miR-29b-3p靶向FoxO3基因改善非酒精性脂肪肝预后的初步研究

MiR-29b-3p targeting FoxO3 gene improves the prognosis of non-alcoholic fatty liver disease

摘要:

目的： 探讨miR-29b-3p靶向FoxO3对非酒精性脂肪肝（non-alcoholic fatty liver disease，NAFLD）的作用。方法： 人肝细胞HL-7702（L02）通过miR-29b-3p抑制剂或NC抑制剂或pcDNA FoxO3转染，经棕榈酸（palmitic acid，PA）处理后，采用油红O染色测定脂质积累；全自动生化分析仪检测甘油三酯（triglycerides，TG）、胆固醇（total cholesterol，TC）含量；ELISA检测超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）、丙二醛（malondialdehyde，MDA）含量；采用RT-qPCR检测叉头转录因子O亚型3（forkhead box class O3，FoxO3）、miR-29b-3p mRNA表达；Western blot检测FoxO3蛋白表达；双荧光素酶实验验证miR-29b-3p靶向FoxO3。结果： 与对照组比较，PA组TG（2.05±0.22）mmol/L、TC（7.80±1.23）mmol/L、TNF-α（149.67±8.96）%、IL-6（157.00±18.33）%、IL-8（177.33±24.58）%、miR-29b-3p（161.00±13.0）%、MDA（0.456±0.028）nmol/mL及油红O染成红色的脂滴数量明显升高，SOD（12.91±0.53）ng/mL、GSH-Px（0.515±0.038）ng/mL含量及FoxO3（65.93±4.56）%表达明显降低；miR-29b-3p靶向FoxO3；与PA组比较，PA+miR-29b-3p inhibitor组、PA+miR-29b-3p inhibitor + pcDNA-FoxO3组可明显降低TG、TC、TNF-α、IL-6、IL-8、miR-29b-3p、MDA含量及油红O染成红色的脂滴数量，明显升高SOD、GSH-Px含量。结论： miR-29b-3p靶向FoxO3可减轻PA诱导的L02细胞脂质积累和炎症。

Abstract:

Objective: To investigate the effect of miR-29b-3p targeting FoxO3 on non-alcoholic fatty liver disease (NAFLD). Methods: Human hepatocytes HL-7702 (L02) were transfected with miR-29b-3p inhibitor, NC inhibitor or pcDNAFoxO3. After palmitic acid (PA) treatment, oil red O staining was used to determine lipid accumulation, fully automatic biochemical analyzer to detect the content of triglycerides (TG) and total cholesterol (TC), ELISA to detect the content of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA), RT-qPCR to detect the expression of forkhead box class O3 (FoxO3), miR-29b-3p mRNA, Western blot to detect the expression of FoxO3 protein, and dual luciferase experiments to verify miR-29b-3p targeting FoxO3. Results: Compared with the control group, the expression of TG (2.05 ± 0.22) mmol/L, TC (7.80 ± 1.23) mmol/L, TNF-α (149.67 ± 8.96) %, IL-6 (157.00 ± 18.33) %, IL-8 (177.33 ± 24.58) %, miR-29b-3p (161.00 ± 13.0) %, MDA (0.456 ± 0.028) nmol/mL and the number of lipid droplets dyed red by Oil Red O in the PA group was significantly increased; the content of SOD (12.91 ± 0.53) ng/mL, GSH-Px (0.515 ± 0.038) ng/mL and the expression of FoxO3 (65.93 ± 4.56) % were significantly reduced; miR-29b-3p targeted FoxO3. Compared with PA group, PA + miR-29b-3p inhibitor group, PA + miR-29b-3p inhibitor + pcDNA-FoxO3 group significantly reduced the content of TG, TC, TNF-α, IL-6, IL-8, miR-29b-3p, MDA and the number of oil droplets dyed red by Oil Red O, and significantly increased the content of SOD and GSH-Px. Conclusion: MiR-29b-3p targeting FoxO3 can reduce PA-induced lipid accumulation and inflammation in L02 cells.