HSG细胞中MBD2对AQP5表达的调控作用

Role of methyl-CpG-binding domain protein 2 in regulating aquaporin-5 expression in human submandibular gland cells in vitro

目的探讨人下颌下腺细胞(human submandibular gland,HSG)中,甲基化CpG结合域蛋白2(menthyl-CpG-binding domain protein 2,MBD2)是否影响水通道蛋白调控-5(aquaporin 5,AQP5)的表达,为干燥综合征(Sj?gren’s syndrome,SS)的治疗提供新的思路。方法体外培养HSG细胞,通过AQP5的启动子质粒与MBD2-siRNA或MBD2过表达质粒共转染HSG细胞,利用双荧光素酶报告基因系统检测AQP5启动子的活性;再用MBD2-siRNA或MBD2过表达质粒转染HSG细胞,利用RT-PCR及Western blot技术检测MBD2和AQP5在mRNA水平及蛋白水平上表达的改变。结果①MBD2-siRNA转染HSG细胞后,AQP5 pro活性分别上调49.30%和58.35%(P<0.05), AQP5在mRNA水平上表达上调35.40%和471.19%(P<0.05),蛋白水平上调56.16%和82.78%(P<0.05)。结果表明:MBD2-siRNA转染HSG细胞后,AQP5启动子活性增加,AQP5表达上调。②MBD2过表达质粒转染HSG细胞后,AQP5 pro活性分别下调25.36%、30.52%及34.78%(P<0.05), AQP5在mRNA水平上表达下调8.06%和87.60%(过表达质粒1μg组与对照组相比无统计学意义,P>0.05;过表达质粒2μg组与对照组相比差异有统计学意义P<0.05),在蛋白水平上表达下调44.50%和53.18%(P<0.05)。实验结果表明:MBD2转染ASG细胞后,AQP5启动子活性降低,AQP5的表达下降(P<0.05)。结论 MBD2可以下调AQP5的表达,这与SS患者的AQP5表达趋势一致,因此MBD2可能成为干燥综合征基因治疗的新靶点。

Objective To study whether methyl-CpG-binding domain protein 2(MBD2)affects the expression of aquaporin-5(AQP5)in human submandibular gland(HSG)cells,thereby exploring a new approach to treatment of Sjogren’s syndrome(SS).Methods Cultured HSG cells were co-transfected with the promoter plasmid of AQP5 and the plasmids for MBD2 silencing(MBD2-siRNA)or overexpression.Luciferase report gene assay was used to detect the transcription level of AQP5 in the transfected cells.In HSG cells transfected with the MBD2-siRNA or the overexpression plasmids,RT-qPCR and Western blotting were performed to analyze the changes in MBD2 and AQP5 expression at both the mRNA and protein levels.Results Transfection of the cells with the 2 MBD2-siRNAs respectively increased AQP5 promoter activity by 49.30%and 58.35%(P<0.05)and obviously upregulated the expression of AQP5 by 35.40%and 471.19%at the mRNA level(P<0.05)and by 56.16%and 82.78%at the protein level(P<0.05).Transfection of the cells with the three MBD2 overexpression plasmids resulted in significantly lowered AQP5 promoter activity by 25.36%,30.52%and 34.78%(P<0.05);while 1 plasmid produced no significant changes in AQP5 mRNA level,the other 2 plasmids significantly down-regulated AQP5 expression by 8.06%and 87.60%at the mRNA level(P<0.05)and by 44.50%and 53.18%at the protein level(P<0.05).Conclusion MBD2 could down-regulats the expression of AQP5 in HSG cells in vitro,which consistent with the AQP5 expression about patients with SS,and this finding indentifies MBD2 as a potential target in gene therapy for the syndrome.