微RNA-146b信号转导及转录激活因子1与3在男性原发性痛风性关节炎中的变化及其临床意义

Changes and clinical significance of microRNA-146b and signal transducer and transcriptional activator 1/3 in male primary gouty arthritis

目目的探讨微RNA-146b(miR-146b)、信号转导及转录激活因子(STAT)1与STAT3在原发性痛风性关节炎(GA)患者PBMCs中的表达变化及可能的临床意义。方法收集120例男性原发性GA[含57例急性期(AG)、63例间歇期(IG)]及66名健康体检者(HC组)的外周血标本、临床资料及实验室检查指标,采用实时荧光定量PCR技术(RT-qPCR)检测PBMCs中miR-146b、STAT1与STAT3的表达水平,比较其在3组间差异并与临床指标间进行相关性分析;构建受试者工作特征曲线(ROC)评估其在GA中的诊断效能。18名健康体检者的PBMCs经100μg/ml的MSU刺激3h模拟急性痛风炎症环境后,RTqPCR检测IL-1β和miR-146b、STAT1与STAT3的转录变化,蛋白质印迹法检测IL-1β、STAT1与STAT3蛋白及磷酸化蛋白表达变化。符合正态计量资料采用t检验、单因素方差分析及LSD-t检验,非正态分布数据采用Kruskal-WallisH检验和Mann-Whitney U检验,变量间相关采用Spearman相关分析,ROC来评估诊断价值。结果果①miR-146b、STAT1、STAT3在3组表达中差异均有统计学意义(F=7.02、19.52、17.07,P均<0.001),miR-146b在原发性GA组(0.32±0.28)显著高于HC组(0.19±0.18),而STAT1(0.019+0.012)、STAT3(0.014±0.010)则显著低于HC组[(0.038±0.029),(0.025±0.016),t=2.96,6.26,5.56,P均<0.01];进一步亚组分析显示miR-146b在AG与IG组的表达均高于HC组[(0.26±0.17),(0.38±0.35),(0.19±0.18),t=2.09,3.30,P均<0.05],但AC组低于IG组(t=2.02,P<0.05);STAT1mRNA在AG与IG组的表达均低于HC组[(0.020±0.012),(0.019±0.012),(0.038±0.029),t=4.89,4.56,P均<0.001],而AG和1G组间差异无统计学意义(t=0.24,P>0.05);STAT3mRNA在AG与IG组的表达均低于HC组[(0.016+0.012),(0.013±0.008),(0.025±0.016),t=5.64,3.33,P均<0.01],且AG组高于IG组(t=2.12,P<0.05)。②Spearman相关分析显示:GA中miR-146b的表达与同型半胱氨酸呈负相关(r=-0.37,P=0.014);STAT1与血肌酐呈负相关(r=-0.29,P=0.019),与肾小球滤过率估算值呈正相关(r=0.25,P=0.047)STAT3与HDL-C呈负相关(r=-0.27,P=0.033)。③ROC曲线显示:miR-146b、STAT1、STAT3的ROC曲线下面积(95%CI)AUC分别为0.679(0.582,0.776)、0.710(0.629,0.791)、0.705(0.626,0.783),三者联合为0.836(0.765,0.907),提示对痛风诊断有一定价值(P均<0.001)。④18例HC的PBMCs经MSU刺激3h后,与空白对照组和阴性对照组相比,miR-146b表达显著降低(H=14.44,P=0.003),而IL-1β、STAT1、STAT3mRNA表达显著升高(H=26.44、27.26、15.90,P均<0.001)。且模型组的IL-1β、STAT、STAT3蛋白及磷酸化蛋白表达明显高于空白对照组,差异均有统计学意义(t=9.97、6.63、7.48、11.25、6.28,P均<0.01)。结论miR-146b、STAT1、STAT3在GA的异常表达与部分临床指标相关,提示可能参与了痛风免疫炎症反应和代谢的调节,具体机制值得进一步研究。

