剂盒检测SET蛋白与组蛋白H4的结合。分别转染空载腺病毒(Ad H1-si RNA/NS,对照组)和SET干涉腺病毒(Ad H1-si RNA/SET,干涉组),比较细胞增殖情况,荧光显微镜观察GC-2spd细胞中SET蛋白的表达;用实时定量逆转录聚合酶链反应(Real time RT-PCR)和蛋白质印迹(Western blot)检测m RNA以及蛋白的表达。结果:免疫荧光显示精母细胞株GC-2 spd阳性表达LDHC和TESK1;SET蛋白表达于GC-2细胞的胞核和胞质中,免疫共沉淀提示SET蛋白与组蛋白H4结合。转染SET干涉腺病毒后,核内和胞质中SET蛋白表达明显降低(P<0.01)。与对照组相比,干涉组细胞增殖明显降低,组蛋白H4乙酰化水平明显增高(P<0.05);但组蛋白乙酰化酶(HATs)和去乙酰化酶(HDACs)m RNA表达水平差异无统计学意义(P>0.05)。结论:精母细胞核内和胞浆中均表达SET蛋白;SET蛋白降调,则抑制GC-2spd细胞增殖。SET蛋白可能通过与组蛋白H4相互作用调节其乙酰化,参与调节精子形成。

Objective： To investigate the effects of SET protein on the regulation of spermiogenesis, the proliferation and histone acetylation of spermatocyte cell line, GC-2 spd, were observed. Methods： The GC-2 spd cell was cultured in vitro, and identified by two markers of spermatocytes, lactate dehydrogenase C （LDHC） and Testis-Specific Kinase 1 （TESK1） . The combination of SET and H4 was detected by an immunoprecipitation kit. The cells were transfected with AdH1-siRNA/SET to knockdown the expression of SET protein, AdH1-siRNA/NS as control. The cell proliferation was compared between two groups. The expressions of SET protein, histone acetyltransferases and histone deacetylases were detected using realtime RT-PCR and Western Blot. Results：Two markers of spermatocytes, LDHC and TESK1, were positively expressed in the GC-2 spd cell. SET protein was expressed in both cytoplasm and nucleus of GC-2 spd cell. Immunoprecipitation showed that SET protein combined with H4. The expression of SET protein in the cytoplasm and nucleus of the cells transfected cells with AdH 1 - siRNA/SET was significantly decreased （ P 〈0.01）, while the cell proliferation significantly inhibited. Interestingly, the acetylation level of H4 was significantly increased in the transfected cells （ P 〈 0.05）. However, there were no significantly differences in the expressions of HATs and HDACs （ P 〉0.05）. Conclusions：SET protein was expressed in both cytoplasm and nucleus of spermatocytes, the knockdown expression of SET protein inhibited the cell proliferation. SET protein participated in the regulation of spermiogenesis, probably by regulating the acetylation level of histone H4.