LncRNA CTB-147C22.9在银屑病发病中的作用及机制研究.

The precise etiology of psoriasis remains obscure. The molecular basis for excessive proliferation and abnormal differentiation in keratinocytes is one of the key factors toward clarifying the pathogenesis of psoriasis. Using RNA-seq profiling analysis, study first found a large amount of long non-coding RNA (lncRNA) related to psoriasis. Some of those are involved in the formation of epidermal differentiation complex, and others have a direct influence on the regulation of cytokines. However, there exists a dearth of information regarding the detailed mechanisms of lncRNA in the pathogenesis of psoriasis. Our preliminary data suggests that lncRNA CTB-147C22.9 may be related to the development of psoriasis. Interference of this lncRNA significantly inhibited the proliferation and migration of keratinocytes. Thus, we use lncRNA CTB as the focus of the study. We plan on investigate the mechanisms of lncRNA CTB in psoriasis through in vitro and in vivo experiments, including Imiquimod and IL-23 induced psoriasis mouse model and transgenic animal models, as well as a relatively large cohort of psoriasis patient samples. Our results will also provide new insights into lncRNA CTB as potential therapeutic targets.

银屑病的发病机制尚不完全明确，研究角质形成细胞过度增殖和异常分化相关分子机制是阐明银屑病发病机制的关键。本研究前期采用RNA-Seq技术检测分析银屑病相关长链非编码RNA（lncRNA），发现大量可能与银屑病密切相关的lncRNA，这些lncRNA部分参与表皮分化复合物的形成，部分直接影响细胞因子调控。然而，lncRNA在银屑病中是否并如何发挥作用还有待深入研究。我们前期研究筛选得到LncRNA CTB-147C22.9可能与银屑病的发生发展密切相关，干扰此lncRNA能显著抑制角质形成细胞的的增殖和迁移能力。本项目将LncRNA CTB作为研究银屑病发病机制的切入点，通过体外细胞实验，咪喹莫特和IL-23诱导银屑病小鼠模型以及相关转基因修饰动物模型并检测一定数量的病人标本，深入探索LncRNA CTB在银屑病发病中的作用机制并明确LncRNA CTB可否成为治疗银屑病的靶点。

本课题首先通过高通测序和细胞水平筛选方法获得候选lncRNA，接下来结合生物信息学分析和体内外研究方法探讨其在银屑病角质形成细胞功能紊乱中作用机制。主要的研究结果如下：1.通过对银屑病样本进行全转录组高通量测序结合体外细胞水平上IL-17和TNFα刺激HaCaT细胞，筛选出能够显著响应TNFα信号的候选lncRNA,即lnc-SYCE3-1:2。TNFα能够稳定上调lnc-SYCE3-1:2的表达，在采用IKK抑制剂后能够逆转此过程。2.结合生物信息学分析和PCR验证，发现lnc-SYCE3-1:2的ceRNA调控机制，即“lnc-SYCE3-1:2>has-miR-3120-5p>TNFSFs/TNFRSFs”。3. 结合生物信息学预测和RNA-Pulldown实验，我们发现lnc-SYCE3-1:2能够与上皮细胞特异性可变剪切调节因子ESRP2相互结合，从而抑制ESRP2对于Hippo通路中NF2和YAP的剪切作用，分别形成NF2和YAP1的neonatal异构体。而已知的是NF2 neonatal异构体不能与LATS1结合，从而阻断了Hippo通路中MST1对于LATS1的磷酸化，进而激活YAP的入核和转录辅助作用。4.RNA-seq数据以及PCR检测都发现Hippo-YAP调控的促上皮细胞分裂增殖基因AREG、BIRC5、EPGN、PLAU、CCNE1等在银屑病中表达上调。5.在HaCaT细胞上过表达lnc-SYC3-1:2能够抑制Hippo通路中LATS1的磷酸化，进而抑制YAP磷酸化和YAP的入核作用；此外过表达lnc-SYC3-1:2能够显著上调Hippo-YAP调控的促上皮细胞分离增殖基因AREG、CTGF、CYR61、EPGN、PLAU的表达，下调角质形成细胞分化标志基因keratin1的表达。6.在HaCaT细胞上沉默lnc-SYCE3-1:2能够显著逆转TNFα抑制Hippo通路磷酸化作用以及促进YAP入核作用。7. 临床上采用拮抗TNFα生物制剂-Infliximab治疗银屑病患者，与治疗前相比，lnc-SYCE3-1:2表达水平被抑制至正常人皮肤样本水平；此外银屑病中高表达的Hippo-YAP信号靶基因经过治疗后恢复至正常人水平。我们的研究结果提示lnc-SYCE3-1:2有望作为银屑病的生物标志物和临床上替代TNFα单抗的银屑病治疗靶点。