微RNA-129-5p靶向Fas相关死亡功能域蛋白基因对体外培养人椎间盘髓核细胞凋亡的影响

目的研究微RNA-129-5p(miR-129-5p)靶向Fas相关死亡功能域蛋白(FADD)基因对人椎间盘髓核细胞(hNPC)凋亡的影响,并探讨其作用机制。方法运用脂质体法将miR-219-5p抑制子转染至hNPC中抑制miR-219-5p表达,将pcDNA-FADD转染至hNPC中使FADD过表达,将miR-219-5p抑制子和FADD siRNA共转染至hNPC中抑制miR-219-5p和FADD表达。采用流式细胞术检测各组细胞凋亡率,蛋白质印迹法检测抑制miR-219-5p表达后hNPC细胞中FADD、半胱氨酸蛋白酶-3(Caspase-3)、Bcl-2和Bax蛋白表达量。通过Targetscan软件预测miR-129-5p和FADD靶向结合位点,采用双荧光素酶报告基因实验检测二者的靶向关系。结果抑制miR-219-5p、过表达FADD均可明显促进hNPC凋亡。Targetscan软件预测发现FADD 3′-UTR上存在miR-219-5p的结合位点,双荧光素酶报告基因实验证实miR-129-5p和FADD具有靶向结合关系。抑制miR-219-5p的表达后,hNPC中FADD表达上调,同时促凋亡蛋白Caspase-3表达上调,抑凋亡蛋白Bcl-2表达下调。抑制FADD可逆转miR-219-5p低表达对hNPC凋亡的促进作用。结论低表达miR-219-5p可促进hNPC凋亡,其机制可能与miR-129-5p靶向FADD有关。

Effect of microRNA-129-5p targeting Fas-associated death domain protein gene on apoptosis of human nucleus pulposus cells in vitro

Objective To study the effect of microRNA-129-5p(miR-129-5p) targeting Fas-associated death domain protein(FADD) gene on the apoptosis of human nucleus pulposus cell(hNPC) and to explore its mechanism. Methods miR-219-5p inhibitor was transfected into hNPCs by liposome method to inhibit the expression of miR-219-5p. pcDNA-FADD was transfected into hNPCs to overexpress FADD. miR-219-5p inhibitor and FADD siRNA were co-transfected into hNPCs to inhibit the expression of miR-219-5p and FADD. The apoptosis rate of hNPCs was detected by flow cytometry. The expression of FADD,Caspase-3,Bcl-2 and Bax in hNPCs were detected by Western blotting after inhibiting the expression of miR-219-5p. The binding sites of miR-129-5p and FADD were predicted by Targetscan software,and the target relationship between them was detected by dual-luciferase reporter gene assay. Results Inhibition of miR-219-5p and over-expression of FADD significantly promoted the apoptosis of hNPCs. Targetscan software predicted that there was a binding site of miR-219-5p on FADD 3''-UTR. Dual-luciferase reporter gene assay confirmed that miR-129-5p and FADD had a targeted binding relationship. After inhibiting the expression of miR-219-5p,the expression of FADD and caspase-3 were up-regulated and Bcl-2 down-regulated in hNPCs. Inhibition of FADD can reverse the effect of miR-219-5p on apoptosis of hNPC. Conclusion Low expression of miR-219-5p can promote the apoptosis of hNPCs,and its mechanism may be related to the miR-129-5p targeting FADD.