多巴胺通过促进RNF20降解抑制神经细胞中组蛋白H2B泛素化

表观遗传修饰参与了药物成瘾的形成过程,而在药物成瘾过程中组蛋白泛素化水平的变化仍未可知。药物成瘾过程中常表现为多巴胺(dopamine, DA)表达量的升高,因此本研究欲探讨多巴胺升高对神经细胞组蛋白泛素化的影响及其机制。Western印迹结果显示,在终浓度0.8 mmol/L的多巴胺作用8 h后,人神经母细胞瘤细胞系SH-SY5Y细胞中环指蛋白20(ring finger protein 20, RNF20)表达量降低(0.29±0.032 vs. 1.0±0.025,P<0.0001),泛素化组蛋白H2B(H2Bub1)表达量下降(0.28±0.032 vs. 1.0±0.017,P<0.0001)。但是RT-PCR结果显示,多巴胺处理SH-SY5Y细胞后,RNF20在mRNA水平的表达无明显变化。在SH-SY5Y细胞中沉默RNF20的表达,H2Bub1在蛋白质水平的表达明显降低(0.20±0.069 vs. 1.0±0.060,P=0.001)。在加入多巴胺的基础上,分别加入蛋白酶体抑制剂MG132、自噬体形成抑制剂3-MA以及空泡型H^+-ATP酶特异性抑制剂Baf-A1等药物来检测RNF20的降解途径,结果发现,加入MG132、3-MA以及Baf-A1后,RNF20表达量均比DA处理组显著上升(1.51±0.095,P=0.0003; 0.89±0.075,P=0.0021; 2.74±0.099,P<0.0001;vs. 0.27±0.044)。上述结果表明,在SH-SY5Y细胞中,RNF20对H2Bub1具有调控作用,多巴胺可通过泛素化及自噬两种途径促进RNF20降解,从而抑制组蛋白H2B泛素化。

Dopamine Inhibits the Ubiquitination of Histone H2B in Nerve Cells by Promoting RNF20 Degradation

Epigenetic modification is involved in the formation of drug addiction, however, it is still unclear whether the level of histone ubiquitination is affected in the process of drug addiction. In the process of drug addiction, the production of dopamine is often increased, but the effect of dopamine on histone ubiquitination remains unknown. This study focused on the effect of dopamine on histone ubiquitination during drug addiction. Western blot showed that after treatment of 0.8 mmol/L dopamine for 8 h, the production of ring finger protein 20 (RNF20) in SH-SY5Y human neuroblastoma cells was decreased (0.29 ± 0.032 vs. 1.0 ± 0.025, P＜ 0.0001)，and the production of ubiquitinated histone H2B (H2Bub1) also decreased (0.28 ± 0.032 vs. 1.0 ± 0.017, P＜ 0.0001). However, RT-PCR results showed that there were no significant differences in mRNA levels of RNF20 expression after SH-SY5Y cells were treated with dopamine. Silence of RNF20, significantly decreased the production of H2Bub1 at the protein level in SH-SY5Y cells (0.2 ± 0.069 vs. 1.0 ± 0.060, P =0.001). Combined with dopamine, proteasome inhibitor MG132, autophagy inhibitor 3-MA, or vacuolar H⁺-ATPase specific inhibitor Baf-A1 were added respectively to detect the degradation of RNF20. The results showed that the production of RNF20 increased significantly in groups treated with DA combined with MG132, 3-MA and Baf-A1 as compared with those treated only with DA (1.51 ± 0.095, P=0.0003; 0.89 ± 0.075, P=0.0021; 2.74 ± 0.099, P＜ 0.0001 vs. 0.27 ± 0.044). These results indicated that dopamine inhibited histone H2B ubiquitination by degrading RNF20 through ubiquitination and autophagy in SH-SY5Y cells.