摘要
目的探讨脂联素通过微小RNA221(miR-221)靶向调控金属蛋白酶组织抑制因子3(Timp3)基因对高糖诱导的小鼠肾系膜细胞增殖的影响及作用机制。方法在体外高糖条件下培养小鼠。肾系膜细胞,分别加入脂联素以及转染miR-221抑制物、miR-221模拟物、小干扰RNA-Timp3(Si—Timp3)等干预因素。根据实验目的及干预因素不同将细胞分组为:正常对照组、高糖组、高糖+脂联素组、高糖+miR-221抑制物转染组及转染对照组、Si-Timp3转染组及转染对照组、miR-221模拟物转染组及转染对照组、高糖+脂联素+miR-221模拟物转染组及转染对照组。噻唑蓝法检测细胞的增殖活性,实时荧光定量聚合酶链反应(RT—PCR)分析miR-221、Timp3的表达水平,Westernblotting法检测Timp3的蛋白表达,荧光素酶报告基因实验检测Timp3的3'-UTR活性。两组间比较采用t检验。结果高糖组促进小鼠肾系膜细胞增殖明显,上调miR.221和抑制Timp3的表达(分别为3.20±0.28比1、0.67±0.07比1,t=13.69、8.35,均P〈0.05),而脂联素加人后该作用减弱(t=7.60、4.12,均P〈0.05)。与高糖+miR-221抑制物对照组比较,转染miR-221抑制物后,Timp3的mRNA表达水平上调(0.78±0.03比0.38±0.04,t=14.57,P〈0.05),细胞增殖减弱。与Si.Timp3对照组比较,转染Si-Timp3后,细胞增殖活性增强(1.72±0.06比1.04±0.12,t=8.76,P〈0.05)。与高糖+脂联素+miR-221minie转染对照组比较,加入脂联素及转染miR-221模拟物后,Timp3的mRNA表达水平下降(O.72±0.09比1.40±0.06,t=10.93,P〈0.05),细胞增殖加强。结论高糖是诱导小鼠肾系膜细胞增殖、上调miR-221水平、抑制Timp3基因表达的重要因素,脂联素可逆转该作用,其机制可能是通过miR-221/Timp3途径,靶向负调控Timp3基因的表达来实现的。
Objective To investigate the inhibiting effect and mechanism of adiponectin on high glucose- induced mouse mesangial cells proliferation through microRNA221(miR-221)/tissue inhibitor of metalloproteinase 3(Timp3) pathway. Methods Mouse mesangial cells cultured in vitro were divided into the following groups: normal control group, high glucose group, high glucose ± adiponectin group, high glucose±adiponectin±miR-221 inhibitor transfected group and control group, small interfering RNA-Timp3 (Si-Timp3) transfected group and control group, miR-221 mimic transfected group and control group, high glucose ± adiponectin ± miR- 221 mimic transfected group and control group. Mouse mesangial cells proliferation was measured by MTT assay. The level of miR- 221 and Timp3 mRNA expressions were measured by quantitative real-time polymerase chain reaction (qRT-PCR) ; Western blotting was performedto detect the expression of Timp3 protein. Dual luciferase reporter assay was used to confirm the activity of 3'-UTR in Timp3. Differences between two groups were compared by LSD-t test. Results High glucose could significantly promote the proliferation of mouse mesangial cells, up- regulate the expression of miR-221 and inhibit Timp3 (3,20±.28vsl, 0.67±0.07vsl, t=13.69, 8.35, all P〈0.05). Adiponectin could reverse the effect (t=7.60, 4.12, all P〈0.05). Compared to high glucose±adiponectin±miR-221 inhibitor transfected control group, miR-221 inhibitor up-regulated the mRNA expression of Timp3 (0.78± 0.03 vs 0.38±0.04, t=14.57, P〈O.05). The cell proliferation was increased significantly in Si-Timp3 transfected group (1.72±0.06 vs 1.04±0.12, t=8.76, P〈0.05). The mRNA level of Timp3 was down-regulated in high glucose±adiponectin±miR-221 mimic transfected group compared to control group (0.72±0.09 vs 1.40±0.06, t= 10.93, P〈0.05 ). Conclusions The findings suggest that high glucose is the important factor to improve mouse mesangial cells growth, up-regulate miR-221 but down-regulate Timp3. Adiponectin can reverse the effect through miR-221 and Timp3 pathway by negatively regulates Timp3 expression in high glucose-induced mouse mesangial cells.
出处
《中华糖尿病杂志》
CAS
CSCD
2016年第11期681-686,共6页
CHINESE JOURNAL OF DIABETES MELLITUS
基金
福建省医学创新课题(2015-CX-13)