高糖环境下miR-96-5p对人角质形成细胞的影响及相关机制研究

Effects of miR-96-5p on high-glucose-induced keratinocytes and the underlying mechanism

目的探讨高糖环境下miR-96-5p对人角质形成细胞的影响及相关机制。方法分离人角质形成细胞以50 mmol/L葡萄糖终浓度模拟高糖环境。通过转染miR-96-5p模拟物和抑制剂构建miR-96-5p过表达和抑制组,分为空白组、正常培养组、高糖培养组、高糖培养+miR-96-5p minic组、高糖培养+miR-96-5p inhibitor组。采用划痕实验观察细胞迁移水平,采用CCK-8实验观察细胞增殖水平,采用Transwell实验检测细胞侵袭能力。采用Western blot检测BNIP3、FAK、FAK磷酸化蛋白(p-F AK)蛋白的表达水平。结果50 mmol/L高糖处理后角质细胞折光性降低,同时细胞中出现空洞。与空白组和正常培养组比较,高糖培养组、高糖培养+miR-96-5p minic组和高糖培养+miR-96-5p inhibitor组的各时间点细胞迁移量明显更低(P<0.05),且高糖培养+miR-96-5p inhibitor组细胞迁移量明显高于高糖培养组和高糖培养+miR-96-5p minic组(P<0.05);与空白组和正常培养组比较,高糖培养组、高糖培养+miR-96-5p minic组和高糖培养+miR-96-5p inhibitor组的各时间点细胞增殖量明显更低(P<0.05),且高糖培养+miR-96-5p inhibitor组细胞增殖明显高于高糖培养组和高糖培养+miR-96-5p minic组(P<0.05);与空白组和正常培养组比较,高糖培养组、高糖培养+miR-96-5p minic组和高糖培养+miR-96-5p inhibitor组的各时间点细胞侵袭量明显更低(P<0.05),且高糖培养+miR-96-5p inhibitor组细胞侵袭明显高于高糖培养组和高糖培养+miR-96-5p minic组(P<0.05);与空白组和正常培养组比较,高糖培养组、高糖培养+miR-96-5p minic组和高糖培养+miR-96-5p inhibitor组的各时间点BNIP3、FAK、p-F AK蛋白表达量明显更低(P<0.05),且高糖培养+miR-96-5p inhibitor组的BNIP3、FAK、p-F AK蛋白表达量明显高于高糖培养组和高糖培养+miR-96-5p minic组(P<0.05)。结论miR-96-5p可通过靶向调控BNIP3/FAK信号通路的表达从而影响角质细胞的增殖、侵袭和迁移能力,进而发挥影响糖尿病足创面愈合的作用。

Objective To investigate the effects of miR-96-5p on high-glucose-induced keratinocytes and the underlying mechanism.Methods Human keratinocytes were isolated and treated in the culture medium containing 50mmol/L glucose to simulate the hyperglycemic environment.Overexpression and knockdown of miR-96-5p were achieved by transfection of miR-96-5p mimic and inhibitor,respectively.Cells were treated with blank control,normal induction,high-glucose induction,high-glucose induction plus transfection of miR-96-5p mimic,and high-glucose induction plus transfection of miR-96-5p inhibitor.Cell migration,proliferation and invasion were detected by scratch assay,CCK-8 assay,and Transwell assay,respectively.Western blot was used to detect the expression levels of Bcl-2/adenovirus E1B 19-kDa interacting protein 3(BNIP3),focal adhesion kinase(FAK)and phosphorylated FAK(p-FAK).Results The induction of 50mmol/L glucose in keratinocytes results in decreased refraction and the presence of cellular cavity.Compared with cells treated with blank control and normal induction,the number of migratory and invasive cells,and proliferative rate at eat time point were significantly lower in those induced with high-glucose,high-glucose induction plus transfection of miR-96-5p mimic,and high-glucose induction plus transfection of miR-96-5p inhibitor(all P<0.05),which were significantly higher in cells with high-glucose induction plus transfection of miR-96-5p inhibitor than those induced with high-glucose,and high-glucose induction plus transfection of miR-96-5p mimic(all P<0.05).Compared with cells treated with blank control and normal induction,protein levels of BNIP3,FAK and p-FAK were significantly lower in those induced with high-glucose,high-glucose induction plus transfection of miR-96-5p mimic,and high-glucose induction plus transfection of miR-96-5p inhibitor(P<0.05),which were significantly higher in cells with high-glucose induction plus transfection of miR-96-5p inhibitor than those induced with high-glucose,and high-glucose induction plus transfection of miR-96-5p mimic(P<0.05).Conclusion MiR-96-5p can affect the proliferation,invasion and migration of keratinocytes by activating the BNIP3/FAK signaling pathway,and thus influencing the healing of diabetic foot wounds.