【作者】 杨洁；

【导师】 张育； 沈维干；

【作者基本信息】 扬州大学 ， 内科学（风湿免疫）， 2021， 硕士

【摘要】 研究背景与目的:类风湿关节炎(Rheunmatoid arthritis,RA)是一种以慢性滑膜炎症和关节外病变为特征的全身自身免疫性疾病。类风湿关节炎成纤维样滑膜细胞(Rheumatoid arthritis Fibroblast-like synoviocyte,RA-FLS)作为RA软骨破坏的关键效应细胞,受RA患者关节内炎性环境刺激表现出增殖异常、迁移及侵袭能力增强等肿瘤细胞样的特性,在RA的发病和发展过程中起着至关重要的作用。长链非编码RNA(longnon-coding RNA,lncRNA)是一组长度大于200 nt的RNA转录本。有研究表明,N6-甲基腺嘌呤(N6-methyladenosine,m6A)修饰的lncRNA在多种肿瘤中能够通过RNA-蛋白结合、RNA-RNA结合或ceRNA机制等方式影响靶基因的功能。人lncRNAJPX定位于Xq13.2,是X染色体失活特异性转录本(X-inactive-specific transcript,XIST)的激活因子,是X染色体失活的分子开关,迄今为止JPX与RA的关系还未见报道。本实验通过探讨JPX对RA-FLS增殖、迁移及侵袭的影响及其可能作用机制,加深我们对RA发病机制的理解,以期为RA的治疗提供更多有价值的新靶标。研究方法:1、构建稳定表达JPX的RA-FLS细胞模型和干扰表达JPX的RA-FLS细胞模型,并通过RT-qPCR方法进行验证。2、采用CCK-8实验、EdU染色实验和细胞克隆形成实验检测过表达/干扰表达JPX对RA-FLS细胞增殖的影响。3、采用伤口愈合实验、Transwell细胞迁移及侵袭实验检测过表达/干扰表达JPX对RA-FLS细胞迁移和侵袭的影响;采用罗丹明标记的鬼笔环肽对RA-FLS细胞中的肌动蛋白细胞骨架进行免疫染色,观察JPX对RA-FLS中肌动蛋白细胞骨架纤维装配的影响。4、提取稳定过表达JPX组细胞和对照组细胞的总RNA,利用RNA-seq检测细胞中转录组学的改变,并通过RT-qPCR和Western Blot实验进行验证。5、采用RNA荧光原位杂交实验(RNA FISH)观察JPX在RA-FLS中的分布。6、通过慢病毒介导的方法构建稳定表达带有Slm(4x)标签的JPX的RA-FLS细胞模型,并采用RT-qPCR方法对RA-FLS中JPX的表达进行验证。利用RNA pull-down实验检测稳定表达带有S1m(4x)标签的JPX的RA-FLS细胞中JPX的可能结合蛋白,一部分RNA pull-down产物电泳、蛋白银染后送去公司做LC-MS/MS检测,另一部分做Western Blot实验进行验证。7、利用RNA dot blot实验检测过表达/干扰表达JPX组细胞和对照组细胞中RNA m6A修饰水平。8、采用Me-RIP-qPCR方法检测JPX以及上皮间质转化(EMT)相关基因Vimentin、N-cadherin、Snail 和 E-cadherin 在 RA-FLS 中的 RNA m6A 修饰水平。研究结果:1、RT-qPCR实验检测表明过表达/干扰表达JPX的RA-FLS细胞模型构建成功。2、CCK-8实验、EdU染色实验和细胞克隆形成实验结果显示,过表达JPX可以促进RA-FLS的增殖,而干扰表达JPX可以抑制RA-FLS的增殖。3、伤口愈合实验、Transwell细胞迁移及侵袭实验结果显示,过表达JPX可以显著促进RA-FLS的迁移与侵袭,干扰表达JPX可以显著抑制RA-FLS的迁移与侵袭。细胞免疫荧光染色实验显示,过表达JPX可以减少RA-FLS细胞中的张力纤维数目,增加片状伪足和丝状伪足的形成,干扰表达JPX可以减少RA-FLS细胞中片状伪足和丝状伪足的形成。4、RNA-seq检测差异基因表达分析结合RT-qPCR和Western Blot实验显示,与对照组相比,过表达JPX组细胞中与EMT相关的基因Vimentin、N-cadherin和Snail的表达显著上调,E-cadherin的表达显著下调,同时与RNA m6A修饰相关的基因METTL14和YTHDC2的表达显著上调。5、RNA FISH结果显示,JPX在RA-FLS的细胞核与细胞质中均有分布,但主要分布于细胞核中。6、成功构建稳定表达带有Slm(4x)标签的JPX的RA-FLS细胞模型。细胞内RNA pull-down沉淀物的蛋白质谱检测分析显示,JPX的结合蛋白中含有CTCF,同时我们通过Western Blot实验检测发现RNA pull-down沉淀物中含有CTCF。7、RNA dot blot实验结果显示,与对照组相比,过表达JPX可以显著上调RA-FLS中RNA m6A修饰水平,干扰表达JPX可以明显降低RA-FLS中RNA m6A修饰水平。8、Me-RIP-qPCR 实验结果显示,RA-FLS 中 JPX、Vimentin、N-cadherin 和 Snail的RNA m6A修饰水平上升,E-cadherin的RNA m6A修饰水平没有明显改变。结论:1、过表达JPX可以促进RA-FLS的增殖、迁移与侵袭,干扰表达JPX可以抑制RA-FLS的增殖、迁移与侵袭;2、JPX可能通过上调METTL14的表达,增加RA-FLS中RNA m6A修饰水平;3、JPX可能通过结合CTCF,上调YTHDC2的表达,调控m6A修饰的EMT相关基因mRNA的翻译,从而促进RA-FLS的迁移和侵袭。更多还原

