miR-10b对滑膜成纤维细胞增殖、侵袭和迁移能力的影响及分子机制

Effects and Molecular Mechanisms of miR-10b on the Proliferation, Invasion and Migration of Fibroblast-Like Synovial Cells

目的:探讨miR-10b对类风湿性关节滑膜成纤维细胞(RA-FLS)炎性因子分泌、增殖、侵袭和迁移的影响及其分子机制。方法:首先,分离原代培养FLS细胞并进行microarray,筛选RA和OA中差异表达的miRNA分子。然后,用real-time PCR对筛选得到的结果进行验证,进而通过生物信息学分析、细胞转染等方法明确miR-10b在FLS细胞中下调的分子机制。最后,采用MTT比色法、划痕实验和Transwell实验等检测miR-10b对其FLS细胞增殖、侵袭和迁移水平的影响。结果:与OA FLS相比,芯片筛选发现176条miRNA在RA-FLS中上调,204条下调;其中,miR-10b在RA-FLS细胞中受肿瘤坏死因子(TNF-α)等多种炎性因子以NF-κB依赖的方式进行调控;miR-10b的下游靶基因为TAK1和TLR4,通过对这两个靶基因的调控,miR-10b一方面可以促进TNF-α的分泌和NF-κB的活化入核,从而激发TNF-α→NF-κB→miR-10b→TNF-α/NF-κB环路;另一方面促进FLS表面TLR4的表达以及LPS对于FLS的刺激作用,激发LPS→NF-κB→miR-10b→TLR4环路。此外,miR-10b的下调可促进FLS细胞的增殖、侵袭和迁移。结论:miR-10b在RA-FLS细胞中显著下调,其通过参与信号环路的调节可影响FLS细胞的炎性细胞因子分泌及其增殖、侵袭和迁移。

Objective： To investigate the effects ofmicroRNA （miR）-10b on the inflammatory cytokine secretion, proliferation, invasion and migration of RA-FLS and the underlying mechanisms. Methods： Primary FLS cells were isolated for miRNA microarray to screen differentially-expressed miRNAs between RA and OA. Then, after confirming the microarray data by real-time PCR analysis, the detailed mechanisms of miR-10b down-regulation was analyzed with bioinformatics and cell transfection methods. Finally, real-time PCR, MTT assay, cell disclosure and transwell assays were used to detect the effects of miR-10b on the cytokine production, proliferation, migration and invasion of FLS cells. Results： As compared with OA patients, 176 miRNAs were up-regulated, whereas 204 miRNAs were down-regulated in RA patients. Among them, miR-10b expression in RA FLS was apparently repressed by several inflammatory cytokines like TNF-α in a NF-KB-dependent manner. Luciferase reporter assay and real-time PCR uncovered that TAK1 and TLR4 are target genes of miR-10b. As a result, miR-10b participated in FLS function regulation via these two genes. On one hand, downregulated miR-10b greatly promoted NF-KB activation and TNF-α production through accelerating TAK1 level and then triggered TNF-α NF-κB→miR-10b→TNF-α/NF-κB regulatory circuit. On the other, low level ofmiR-10b enhanced TLR4 expression on FLS and therefore strengthened LPS stimulation. This activated another regulatory circuit LPS→NF-κB→miR-10b→TLR4. Finally, miR-10b significantly promoted FLS proliferation, invasion, migration and pro-inflammatory cytokines production through these two circuits. Conclusion： miR-10b was apparently downregulated in RA-FLS and it greatly promoted pro-inflammatory cytokine secretion, proliferation, migration and invasion of FLS through two different regulatory circuits.