微小RNA-148b通过靶向调控AMPKα1介导高糖诱导大鼠肾小球系膜细胞的内质网应激

MicroRNA-148b influences high glucose-induced endoplasmic reticulum stress in rat mesangial cell by targeting AMPKα1

目的观察高糖刺激大鼠系膜细胞后微小RNA-148b（miR-148b）的表达变化，及其对靶基因腺苷酸活化的蛋白激酶α1（AMPKcd）的调控作用和对细胞外基质蛋白分泌的影响。方法大鼠系膜细胞分3组：正常糖组（5．5mmol／L葡萄糖）、高渗组（5．5mmol／L葡萄糖＋19．5mmol／L甘露醇）和高糖组（25．0mmol／L葡萄糖），实时定量PCR检测miR-148b的表达。转染miR-148b抑制物进一步探讨靶向抑制miR-148b的作用。实时定量PCR检测AMPKα1 mRNA的表达；Western印迹法检测AMPKα1、葡萄糖调节蛋白78（GRP78）、C／EBP同源蛋白（CHOP）、纤连蛋白（FN）、Ⅳ型胶原（ColⅣ）蛋白的表达；免疫荧光法检测ColⅣ的表达。结果高渗组与正常对照组miR-148b表达差异无统计学意义（P〉0．05）；与正常对照组比较，高糖组细胞内miR-148b的表达上调，GRP78、CHOP、FN、ColⅣ表达均显著上调（均P〈0．05），AMPKα1 mRNA及蛋白表达均下调（均P〈0．01）。与高糖组比较，转染miR-148b抑制物的高糖组miR-148b及GRP78、CHOP、FN、ColⅣ蛋白表达均显著下调（均P〈0．05），AMPKcd mRNA及蛋白表达均上调（均P〈0．05）；转染miR-148b阴性对照的高糖组上述指标与高糖组相比差异均无统计学意义（均P〉0．05）。结论高糖可上调体外系膜细胞miR-148b的表达，靶向抑制AMPKα1的表达，从而诱导内质网应激及系膜细胞产生过多细胞外基质蛋白。抑制miR-148b可上调靶基因AMPKα1的表达，从而抑制高糖诱导的系膜细胞内质网应激亢进，减少细胞外基质蛋白的聚积。

Objective To observe the expression of microRNA-148b （miR-148b） induced by high glucose in rat mesangial cells, and to explore its effect on its target gene AMP-activated protein kinase α1 （AMPKα1） and extracellular matrix excretion. Methods Rat mesangial cells were divided into 3 groups： normal glucose （NG, 5.5 mmol/L glucose） group, hypertonic （MA, 5.5 mmol/L glucose＋19.5 mmol/L mannitol） group and highglucose （HG, 25.0 mmol/L glucose） group. MiR- 148b expression was detected by real time PCR. Then miR- 148b inhibitor was transfected to rat mesangial cells. Their protein expressions of AMPKod, glucose regulated protein 78 （GRP78）, C/EBP homologous protein （CHOP）, fibronectin （FN） and collagen Ⅳ were detected by Western blotting. The expression of AMPKα1 mRNA was detected by real time PCR. The expression of collagen Ⅳ was also detected by immunofluorescence. Results Compared with NG group, HG group showed up-regulated miR-148b expression, down- regulated AMPKα1 mRNA and protein expressions, and upregulated CHOP, GRP78, collagen IV and FN expressions （all P 〈 0.05）. HG-induced mesangial ceils with miR-148b inhibitor had upregulated AMPKα1 mRNA and protein expressions, and down- regulated CHOP, GRP78, collagen Ⅳ, FN expressions as compared with HG-induced cells without miR-148b inhibitor （all P 〈 0.05）. Conclusions HG can up-regulate miR-148b expression and down-regulate AMPKα1 expression in rat mesangial cells, then activate endoplasmic reticulum stress to induce extracellular matrix excretion. MiR-148b inhibitor up-regulates AMPKα1 expression, inhibits endoplasmic reticulum stress and reduces extracellular matrix excretion.