微小RNA-106b-5p与JAK-STAT通路相互作用对原发性胆汁性胆管炎巨噬细胞极化的调控机制

A key question on primary biliary cholangitis(PBC) is why inflammatory damage of intrahepatic biliary epithelial cells is persistent and specific in PBC patients, but not in healthy individuals, since there is IBEC apoptosis both in healthy individuals and PBC patients. In previous studies, we found that monocytes/macrophages were polarized to phenotype M1 whose phagocytic ability is weak, with abnormal upregulation of miRNA-106b-5p positively associated with M1, but negatively with M2. This miRNA can target JAK-STAT signaling pathway which play an important role in monocyte/macrophage polarization. Accordingly, we proposed that M1 polarization could be induced by interaction between miRNA-106b-5p and JAK-STAT signaling pathway, leading to inability to timely remove apoptotic IBECs and subsequently secondary necrosis of IBECs, which will trigger a persistent and specific inflammatory response. To confirm these hypotheses, we will explore molecular mechanisms of interaction between miRNA-106b-5p and JAK-STAT signaling pathway and its regulation on monocyte/macrophage polarization in vivo and vitro, by combination of various experiments, including CHiP，luciferase report, inhibition and over-expression, animal experiments, and so on. Our aim is to provide a reference for answering above key question on PBC and finding a potential therapeutic target for PBC.

原发性胆汁性胆管炎（PBC）研究中的一个关键问题是正常人和PBC患者都有肝内胆管上皮细胞（IBEC）凋亡现象，但为何只有后者出现持续特异炎症损伤？我们的前期研究发现PBC中单核巨噬细胞主要向吞噬能力弱的M1极化，microRNA-106b-5p异常升高，与M1正相关，与M2负相关，该microRNA可以作用于JAK-STAT通路，后者在单核巨噬细胞极化中起重要作用。因此，我们推测该microRNA与JAK-STAT通路可以相互作用，调节单核巨噬细胞向M1极化，导致凋亡IBEC无法及时清除，继发坏死，诱发持续特异炎症，并拟在本课题中利用染色质免疫沉淀、荧光素酶报告、抑制和过表达及动物实验等技术，通过体内外研究，探讨microRNA-106b-5p与JAK-STAT通路相互调节及对单核巨噬细胞极化的调控作用及其分子机制。为回答上述PBC关键问题，寻找PBC潜在治疗靶点提供思路借鉴。

原发性胆汁性胆管炎（PBC）研究中一个关键问题是正常人和PBC患者都有肝内胆管上皮细胞（IBEC）凋亡现象，但为何只有后者出现持续特异炎症损伤？本研究围绕这一问题，通过临床、细胞和动物实验探讨了microRNA-106b-5p对单核巨噬细胞吞噬和炎症功能的调节作用及其在PBC发病中的意义，取得了如下结果：一、microRNA-106b-5p在PBC外周血表达明显升高，而且与M2比例呈显著负相关（r=-0.641，P=0.004），与M1有正相关趋势，但统计学上无显著性（r=0.395，P=0.104）。二、通过细胞实验发现，microRNA-106b-5p主要作用于STAT6，而对其它STAT分子没有明显作用。Poly I:C刺激明显上调microRNA-106b-5p表达，抑制STAT6磷酸化，抑制microRNA-106b-5p后，STAT6磷酸化明显增加，抑制STAT6通路后，microRNA-106b-5p表达明显增加。Poly I:C刺激可以抑制单核巨噬细胞吞噬凋亡IBEC，促进巨噬细胞分泌促炎性细胞因子如TNF-α、INF-β和IL-12，抑制其分泌抗炎性细胞因子IL-10，活化T细胞。抑制microRNA-106b-5p可以部分抑制巨噬细胞上述功能，抑制STAT6通路则明显促进上述功能。三、在PBC小鼠模型中成功清除和转回输单核巨噬细胞，并在转回输的单核巨噬细胞中实现了microRNA-106b-5p抑制和过表达。发现清除再转输过表达microRNA-106b-5p单核巨噬细胞的PBC动物模型门管区炎性细胞浸润明显增加，但对门管区巨噬细胞极化标志物iNOS（M1标志物）和Arg1、MRC1、FIZZ1、CHI3L3（M2标志物）的表达没有明显影响。清除再转输过表达microRNA-106b-5p单核巨噬细胞的PBC小鼠CD8+CD44+双阳性T细胞的比例明显增加，血清IL-12水平明显升高，而CD4+CD44+双阳性T细胞的比例没有明显变化。通过上述实验，结合我们以前研究所发现的JAK1是microRNA-106b-5p的一个直接作用靶点，我们初步阐明microRNA-106b-5p可以通过抑制JAK1-STAT6通路抑制单核巨噬细胞对凋亡IBEC的吞噬，导致后者继发性坏死，从而促进巨噬细胞和CD8+T细胞的活化和炎症因子的产生，参与PBC的炎症损伤。