Expression and clinical significance of N6-methyladenosine modification-related genes in peripheral blood mononuclear cells from patients with gouty arthritis
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目的 探讨N6-甲基腺苷(m6A)修饰相关基因在原发性痛风性关节炎(GA)患者外周血单个核细胞(PBMC)中的表达及其可能的作用.方法 收集2023年10月至12月期间在川北医学院附属医院风湿免疫科门诊就诊的急性期痛风患者(AG组)、间歇期痛风患者(IG组)、年龄及性别匹配的健康体检者(HC组)各45例.采用实时荧光定量逆转录聚合酶链反应(RT-qPCR)检测m6A修饰相关基因[甲基转移酶3(METTL3)、甲基转移酶14(METTL14)、Wilms肿瘤1相关蛋白(WTAP)、肥胖相关蛋白(FTO)、AlkB同系物5(ALKBH5)、胰岛素样生长因子2-mRNA结合蛋白(IGF2BP)2、IGF2BP3、含YTH结构域结合蛋白1(YTHDF1)和含YTH结构域蛋白2(YTHDC2)]在3组PBMCs中的表达水平,并与临床指标进行相关分析.计量资料符合正态分布采用方差分析或t检验,非正态检验用Kruskal-Wallis H检验和Mann-Whitney U检验分析数据.采用受试者特征曲线(ROC)评估m6A修饰相关基因对诊断GA的价值.结果 ① IGF2BP2(Z=-3.59,P＜0.001)、WTAP(Z=-5.25,P＜0.001)、METTL14(Z=-3.62,P＜0.001)、YTHDF1(Z=-2.12,P=0.034)、YTHDC2(Z=-2.00,P=0.045)在痛风组(AG+IG患者)与HC组表达差异均有统计学意义.其中,IGF2BP2 在 GA 组[28.08(17.99,47.06)×10-4]的表达显著高于 HC 组[19.23(12.90,25.78)×10-4],WTAP、METTL14、YTHDF1 和 YTHDC2 在 GA 组[298.61(213.61,377.80)×10-4,9.94(6.43,13.46)×10-4,52.63(28.22,72.77)×10-4,40.24(20.74,73.32)×10-4]的表达均显著低于 HC 组[398.45(339.88,454.89)×10-4,13.27(11.07,15.85)×10-4,64.43(43.61,87.10)×10-4,53.11(36.37,79.28)×10-4];进一步亚组分析发现IGF2BP2、WTAP、METTL14、YTHDF1、YTHDC2 在 3 组间表达中差异均有统计学意义(H=19.62、31.73、13.14、16.64、28.90,P均≤0.001),WTAP 和 METTL14 在 AG 组[311.13(234.96,426.67)×10-4(Z=-3.27,P=0.001),9.64(5.21,15.21)×10-4(Z=-2.71,P=0.008)]与 IG 组[272.27(203.29,347.95)×10-4(Z=-5.78,P＜0.001),10.40(6.88,12.88)×10-4(Z=-3.54,P=0.003)]的表达均低于 HC 组[398.45(339.88,454.89)×10-4,13.27(11.07,15.85)×10-4],但 AG 与 IG 之间差异无统计学意义(P＞0.05);YTHDF1 和 YTHDC2 在 AG 组[38.10(16.19,56.78)×10-4,24.31(14.35,42.77)×10-4]均显著低于 IG 组[64.13(48.28,74.40)×10-4(Z=-3.54,P＜0.001),65.49(39.89,91.23)×10-4(Z=-4.96,P＜0.001)]和 HC 组[64.43(43.61,86.92)×10-4(Z=-3.51,P＜0.001),53.11(36.37,79.28)×10-4(Z=-4.25,P＜0.001)],但 IG 与 HC 组间差异无统计学意义(P＞0.05);IGF2BP2 在 AG 组[25.32(16.40,40.43)×10-4]和 HC 组[19.23(12.90,25.78)×10-4]均显著低于 IG 组[31.10(22.60,49.58)×10-4](Z=-2.46,P=0.014;Z=-4.54,P＜0.001),但 AG 和 HC 比较差异无统计学意义(P＞0.05).②Spearman相关分析显示:在GA患者中,IGF2BP2、METTL14和YTHDF1的表达分别与血浆葡萄糖(r=0.22,P=0.037)、血尿酸(r=0.38,P=0.003)和总胆固醇(r=0.23,P=0.034)呈正相关,WTAP与血浆葡萄糖呈负相关(r=-0.25,P=0.020).③5个差异基因联合预测GA的ROC曲线下面积(95％CI)为0.90(0.84,0.95).结论 m6A修饰相关基因在GA中存在异常表达且与GLU和UA等临床指标相关,推测其可能参与GA的发病及对评估GA患者代谢有一定的参考价值.
