糖尿病肾病小鼠肾组织与细胞中miR-92b表达及其对纤维化的影响

Expression of microRNA-92b in renal tissues and cells of mice with diabetic nephropathy and its effect on fibrosis

目的观察糖尿病肾病(DN)小鼠及人肾小管上皮细胞中微小RNA 926(miR-92b)的表达变化,并探讨其对肾纤维化的影响与机制。方法 (1)小鼠实验:选取8周龄雄性小鼠12只,随机分为Dia组6只和Con组6只,Dia组腹腔内一次性注射STZ溶液,5个月后即制备成功DN模型; Con组注射等量柠檬酸-柠檬酸钠缓冲液。取两组小鼠肾组织,采用RT-PCR法检测miR-92b表达,Western blotting法检测磷脂酰肌醇-3激酶-蛋白激酶(PI3K/Akt)相关蛋白Akt、p-Akt和纤维链接蛋白(FN)蛋白表达。(2)细胞实验:培养人肾小管上皮细胞株HK-2,分为高糖组、低糖组、高渗对照组,分别用25 mmol/L右旋葡萄糖、5 mmol/L右旋葡萄糖、25 mmol/L左旋葡萄糖刺激24 h,采用RT-PCR法检测miR-92b表达,Western blotting法检测PI3K/Akt信号通路相关蛋白Akt、p-Akt和FN蛋白表达。将HK-2细胞分为DG组、HG组、HG+miR-92b组和HG+Neg组,DG组用25 mmol/L左旋葡萄糖刺激24 h,HG组、HG+miR-92b组和HG+Neg组用25 mmol/L右旋葡萄糖刺激24 h;然后采用细胞瞬时转染技术,将miR-92b转染至HG+miR-92b组细胞中,将miRNEG对照转染至HG+Neg组细胞中,HG组、DG组不转染。转染24 h后收集细胞,采用Western blotting法检测Akt、p-Akt和FN蛋白表达。结果与对照组相比,糖尿病肾病模型组小鼠肾组织和细胞中miR-92b的表达量降低,p-Akt/Akt和FN/β-actin相对蛋白表达量升高(P均<0. 05)。与DG组相比,p-Akt/Akt和FN/β-actin相对蛋白表达量在HG组HK-2细胞中增高(P均<0. 05);与HG组相比,HG+92b组p-Akt/Akt和FN/β-actin相对蛋白表达量均降低(P均<0. 001)。结论 DN小鼠及细胞中miR-92b表达水平降低,PI3K/Akt信号通路被激活和FN mRNA表达水平升高,过表达miR-92b可抑制PI3K/Akt信号通路、降低FN蛋白表达,从而减轻纤维化。

Objective To observe the expression of miR-92b in the diabetic nephropathy (DN) mice and human renal tubular epithelial cells, and to explore its effect on renal fibrosis. Methods ① Mouse experiment: 12 male mice of 8 weeks old were randomly divided into the Dia group and Con group,with 6 in each. Mice in the Dia group received the single intraperitoneal injection of STZ solution, and the DN models were successfully prepared after 5 months;the mice in the Con group were injected with an equal amount of citric acid-sodium citrate buffer. The kidney tissues of the two groups were taken. The expression of miR-92b was detected by RT-PCR, and the expression levels of phosphatidylinositol 3-kinase-protein kinase (PI3K/Akt)-related proteins Akt, p-Akt and fibronectin (FN) were detected by Western blotting.② Cell experiment: Human renal tubular epithelial cell line HK-2 was cultured and divided into high glucose group, low glucose group and hypertonic control group. Cells in the three groups were respectively stimulated with 25 mmol/L dextrose, 5 mmol/L dextrose, and 25 mmol/L L-glucose for 24 h. The expression of miR-92b was detected by RT-PCR, and the expression of PI3K/Akt-related proteins Akt, p-Akt and FN was detected by Western blotting. HK-2 cells were divided into the DG group, HG group, HG+miR-92b group, and HG+Neg group. Cells in the DG group were stimulated with 25 mmol/L L-glucose for 24 h, while cells in the HG group, HG+miR-92b group, and HG+Neg group were stimulated with 25 mmol/L dextrose for 24 h. Then, miR-92b mimics was transfected into HG+miR-92b cells by cell transient transfection technique, and miRNEG control was transfected into HG+Neg cells. Cells in the HG group and DG group were not transfected. Cells were harvested after 24-hour transfection. The expression of Akt, p-Akt and FN protein was detected by Western blotting. Results Compared with the control group, the expression of miR-92b in the kidney tissues and cells of the DN model group decreased significantly, and the relative protein expression levels of p-Akt/Akt and FN/β-actin increased significantly (all P <0.05). Compared with the DG group, the relative protein expression levels of p-Akt/Akt and FN/β-actin in cells of HG group increased (all P <0.05). Compared with HG group, the relative protein expression levels of p-Akt/Akt and FN/β-actin in the HG+92b group decreased significantly (all P <0.001). Conclusion The miR-92b expression decreases, PI3K/Akt signaling pathway is activated and the expression of FN mRNA increases in DN mice and cells. Overexpression of miR-92b can inhibit PI3K/Akt signaling pathway, decrease FN protein expression, and thus reduce DN fibrosis.