幼年特发性关节炎患者外周血单个核细胞中miR-21miR-19a与JA刚STAT通路的表达与意义

The expression level and value of miR-21, miR-19a and JAK/STAT pathway in peripheral monocytes of juvenile idiopathic arthritis

目的探讨miR-21、miR-19a在幼年特发性关节炎（JIA）发病中的作用及与JAK／STAT通路关键靶基因（SOCS3、STAT3）的相互关系。方法收集广州市妇女儿童医疗中心确诊的33例活动期JIA患儿（病例组，其中全身型组20例，关节型组13例）；健康对照组20名。Fieoll法分离PBMCs，提取和纯化miRNA，反转录为cDNA；通过Targetscan和RNA22共同预测靶基因，设计引物，miR-21、miR-19a以u6为内参，STAT3、SOCS3、IL-6、TNF-α mRNA以β肌动蛋白为内参，应用荧光定量PCR分别检测其组间表达，计算标准化后的2^-△△CT值表示miRNA的相对表达含量，非参数检验统计2组数据2^-△△CT的差异。结果病例组与对照组miR-21表达水平低于对照组，差异有统计学意义（Z=2．11，P=0．036）；其中，对照组miR-21相对表达量为全身型组的7．7（7～8．5）倍、关节型的6．49（6～7）倍，差异有统计学意义（Z=2．615，P=0．0149；Z=2．654，P=0．0291）；全身型组与关节型组间miR-21表达水平差异无统计学意义（Z=0．221，P=0．8271）。病例组与对照组miR-19a表达水平低于对照组，差异有统计学意义（Z=2．41，P=0．014）；其中，对照组miR-19a相对表达量为全身型组的11．3（10-12．1）倍、关节型的12．2（12～13．5）倍，差异有统计学意义（Z=2．334，P=0．0157；Z=2．414，P=0．0266）；全身型组与关节型组间miR-19a表达水平差异无统计学意义（Z=O．538，P=O．596）。软件预测显示与JAK／STAT通路相关的STAT3，SOCS3、TNF—α分别为miR．21、mill-19a的靶基因。荧光定量PCR示病例组（全身型组、关节型组）STAT3[6．24（2．81，7．54）与3．97（1．81，5．75），P=0．001，0．008j、TNF-α[3．03（2．07，3．80）与3．42（2．46，4．68），P=0．002，0．001]、IL-6[4．75（3．59，6．32）与3．52（2．31，7．51），P=0．006，0．036]、SOCS3p2．54（1．77，4．00）与3．57（1．95，3．83），P=0．003，0．001[mRNA相对表达量较对照组高；病例组（全身型组、关节型组）STAT3mRNA相对表达量与miR-21呈负相关（r=-O．5854，P=0．0067；r=-0．6134，P=0．0447），病例组（全身型组、关节型组）TNF-α、SOCS3mRNA相对表达量与miR-19a呈现负相关，全身型组TNF．d（r=-0．6642，P=0．0014）、SOTS3（r=-0．7903，P=0．0001）。关节型组TNF-α（r=-0．6261，P=0．0393）、SOCS3（r=-0．8824，P=0．003）。结论JIA患者与健康人相比，PBMCs中miR-21、miR-19a表达水平的降低，其靶基因STAT3、SOCS3、TNF-α mRNA表达升高，提示JAK／STAT通路激活，miR-21、mill-19a水平降低与该通路的激活有关。

Objective To explore the expression of miR-21 and miR-19a in juvenile idiopathic arthritis （JIA） and the relationship among the key target genes （SOCS3, STAT3） in JAK/STAT pathways. Methods The venous blood from 33 cases of active JIA in Guangzhou Women and Children Medical Center were collected. All cases were divided into two groups： the systemic group （n=20）, polyarthritis group （n=13）.Twenty subjects were used as the normal control group. Peripheral blood mononuclear cells （PBMCs） were extracted and separated with Ficoll. miRNA was extracted and purified and real-time quantitative polymerase chain reaction （RT-PCR） was used to obtain cDNA. Target genes of miRNA were detected through Targetscan and RNA22. U6 was used for reference of miR-19a, miR-21 and β-actin were used for STAT3, SOCS3, IL-6, TNF-α mRNA. All the expression were detected by fluorescence quantitative PCR among the groups and calculated the result in standardized 2^-△△CT value, non-parametric test was used to test the differences. Results The expression of miR-2l were significantly reduced in the case group than the control group （Z=2.11, P= 0.036）, in which miR-21 was 7（7-8.5） times reduced than the SJIA group, 6.49 （6-7） times than the pJIA group, the difference was statistically significant （Z=2.615, P=0.014 9; Z=2.654, P=0.0291）. But no significant difference of miR-21 expression could be found between the SJIA and PJIA groups （Z=0.221, P =0.827 1）. The expression of miR-19a was significantly reduced in the case group than the control group （Z=2.41, P=0.014）, in which miR-19a was 11.3 （10-12.1） times to the SJIA group, 12.2 （12-13.5） times to the pJIA group, the difference was statistically significant （Z=2.334, P=0.015 7; Z=2.414, P=0.026 6）. But no significant difference could be detected in the miR-21 expression between the SJIA and the PJIA groups （Z=0.538, P=0.596）. Software estimated that STAT3, SOCS3, TNF-α were the target genes of miR-21 and miR-19a in the JAK/STAT pathways respectively. Fluorescence quantitative PCR had shown that mRNA expression of STAT3 [6.24（2.81, 7.54） and 3.97（1.81, 5.75）, P=0.001, 0.008], TNF-α [3.03（2.07, 3.80） and 3.42（2.46, 4.68）, P=0.002, 0.001], IL-6[4.75（3.59, 6.32） and 3.52（2.31, 7.51）, P=0.006, 0.036], SOCS312.54（1.77, 4.00） and 3.57（1.95, 3.83）, P= 0.003, 0.001] was higher in the case Group （SJIA group, the PJIA group） than the control group; STAT3 mRNA expression was negatively correlated with the miR-21 （r=-0.585 4, P=0.006 7; r=-0.613 4, P=0.044 7） and there was statistically significant difference. TNF-α, SOCS3 mRNA expression in the case group （SJIA group, PJIA group） was negatively correlated with the miR-19a. TNF-α （r=-0.664 2, P=0.001 4）, SOCS3 （r=-0.790 3, P=0.000 1） of the SJIA group,was higher than those of the PJIA group TNF-α （r=-0.626 1, P=0.039 3）, SOCS3 （r=-0.8824,/）--0.003）, the difference was significant. Conclusion The expression of miR-21, miR-19a in PBMC in the JIA patients are lower than the control group. The high expression of the target genes, miR-21, miR-19a of STAT3, SOCS3, TNF-a suggest that these genes might associate with, activating of JAK/STAT pathway.