Objective To This study was to investigate the expression and possible clinical significance of microRNA-146b(miR-146b)and signal transducer and transcriptional activator 1 and 3(STAT1/3)in peripheral blood mononuclear cells(PBMCs)of patients with primary gouty arthritis(GA).Methods The peripheral blood samples,clinical data and laboratory indexes of 120 male cases of GA[including 57 cases of acute(AG group)and 63 cases of intermittent(IG group)]and 66 healthy subjects(HC group)were collected.The expression levels of miR-146b and STAT1/3 in PBMCs were detected by real-time fluorescence quantitative PCR(RT-qPCR).The differences among the three groups were compared and the correlation between them and clinical indexes was analyzed.The receiver operating characteristic curve(ROC)was constructed to evaluate its diagnostic value in GA.After the PBMCs of 18 healthy subjects were stimulated by 100μg/ml MSU for 3 hours to simulate acute gout inflammatory environment,the transcriptional changes of IL-1β,miR-146b and STAT1/3 were detected by RT-qPCR,and the expressions of IL-1β,STAT1/3 protein and phosphorylated protein were detected by Western blotting.T test or one-way ANOVA and LSD-t test were used in accordance with the normal measurement data,Kruskal-Wallis H and Mann-Whitney U test were used in the non-normal data,Spearman correlation analysis was used in the correlation between variables,and the diagnostic value was evaluated by the receiver working characteristic curve ROC.Results ①There were statistical differences in the expression of miR-146b,STAT1 and STAT3 among the three groups(F=7.02、19.52、17.07,all P<0.001).The expression of miR-146b in gout group[(0.32±0.28)]was significantly higher than that in HC group(0.19±0.18)(t=2.96,P=0.003),while STAT1(0.019±0.012)and STAT3(0.014±0.010)were significantly lower than those in HC group(0.038±0.029),(0.025±0.016)(t=6.26,5.56,both P<0.001).Further subgroup analysis showed that the expression of miR-146b in AG and IG groups was higher than that in HC group[(0.27±0.17),(0.38±0.35),(0.19±0.18),t=2.09,3.30,both P<0.05],but that in AG group was lower than that in IG group(t=2.02,P<0.05).The expression of STAT1 mRNA in AG and IG groups was lower than that in HC group[(0.020±0.012),(0.019±0.012),(0.038±0.029),t=4.89,4.56,both P<0.001],but there was no statistical significance between AG and IG groups(t=0.24,P>0.05).The expression of STAT3 mRNA in AG and IG groups was lower than that in HC group[(0.016±0.012),(0.012±0.008),(0.025±0.016),t=5.64,3.33,both P<0.01],and the expression of STAT3 mRNA in AG group was higher than that in IG group(t=2.12,P<0.05).②Spearman correlation analysis showed that the expression of miR-146b in GA was negatively correlated with HCY(r=-0.37,P=0.014),STAT1 was negatively correlated with Crea(r=-0.29,P=0.019),positively correlated with eCFR(r=-0.25,P-0.047),and STAT3 was negatively correlated with HDL-C(r=-0.27,P=0.033).③ROC curve showed that the AUC(95%CI)of miR-146b,STAT1 and STAT3 were 0.679(0.582,0.776),0.710(0.629,0.791)and 0.705(0.626,0.783),and the combined AUC(95%CI)of the three was 0.836(0.765,0.907).④Compared with blank control group and negative control group,the expression of miR-146b in PBMCs of 18 cases of HC was significantly decreased(H=14.44,P=0.003),while the expression of IL-1β,STAT1 and STAT3 mRNA was significantly increased after 3 h of MSU stimulation(H=26.44、27.26、15.90,all P<0.001).The expression of IL-1β,STAT1 and STAT3 protein and phosphorylated protein in the model group were significantly higher than those in the blank control group,and the differences were statistically significant(t=9.97.6.63、7.48、11.25、6.28,all P<0.01).Conclusion The abnormal expression of miR-146b and STAT1/3 in GA is related to some clinical indicators,suggesting that it may be involved in the regulation of gout immune inflammatory response and metabolism,and the specific mechanism is worth further study.