微小RNA-148a-3p对强直性脊柱炎患者T细胞自噬及炎症应答的影响及其机制

Effect of microRNA-148a-3p on cell autophagy and inflammatory response of T cells in ankylosing spondylitis and possible mechanism

目的探讨微小RNA（miRNA，miR）-148a-3p对强直性脊柱炎患者T细胞自噬及炎症应答的影响及其作用机制。方法采用实时定量反转录聚合酶链反应（RT-qPCR）检测强直性脊柱炎（AS）患者T细胞中miR-148a-3p和DNA甲基转移酶1（DNMT1）的表达水平。利用Western blot检测自噬标志分子[微管相关蛋白1轻链3（LC3）、Beclin-1和自噬相关基因5（ATG5）]的蛋白表达量。利用双荧光素酶报告系统、RT-qPCR及Western blot法验证miR-148a-3p与DNMT1的靶向作用关系。结果与正常对照组比较，AS患者T细胞中miR-148a-3高表达，而DNMT1表达水平较低。miR-148a-3p过表达显著增加了炎性因子白细胞介素（IL）-6、IL-17和IL-23的表达水平（4.23±0.34比1.06±0.15，P＝0.000；2.87±0.29比1.02±0.17，P＝0.000；3.12±0.32比1.07±0.21，P＝0.000）；反之，抑制miR-148a-3p的表达，细胞因子表达水平则明显降低（0.41±0.14比0.97±0.13，0.52±0.10比0.95±0.14，0.45±0.12比0.98±0.18；P＝0.008）。此外，过表达miR-148a-3p明显降低自噬标志基因LC3Ⅱ/LC3Ⅰ、ATG5和Beclin-1的表达水平（0.44±0.13比1.06±0.16，P＝0.003；0.38±0.11比1.07±0.16，P＝0.002；0.42±0.12比1.03±0.14，P＝0.003）；相反，下调miR-148a-3p则增加自噬水平（2.89±0.28比1.03±0.17，P＝0.000；3.31±0.29比0.98±0.18，P＝0.000；3.68±0.31比1.02±0.15，P＝0.000）。双荧光素酶报告基因检测结果显示，miR-148a-3p与DNMT1存在直接的靶标关系（0.44±0.07比1.00±0.12，P＝0.002；1.68±0.17比1.00±0.11，P＝0.004）；且miR-148a-3p过表达抑制DNMT1的mRNA（0.38±0.17比0.98±0.23，P＝0.011）和蛋白（0.41±0.17比0.97±0.25，P＝0.014）表达水平，反之则促进DNMT1的mRNA（3.83±0.29比1.04±0.21，P＝0.000）和蛋白（3.46±0.28比1.02±0.20，P＝0.000）表达。结论miR-148a-3p其可以通过靶向调控DNMT1的表达来调节成T细胞自噬及炎症应答。

ObjectiveTo investigate the effects of microRNA （miRNA, miR）-148a-3p on autophagy and inflammatory response of T cells from ankylosing spondylitis （AS） and the potential underlying mechanism.MethodsReal-time quantitative reverse transcriptase-polymerase chain reaction （RT-qPCR） was used to detected the expression level of miR-148a-3p and DNA methy transferas 1 （DNMT1） in AS. Western blotting was used to determine the protein expression of autophagy related proteins microtubule-associated protein 1 light chain 3 （LC3）, Beclin-1 and autophagy related gene 5 （ATG5）. The targeting effect of miR-148a-3p on DNMT1 gene was verified by the dual-luciferase reporter assay system, Western blotting and RT-qPCR.ResultsThe expression of miR-148a-3p was highly expressed and DNMT1 was significantly downregulated in T cell from AS patients compared with normal group. Additionally, forced expression of miR-148a-3p obviouslyincreased the expression of inflammatory factors IL-6, IL-17 and IL-23 （4.23±0.34 vs. 1.06±0.15, P=0.000; 2.87±0.29 vs. 1.02±0.17, P=0.000; 3.12±0.32 vs. 1.07±0.21, P=0.000）, whereas inhibition the expression of miR-148a-3p strongly attenuated the level of these cytokines （0.41±0.14 vs. 0.97±0.13, P=0.002; 0.52±0.10 vs. 0.95±0.14, P=0.006; 0.45±0.12 vs. 0.98±0.18, P=0.008）. Moreover, introduction of miR-148a-3p dramatically inhibited the expression of autophagy proteins LC3Ⅱ/LC3Ⅰ, ATG5 and Beclin-1 （0.44±0.13 vs. 1.06±0.16, P=0.003; 0.38±0.11 vs. 1.07±0.16, P=0.002; 0.42±0.12 vs. 1.03±0.14, P=0.003）, while downregulating miR-148a-3p significantly promoted autophagy （2.89±0.28 vs. 1.03±0.17, P=0.000; 3.31±0.29 vs. 0.98±0.18, P=0.000; 3.68±0.31 vs. 1.02±0.15, P=0.000）. More importantly, our study explored thatDNMT1 was a direct and functional target of miR-148a-3p （0.44±0.07 vs. 1.00±0.12, P=0.002; 1.68±0.17 vs. 1.00±0.11, P=0.004）. RT-qPCR and Western blotting analysis further validated that miR-148a-3p negatively regulated the mRNA （0.38±0.17 vs. 0.98±0.23, P=0.011; 3.83±0.29 vs. 1.04±0.21, P=0.014） and protein （0.41±0.17 vs. 0.97±0.25, P=0.000; 3.46±0.28 vs. 1.02±0.20, P=0.000） expression of DNMT1.ConclusionOur findings demonstrated that miR-148a-3p is involved in AS by regulating cell autophagy and inflammatory response through targeting and regulating DNMT1 expression, which provides a potential target for treatment of AS in future.