Objective To investigate the expression of N6-methyladenosine(m6A)modification-related genes and their possible roles in peripheral blood mononuclear cells(PBMCs)of patients with primary gouty arthritis(GA).Methods Forty-five patients each with acute gout(AG),intermittent gout(IG),and age-and gender-matched healthy controls(HC)were collected from the outpatient clinic of the Department of Rheumatology and Immunology of the Affiliated Hospital of Chuanbei Medical College between October and December of 2023.The expression levels of m6A modification-related genes(METTL3、METTL14、WTAP、FTO、ALKBH5、IGF2BP2、IGF2BP3、YTHDF1、YTHDC2)in PBMCs among the 3 groups were detected by RT-qPCR and correlation analysis with clinical indicators was performed.Measurements conforming to normal distribution were analyzed using ANOVA or t-tests,and data were analyzed using the Kruskal-Wallis H-test and Mann-Whitney U-test for data that is not-normaly distributed.The value of m6A modification-related genes for the diagnosis of GA was evaluated using subject characterization curve ROC.Results ①There were statistically significant differences in the expression of IGF2BP2(Z=-3.59,P＜0.001)、WTAP(Z=-5.25,P＜0.001),METTL14(Z=-3.62,P＜0.001)、YTHDF1(Z=-2.12,P=0.034)and YTHDC2(Z=-2.00,P=0.045)in the disease group and the normal control group.Among them,the expression of IGF2BP2 in the GA group[28.08(17.99,47.06)×10-4]was significantly higher than that in the HC group[19.23(12.90,25.78)×10-4],and the expressions of WTAP、METTL14、YTHDF1 and YTHDC2 in the GA group[298.61(213.61,377.80)×10-4,9.94(6.43,13.46)×10-4,52.63(28.22,72.77)×10-4,40.24(20.74,73.32)×10-4]were significantly lower than those in the HC group[398.45(339.88,454.89)×10-4,13.27(11.07,15.85)×10-4,64.43(43.61,87.10)×10-4,53.11(36.37,79.28)×10-4].Further subgroup analysis revealed statistically significantdifferences in the expression of IGF2BP2、WTAP、METTL14、YTHDF1 and YTHDC2 among the 3 groups(H=19.62、31.73、13.14、16.64、28.90,all P≤0.001).The expressions of WTAP and METTL14 in the AG group[311.13(234.96,426.67)×10-4,Z=-3.27,P=0.001;9.64(5.21,15.21)×10-4,Z=-2.71,P=0.008]and IG group[272.27(203.29,347.95)×10-4,Z=-5.78,P＜0.001;10.40(6.88,12.88)×10-4,Z=-3.54,P=0.003]were lower than those in the HC group[398.45(339.88,454.89)×10-4,13.27(11.07,15.85)×10-4].However,there was no significant difference between AG and IG group(P＞0.05).Both YTHDF1 and YTHDC2 were significantly lower in the AG group[38.10(16.19,56.78)×10-4,24.31(14.35,42.77)×10-4]than those in the IG group[64.13(48.28,74.40)×10-4(Z=-3.54,P＜0.001,65.49(39.89,91.23)×10-4(Z=-4.96,P＜0.001)]and HC group[64.43(43.61,86.92)×10-4(Z=-3.51,P＜0.001),53.11(36.37,79.28)×10-4(Z=-4.25,P＜0.001)].But there was no statistically significant difference between IG and HC groups(P＞0.05);IGF2BP2 was significantly lower in the AG group[25.32(16.40,40.43)×10-4,Z=-2.46,P=0.014]and HC grpup[19.23(12.90,25.78)×10-4,Z=-4.54,P＜0.001]than in the IG group[31.10(22.60,49.58)×10-4],but the comparison between AG and HC showed no statistically significant difference(P＞0.05).(2)Spearman correlation analysis showed that in GA patients,the expression of IGF2BP2、METTL14 and YTHDF1 was positively correlated with plasma glucose、blood uric acid(sUA)and total cholesterol level respectively(r=0.22,P=0.037;r=0.38,P=0.003;r=0.23,P=0.034),and WTAP was negatively correlated with GLU(r=-0.25,P=0.020).(3)The ROC curve for the joint prediction of the five differential genes showed that the 95％CI for area under the curve in GA was 0.90(0.84,0.95).Conclusion The m6A modification-related genes are abnormally expressed in GA and are correlated with clinical indicators such as GLU and UA,which are hypothesized to be involved in the pathogenesis of GA and have a certain reference value for the evaluation of metabolism in GA patients.
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