miR-146a对氢醌抑制HL-60细胞分化的调控作用

目的： 观察氢醌对HL-60细胞向单核细胞分化的影响，并初步探讨miR-146a的调控作用。方法：设miR-146a-5p 抑制剂和阴性对照处理的HL-60细胞，分别以不同浓度(0、0.5、1.0、2.5和5.0 μmol/L)的氢醌处理3 h后，接种于含豆蔻酰佛波醇(PMA)的培养基中，培养72 h诱导其分化。采用实时荧光定量PCR检测细胞内miR-146a及其靶基因TRAF6的表达量，流式细胞术检测CD11b和CD14的表达，Western blot检测TRAF6蛋白及其下游调控因子IκBα蛋白的表达水平。结果：与对照组相比(0 μmol/L)， 氢醌可抑制PMA诱导的HL-60细胞CD11b和CD14的表达，5.0 μmol/L的氢醌使其表达量分别减少44%和38% (P均<0.01)；同时miR-146a的表达比对照组升高近2倍(P<0.01)；TRAF6 mRNA的表达下降约40%，其蛋白表达下降74%(P均<0.01)；磷酸化IκBα蛋白的表达减少约45%，IκBα总蛋白表达升高约73%(P均<0.01)。当抑制miR-146a的表达后，氢醌对TRAF6、IκBα和磷酸化IκBα蛋白表达的影响不明显。结论：miRNA-146a是氢醌影响HL-60细胞分化的调控因子之一。

The regulatory role of miR-146a in the differentiation of HL-60 cells inhibited by hydroquinone

OBJECTIVE： To study the effects of hydroquinone（HQ） on monocytic differentiation of HL-60 cells and explore the regulatory role of miR-146a in the mechanism of toxicity. METHODS：Using the model of monocytic differentiation induction with phorbol 12-myristate 13-acetate （PMA）,HL-60 cells were transfected with miR-146a-5p inhibitor or negative control and then treated with various doses of hydroquinone（0,0.5,1.0,2.5 and 5.0μmol/L） for 3 h,and cells collected after induction with PMA for 72 h. The expressions of miR-146a and its target gene TRAF6,were measured by real-time quantitative PCR. CD11b and CD14 levels were also determined by flow cytometry. Western blot was used to detect protein expression levels of TRAF6,and its downstream regulatory factor IκBα. RESULTS：Hydroquinone could inhibit the expressions of CD11b and CD14 in HL-60 cells induced by PMA. Compared with control group（0 μmol/L）,5.0 μmol/L of hydroquinone decreased the expressions of CD11b and CD14 by 44% and 38%,respectively （both P〈0.01）;the expression of miR-146a increased nearly 2-fold （P〈0.01）. The expressions of TRAF6 mRNA and protein were decreased by 40% and 74%（P〈0.01）,respectively. The expression of phosphorylated IκBα protein was reduced by approximately 45%,and the total IκBα protein increased nearly 73%（P〈0.01）. After inhibiting the expression of miR-146a,there were no significant effects of hydroquinone on TRAF6,phosphorylated IκBα and total IκBα protein expression. CONCLUSION： miRNA-146a may be one of the regulatory factors in which hydroquinone influence HL-60 cells differentiation.