苦瓜蛋白通过FXR/miR-23b-3p/HNF4α途径抑制载脂蛋白(a)表达

Momordicin 28 inhibits expression of apolipoprotein ( a ) by FXR/miR-23b-3p/HNF4α pathway

目的 研究苦瓜蛋白（MD28）对HepG2细胞载脂蛋白（a）[Apo（a）]表达的影响,探索其作用机制。 方法 离心、超滤、透析、阳离子交换色谱、反相高效液相色谱等方法提取苦瓜蛋白；MTT法检测苦瓜蛋白处理HepG2细胞后细胞存活率；实时荧光定量PCR（qRT-PCR）和Western blot分别检测Apo（a）、法尼酯X受体（FXR）、肝细胞核因子4α（HNF4α）的mRNA和蛋白表达水平；qRT-PCR检测hsa-miR-23b-3p的表达水平,采用小干扰RNA转染沉默FXR。 结果 与对照组相比,0.1、0.2、0.4、0.8及1.6 g/L苦瓜蛋白处理HepG2细胞24 h,细胞存活率无统计学差异；与对照组相比,苦瓜蛋白呈剂量和时间依赖性抑制Apo（a）表达,以1.6 g/L苦瓜蛋白作用48 h的效果最为显著；苦瓜蛋白上调FXR和hsa-miR-23b-3p表达水平,下调HNF4α表达水平,沉默FXR则逆转苦瓜蛋白的上述作用,沉默hsa-miR-23b-3p能逆转苦瓜蛋白对HNF4α的下调作用,但对FXR的表达无影响。 结论 苦瓜蛋白通过FXR/miR-23b-3p/HNF4α途径抑制HepG2细胞Apo（a）的表达,有望成为临床降低脂蛋白（a）的候选药物。

Aim To study the effect of momordicin 28 （MD28） on the expression of apolipoprotein（a） in HepG2 cells and explore its mechanism. Methods The cell viability of HepG2 cells treated with MD28 was determined by MTT assay. MD28 was purified by centrifugation, ultrafiltration, dialysis, cation exchange chromatography and reversed-phase high performance liquid chromatography. The mRNA and protein expression levels of apolipoprotein（a）, farnesyl ester X receptor （FXR） and hepatocyte nuclear factor 4α （HNF4α） were detected by qRT-PCR and Western blot respectively, qRT-PCR was used to detect the expression level of hsa-miR-23b-3p, and small interfering RNA transfection was used to silence FXR. Results Compared with the control group, the cell viability of HepG2 cells treated with MD28 at 0.1,0.2,0.4,0.8 and 1.6 g/L had no significant difference. Compared with the control group, MD28 inhibited apolipoprotein（a） expression in a dose and time dependent manner, the best inhibitory effect was 1.6 g/L MD28 on 48 h. FXR and hsa-miR-23b-3p expression levels were up-regulated by MD28 while HNF4α was down-regulated in HepG2 cells, and silencing FXR reversed above-mentioned effect of MD28, silencing hsa-miR-23b-3p could also reverse the down-regulation effect of MD28 on HNF4α, but had no effect on FXR expression. Conclusion MD28 inhibits the expression of Apo（a） in HepG2 cells through the FXR/miR-23b-3p/HNF4α pathway, and is expected to be a candidate for the clinical reduction of lipoprotein（a）.