达格列净通过miR-140-5p/FNDC5通路调控高糖诱导内皮细胞增殖、凋亡的机制研究

The mechanism underlying the regulatory effect of dapagliflozin on the proliferation and apoptosis of high-glucose-induced endothelial cells through the miR-140-5p/FNDC5 axis

目的探究达格列净(DAPA)调控高糖诱导内皮细胞增殖、凋亡的潜在作用。方法5.5、30 mmol/L葡萄糖处理的内皮细胞设为正常组(NG)、高糖组(HG);达格列净DAPA(20、40、80μmol/L)处理HG细胞。脂质体法将agomiRNA组(转染agomiRNA)、agomiR-140-5p组(转染agomiR-140-5p)、antagomiRNA组(转染antagomiRNA)、antagomiR-140-5p组(转染antagomiR-140-5p)、si-NC组(转染si-NC)、si-FNDC5组(转染si-FNDC5)、pcDNA组(转染pcDNA)、pcDNA-FNDC5组(转染pcDNA-FNDC5)、80μmol/L+agomiRNA组(转染agomiRNA再用80μmol/L处理)、80μmol/L+agomiR-140-5p组(转染agomiR-140-5p再用80μmol/L的DAPA处理)、80μmol/L+agomiR-140-5p+pcDNA组(共转染agomiR-140-5p和pcDNA并用80μmol/L的DAPA处理)、80μmol/L+agomiR-140-5p+pcDNA-FNDC5组(共转染agomiR-140-5p和pcDNA-FNDC5并用80μmol/L的DAPA处理)转染至内皮细胞。实时荧光定量聚合酶链反应(RT-qPCR)实验、蛋白免疫印迹(WB)实验检测细胞miR-140-5p、FNDC5、Ki67、PCNA、caspase-3的表达;细胞计数试剂盒(CCK8)、流式细胞术检测细胞增殖、凋亡;双荧光素酶报告实验检测细胞荧光活性。结果与NG组比较,HG组细胞增殖率显著降低,凋亡率显著升高,Ki67、PCNA蛋白表达显著降低,caspase-3的蛋白表达显著升高,miR-140-5p表达显著升高,FNDC5表达均显著降低(P<0.05)。过表达miR-140-5p或敲减FNDC5具有相似的抑制HG细胞增殖,促进凋亡的作用,而抑制miR-140-5p或过表达FNDC5则具有相反的作用。双荧光素酶报告实验显示,miR-140-5p靶向负调控FNDC5。过表达FNDC5可部分逆转过表达miR-140-5p对HG细胞的增殖、凋亡调控,增强DAPA的作用。结论DAPA可减轻高糖对内皮细胞的增殖抑制和凋亡促进作用,其潜在的作用机制与抑制miR-140-5p/FNDC5通路有关。

Objective To explore the potential role of dapagliflozin(DAPA)in regulating the proliferation and apoptosis of high-glucose-induced endothelial cells.Methods Endothelial cells treated with 5.5mmol/L and 30mmol/L glucose were included into normal group(NG)and high-glucose group(HG).High-glucose-induced endothelial cells were treated with 20,40 and 80μmol/L DAPA.Cells were then divided into AgomiRNA group(transfected with agomiRNA),agomiR-140-5p group(transfected with agomiRNA-140-5p),antamiRNA group(transfected with antamiRNA),antamiR-140-5p group(transfected with antamiRNA-140-5p),si-NC group(transfected with si-NC),si-FNDC5 group(transfected with si-FNDC5),pcDNA group(transfected with pcDNA),pcDNA-FNDC5 group(transfected with pcDNA-FNDC5),80μm+agomiRNA group(transfected with agomiRNA followed by the treatment of 80μmol/L DAPA),80μmol/L+agomiR-140-5p group(transfected with agomiR-140-5p followed by the treatment of 80μmol/L DAPA),80μmol/L+agomiR-140-5p+pcDNA group(co-transfected with agomiR-140-5p and pcDNA followed by the treatment of 80μmol/L DAPA),and 80μmol/L+agomiR-140-5p+pcDNA-FNDC5 group(co-transfected with agomiR-140-5p and pcDNA-FNDC5 followed by the treatment of 80μmol/L DAPA).Cell transfection was performed using Lipofectamine.Expression levels of miR-140-5p,FNDC5,Ki67,PCNA and caspase-3 were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot(WB).Cell counting kit-8(CCK8)and flow cytometry were used to detect cell proliferation and apoptosis,respectively.Dual-luciferase reporter assay was used to detect the targeting relationship between miR-140-5p and FNDC5.Results Compared with those of NG group,we detected significantly reduced proliferation rate,protein levels of Ki67 and PCNA and mRNA level of FNDC5,and significantly enhanced apoptotic rate,protein level of caspase-3 and mRNA level of miR-140-5p in HG groups(all P<0.05).Overexpression of miR-140-5p or knockdown of FNDC5 similarly inhibited the proliferation and stimulated the apoptosis of cells in HG group;while opposite results were achieved by knockdown of miR-140-5p or overexpression of FNDC5.Dual-luciferase reporter assay revealed that miR-140-5p negatively regulated FNDC5.Overexpression of FNDC5 partially reversed the role of overexpressed miR-140-5p in mediating the proliferation and apoptosis of high-fat-induced cells,which significantly strengthened the effect of DAPA.Conclusion DAPA alleviates high-fat-induced inhibited proliferation and promoted apoptosis in endothelial cells by inhibiting the miR-140-5p/FNDC5 axis.