微小RNA-155／微小RNA-21对急性期川崎病Toll样受体4表达的影响

The effects of miR-155/miR-21 on the expression of TLR4 in patients with acute Kawasaki disease

目的探讨单核细胞微小RNA（miR）-155／miR-21对急性期川崎病TLR4表达的可能影响。方法选取急性期川崎病患儿45例。根据是否合并冠状动脉损伤分为冠状动脉损伤（KD-CAL＋）组16例，无冠状动脉损伤（KD—CAL-）组29例。同年龄组健康体检儿童20名为健康对照（Ctrl）组。流式细胞术检测单核细胞TLR4蛋白表达水平，CD14＋免疫磁珠分选单核细胞，实时PCR检测单核细胞miR-155、miR-21表达并检测IL-10、IL-6、IL-1β、TNF—α等mRNA表达水平。EHSA检测MMP-9、高迁移率族蛋白BI（HMGB1）、热休克蛋白（HSP）60血浓度。采用t检验比较2组间差异。结果①急性期川崎病患儿单核细胞TLR4表达明显高于对照组[（12．4±5．1）％与（4．6±1．6）％，t=5．55，P〈0．05]，KD—CAL＋组TLR4表达明显高于KD—CAL-组[（17．1±4．6）％与（9．6±2．7）％，t=5．89，P〈0．05]；②急性期川崎病患儿HMGB1[（10．2±7．6）ng/m与（3．2±1．0）ng/ml，t=3．55，P〈0．05]及HSP60[（56±23）ng/ml与（18±7）ng／ml，t=4．90，P〈0．05]血浓度水平高于对照组，静脉注射丙种球蛋白（IVIG）治疗后二者血浓度明显下降（P〈0．05）；③急性期川崎病患儿单核细胞miR-155[（2．1±2．7）×10^-2与（4．6±3．1）×10^-3，t=2．57，P〈0．05]及miR-21表达（11．6±4．0与8．7±2．2，t=2．78，P〈0．05）明显高于对照组，KD-CAL＋组miR-155表达明显高于KD-CAL-组[（4．2±3．0）×10^-2与（11．0±1．5）×10^-4，t=4．07，P〈0．05]，miR．21表达明显低于KD—CAL-组（8．8±2．5与12．8±4．0，t=3．46，P〈0．05）；④急性期川崎病患儿单核细胞IL-10mRNA表达高于对照组[（34．9±17．5）×10^-4与（1．0±9．4）×10^-4，t=5．62，P〈0．05]，KD-CAL＋组IL-10mRNA表达明显低于KD-CAL-组[（26．6±13．4）×10^-4与（39．5±1．8）×10^-4，t=2．36，P〈0．05]；⑤急性期川崎病患儿TNF-α、IL-6、IL-1β等mRNA表达明显高于对照组（P〈0．05），且KD—CAL＋组明显高于KD-CAL-组（P〈0．05）；⑥急性期川崎病患儿血浆MMP-9浓度高于对照组[（1141±541）pg/ml与（304±94）pg/ml，t=4．83，P〈0．05]，KD-CAL＋组MMP-9血浓度明显高于KD—CAL-组[（1350±625）pg/ml与（945±370）pg/ml，t=2．21，P〈0．05]。结论miR-155过表达，miR-21表达相对不足导致的miR-155／miR-21调控失衡可能是急性期川崎病TLR4异常表达的原因之一，HMGB1、HSP60等内源性配体浓度持续增高也可能参与触发或放大TLR4表达。

Objective To investigate the effects of miR-155/miR-21 on the expression of TLR4 in patients with Kawasaki disease （KD）. Methods Forty-five children with acute KD were enrolled into the study. Children were divided into the group with coronary artery lesions （KD-CAL＋） and the group without coronary artery lesions （KD-CAL-）. The age-matched twenty healthy children were included as the control group. The levels of TLR4 expression were analyzed by flow cytometry. The CD14＋ monocytes in the peripheral blood were separated by CD14＋ immunomagnetic beads. Real-time polymerase chain reaction （PCR） were performed to evaluate the expression of miR-155, miR-21 and interleukin （IL）-10, tumor necrosis factor （TNF）-α, IL-6, IL-1β in CD14＋ monocytes at mRNA level. The concentration of matixmetalloproteinase （MMP）-9, HSP60, high mobility group box-1 protein （HMGB1） were measured by enzyme-linked immunosorbent assay （ELISA）. T test was used to compare the differences between groups and P〈0.05 was considered to be statistically significant. Results The levels of Toll-like receptor 4 （TLR4） expression in children with acute KD were significantly higher than those in the control group [（12.4±5.1）% vs （4.6±1.6）%, t=5.55, P〈0.05], and the levels of TLR4 expression in the KD-CAL＋ group were remarkably elevated compared with the KD-CAL＋ group [（17.1 ±4.6）% vs （9.6±2.7）%, t=5.89, P〈0.05]. The plasma concentrations of HMGBII [（10.2±7.6 vs 3.2±1.0） ng/ml, t=3,55, P〈0.05] and HSP60 [（56±23 vs 18±7） ng/ml, t=4.90, P〈0.05] in the KD group were increased（P〈0.05）, but significantly decreased after treatment with IVIG （P〈0.05）. The levels of miR-155 [（21.0±27.0）×10^-3 vs （4.6±3.1）×10^-3, t=2.57, P〈0.05] and miR-21 （11.6±4.0 vs 8.7±2.2, t=2.78, P〈0.05） expression in the KD group were significantly higher than those in the controls （P〈0.05）. The level of miR-155 expression in the KD-CAL＋ group was apparently higher than that in the KD-CAL- group [（4.2±3.0）×10^-2 vs （11.0±1.5）×10^-4, t=4.07, P〈0.05] and the expression of miR-21 was lower than that in the KD-CAL- group （8.8±2.5 vs 12.8±4.0, t=3.46, P〈0.05）. Compared with the controls, the levels of IL-10 mRNA expression in the KD group were significantly increased [（34.9±17.5） ×10^-4 vs （1.0±9.4） ×10^-4, t =5.62, P〈0.05）, and IL-10 expression in the KD-CAL- group was higher than that in the KD-CAL＋ group [（26.6±13.4）×10^-4 vs （39.5±1.8）±104, t=2.36, P〈0.05]. The plasma concentrations of MMP-9 in the KD group were remarkably higher than those in the controls [（1141±541） pg/ml vs （304±94） pg/ml, t=4.83, P〈0.05], the concentration of MMP-9 in the KD-CAL＋ group was higher than that in the KD-CAL- group [（1 350±625） pg/ml vs （945± 370） pg/ml, t=2.21, P〈0.05]. Conclusion The dysregulation of miR-155 and miR-21 may be one of the reasons that result in over-expression of TLR4 in patients with acute KD, and the persistently increased HSP60 and HMGB1 may trigger or amplify TLR4 expression.