【Abstract】 Research background and purpose:Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by chronic synovitis and extraarticular lesions.Because of the inflammatory environment,Rheumatoid arthritis Fibroblast-like synoviocytes(RA-FLSs),as the key cells of cartilage destruction in RA,show tumor cell-like characteristics such as abnormal proliferation,enhanced migration and invasion features,and play a crucial role in the pathogenesis and development of RA.Long non-coding RNAs(lncRNAs)are non-coding transcripts with a length of more than 200 nt.Studies have shown that IncRNA modified by N6-methyladenosine(m6A)can affect the function of target genes in a variety of tumors through RNA-protein binding,RNA-RNA binding or ceRNA mechanism.Human lncRNA JPX,located at Xq13.2,is an activator of X chromosome inactivation-specific transcript(XIST)and a molecular switch for X chromosome inactivation.However,the relationship between JPX and RA has not been reported.In this study,we explored the effect of JPX on the proliferation,migration and invasion in RA-FLS and its possible mechanism,which would deepen our understanding of the pathogenesis of RA,and provide more valuable novel targets for the treatment in RA.Methods:1.Stably expressing JPX and JPX knockdown in RA-FLS were constructed and verified by RT-qPCR.2.Effects of overexpression/knockdown of JPX on the cell proliferation of RA-FLS were detected by CCK-8 assays,EdU staining assays and cell clone formation assays.3.Effects of overexpression/knockdown of JPX on the migration and invasion of RA-FLS were detected by wound healing assays and transwell cell migration and invasion assays.Effects of overexpression/knockdown of JPX on the assembly of actin filaments in RA-FLS were observed by immunostaining with rhotamine-labeled phalloidin.4.Total RNA was extracted from RA-FLS stably overexpressing JPX and the control cells.The transcriptome changes in cells were detected by RNA-seq,and verified by RT-qPCR and Western Blot.5.The distribution of JPX in RA-FLS was observed by RNA fluorescence in situ hybridization assay(RNA FISH).6.Stably expressing S1m(4x)tagged JPX in RA-FLS was constructed by lentivirus-mediated method,and the expression of JPX in RA-FLS was verified by RT-qPCR.The possible binding proteins of JPX in RA-FLS stably expressing JPX tagged with S1m(4x)were evaluated by RNA pull-down assay.Some of the RNA pull-down products were silver-stained after SDS-PAGE and sent to the company for LC-MS/MS detection,and others were verified by Western Blot.7.The modification of RNA m6A in the overexpression/knockdown JPX group and the control group was detected by RNA dot blot assays.8.The RNA m6A modification of JPX and EMT-related genes such as Vimentin,N-cadherin,Snail and E-cadherin in RA-FLS was detected by Me-RIP-qPCR.Results:1.RT-qPCR results showed that the RA-FLS cell models with overexpression/knockdown of JPX were successfully constructed.2.Results from CCK-8 assays,EdU staining assays and cell clone formation assays showed that overexpression of JPX could promote cell proliferation,while JPX knockdown could inhibit cell proliferation.3.Wound healing assays,transwell cell migration and invasion assays showed that overexpression of JPX could significantly promote the migration and invasion of RA-FLS,and JPX knockdown could significantly inhibit the migration and invasion of RA-FLS.Immunofluorescence staining assays showed that overexpression of JPX could reduce the formation of stress fibers in RA-FLS,and increase the formation of filopodia and lamellipodia,while JPX knockdown could reduce the formation of filopodia and lamellipodia.4.Differential expression of genes in RNA-seq combined with RT-qPCR and Western Blot showed that,compared with the control group,expressions of EMT-related genes such as Vimentin,N-cadherin and Snail were up-regulated,while the expression of E-cadherin was down-regulated in the overexpressed JPX group.Meanwhile,expressions of RNA m6A modification-related genes such as METTL14 and YTHDC2 were up-regulated.5.Results of RNA FISH showed that JPX was distributed both in the nucleus and cytoplasm of RA-FLS,and mainly in the nucleus.6.Stably expressing JPX tagged with S1m(4x)in RA-FLS was successfully constructed.Protein profile analysis of invivo RNA pull-down precipitates showed that the binding proteins of JPX contained CTCF.Meanwhile,results from Western Blot assay showed that the RNA pull-down precipitates contained CTCF.7.Results from RNA dot blot assays showed that compared with the control group,overexpression of JPX could significantly up-regulate the modification of RNA m6A in RA-FLS,while knockdown of JPX could significantly reduce the modification of RNA m6A in RA-FLS.8.Results from Me-RIP-qPCR assays showed that the RNA m6A modification of JPX,Vimentin,N-cadherin and Snail in RA-FLS was increased,while the RNA m6A modification of E-cadherin did not change significantly.Conclusions:1.Overexpression of JPX could promote the proliferation,migration and invasion of RA-FLS,while knockdown of JPX could inhibit the proliferation,migration and invasion of RA-FLS.2.JPX might increase the modification of RNA m6A in RA-FLS by up-regulating the expression of METTL14.3.JPX might up-regulate the expression of YTHDC2 by interacting with CTCF,and regulate the translation of mRNA in EMT-related genes which modified by RNA m6A,thereby promoting the migration and invasion of RA-FLS.更多还原