脂联素调控微小RNA221／金属蛋白酶组织抑制因子3信号对高糖环境下小鼠肾系膜细胞增殖的抑制作用

Inhibiting effect of adiponectin on high glucose-induced mouse mesangial cells proliferation via microRNA221/tissue inhibitor of metalloproteinase 3 signaling regulation

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.