【作者】 张心浩；

【导师】 崔永华；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉—头颈外科学， 2009， 博士

【摘要】 第一部分慢性鼻窦炎和鼻息肉中miRNAs的差异性表达研究背景：慢性鼻窦炎(chronic rhinosinusitis，CRS)和鼻息肉(nasal polyps，NP)是鼻腔鼻窦的慢性炎症性疾病，它的发病机制尚未完全阐明。MicroRNA(miRNA)已被证明广泛参与免疫反应。我们对慢性鼻窦炎伴和鼻息肉及正常人群的miRNA(miR)进行差异性表达的研究，筛选出部分miRNA，验证表达的差异性。方法：采用miRNA芯片(miRNA array)方法对慢性鼻窦炎，鼻息肉和正常鼻粘膜miRNA的表达谱进行了检测分析。根据文献报道，筛选出miR-125b和miR-26a，用实时定量(real-time)逆转录聚合酶链反应(reverse transcription polymerase chainreaction，RT-PCR)和原位杂交进一步验证芯片结果。结果：慢性鼻窦炎组中，有4种miRNAs表达上调，23种miRNAs表达下调；鼻息肉组织中，有7种miRNAs表达上调，69种miRNAs表达下调。实时定量RT-PCR显示miR-125b鼻息肉表达升高，且有统计学意义(P＜0.05)。miR-125b在BEAS-2B细胞上存在表达，miR-26a在BEAS-2B细胞无表达。miR-125b在人体鼻息肉组织的上皮表达上调。结论：miRNA在慢性鼻窦炎和鼻息肉的表达存在差异。miR-125b在鼻息肉中表达明显上调，可能与鼻息肉的发生发展有关。研究其调控方式，可为进一步探索鼻息肉的发病机制提供线索。第二部分MiR-125b表达上调在鼻息肉发病机制中的意义背景：miR-125b已被证明在鼻息肉表达增强。用生物信息学方法预测出4EBP1的3’非编码区与miR-125b有互补系列。4EBP1通过对Ⅰ型IFN信号通路有调控作用。我们对4EBP1是否为miR-125b的直接靶点进行验证，并对miR-125b是否通过调控4EBP1在鼻息肉发病中发挥作用进行了探讨。方法：采用免疫组织化学方法检测4EBP1在慢性鼻窦炎及鼻息肉中的表达情况。将miR-125b的mimic及inhibitor转染BEAS-2B细胞，24小时后采用免疫印迹法(Western blot)检测4EBP1蛋白质表达水平；构建将4EBP1 3’UTR的部分序列嵌入pGL3质粒，以构建pGL3-4EBP1质粒；重组质粒与miR-125b的mimic共转染BEAS-2B细胞，检测荧光素酶吸光度；实时定量RT-PCR用于检测慢性鼻窦炎及鼻息肉中IFN-β和IL-5 mRNA的表达水平有无差异，对IFN-β和IL-5 mRNA表达水平进行相关性分析。结果：免疫组化发现与正常对照比较，4EBP1蛋白质水平在鼻息肉出现下调；体外实验发现，分别上调和下调miR-125b水平后，4EBP1蛋白水平相应出现下降和升高。荧光素酶报告基因检测发现共转染重组有miR-125b互补结合序列的质粒和miR-125b mimic后，荧光素强度明显降低，提示miR-125b能抑制4EBP1的表达。实时定量RT-PCR显示与对照组相比，IFN-β和IL-5 mRNA在鼻息肉表达增强，且有统计学差异(P＜0.05)。慢性鼻窦炎及鼻息肉中IFN-β和IL-5 mRNA水平呈负相关(P＜0.05)。结论：4EBP1是miR-125b的直接靶向调控分子。miR-125b通过介导4EBP1的表达，在鼻息肉的发病中发挥作用。4EBP1表达水平能影响IFN-β产生量，可能对鼻粘膜天然免疫发挥影响。IFN-β在鼻粘膜不仅与天然免疫有关，而且与鼻息肉中Th1／Th2失衡关系密切。更多还原

【Abstract】 PartⅠIdentification of differentially expressed miRNAs in chronicrhinosinusitis with and without nasal polypsBackground: Chronic rhinosinusitis (CRS) is one of the most frequently reportedchronic diseases. Although many causative factors have been implicated in pathogenesis ofCRS, the exact etiology of CRS is still far from completely revealed. MicroRNAs havebeen shown to be involved in various immunological events and several inflamatorydiseases. Therefore, our study was aimed to determine the miRNA expression profile inboth CRS with and without NPs and to identify differentially-expressed miRNAs.Methods: The miRNA array analysis was used to obtain miRNA expression profilingin both CRS with and without NPs. MicroRNA-125b and miR-26a were selected to validatethe array result by real-time PCR and in situ hybridization, which was based on literature.Results: Four miRNAs were up-regulated in CRS without NPs compared withcontrols, while 23miRNAs down-regulated. Seven miRNAs were increased in CRS withNPs compared with controls, while 69 miRNAs reduced. The enhanced level of miR-125bin CRS with NPs and reduced miR-26a were validated by real-time RT-PCR.MicroRNA-125b was expressed in BEAS-2B cells and sinasal epithelium, while theexpression of miR-26a was not detected in BEAS-2B cells.Conclusion: Differentially-expressed miRNAs were identified in CRS with andwithout NPs. The increased expression of miR-125b was associated with CRS with NPs. Afurther study of miR-125b may provide new clues to the pathogenesis of CRS with NPs. PartⅡThe impilcation of the enhanced expression of miR-125b innasal polypsBackground: MicroRNA-125b was demonstrated to be increased in CRS with NP inour previous study. The binding site of miR-125b with 4EBP1 was demonstrated bybioinformatics prediction. 4EBP1 was showed to have influence in I-IFN signaling. Thisstudy was designed to investigate whether 4EBP1 was a direct target of miR-125b and toexplore the the role of miR-125b in pathogenesis of CRS with NPs by regulating 4EBP1.Methods: The expression of 4EBP1 was assessed by means withimmunohistochemistry. Western blot was used to examine 4EBP1 level of BEAS-2B cellsuntreated.or transfected with mimic 125b and inhibitor. 4EBP13’ UTR and its mutant wereinserted into luciferase report plasmid to contruct chimeric vectors. A luciferase reporterassays was performed to investigate direct effect of miR-125b on 4EBP1. IL-5 and IFN-βmRNA level were detected by using real time RT-PCR.Results: The 4EBP1 expression in sinonasal mucosa was significantly inhibited inCRS patients with NPs. Experimentally induced suppression or forced expression ofmiR-125b causes reciprocal alterations in protein expression of 4EBP1 in BEAS-2B cells.Moreover, a significant negative effect on luciferase activity was observed in BEAS-2Bcells con-transfected with construct bearing an intact miR-125b binding sites and mimicwhen compared with control. IL-5 and IFN-βmRNA were showed to be enhanced in CRSpatients with NPs compared with controls. IL-5 level inversely correlated with IFN-βexpression in sinonasal mucosa.Conclusion: 4EBP1 was a direct target of miR-125b. 4EBP1 may play a role insinasal epithelial immunity through modulating production of IFN-β, which was associatedwtih Th1/Th2 unbalance in CRS with NPs. MicroRNA-125b may be involved inpathogenesis of CRS with NPs by targeting 4EBP1.更多还原