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lncRNA 5033413D16Rik对实验性自身免疫性葡萄膜炎Th17细胞活性的抑制作用
李学佳
张开朗
陈思思
魏瑞华
粘红
作者及单位信息
·
DOI： 10.3760/cma.j.cn115989-20230220-00056
Inhibitory effect of lncRNA 5033413D16Rik on the activity of T helper 17 cells in experimental autoimmune uveitis
Li Xuejia
Zhang Kailang
Chen Sisi
Wei Ruihua
Nian Hong
Authors Info & Affiliations
Li Xuejia
Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China
Zhang Kailang
Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China
Chen Sisi
Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China
Wei Ruihua
Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China
Nian Hong
Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China
·
DOI： 10.3760/cma.j.cn115989-20230220-00056
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摘要

目的探讨长链非编码RNA 5033413D16Rik(lncRNA 5033413D16Rik)对实验性自身免疫性葡萄膜炎(EAU)中光感受器间维生素A类结合蛋白 1－20(IRBP 1－20)特异性辅助性T17(Th17)细胞活性的影响。

方法选取SPF级8～10周龄C57BL/6雌性小鼠12只，采用随机数字表法分为EAU组和正常对照组，每组各6只。正常对照组小鼠不接受任何处理，将IRBP 1－20和完全弗氏佐剂充分乳化后主动免疫EAU组小鼠。免疫后13 d，行眼底检查并进行炎症评分，苏木精－伊红染色观察视网膜组织病理学改变；分离EAU小鼠脾脏及淋巴结中T细胞，实时荧光定量PCR检测lncRNA 5033413D16Rik相对表达量。将分离培养的T细胞分为短发夹RNA(shRNA)-5033413D16Rik转染组和shRNA-阴性对照(NC)转染组，分别转染相应shRNA，实时荧光定量PCR验证shRNA-5033413D16Ri的敲低效率。将2个组细胞分别与骨髓源性树突状细胞在Th17极化条件下共培养，实时荧光定量PCR检测2个组共培养细胞中转录因子维甲酸相关核孤儿受体γt(RORγt)、白细胞介素17(IL-17)、IL-23受体(IL-23R)和粒细胞－巨噬细胞集落刺激因子(GM-CSF)mRNA相对表达量；ELISA试剂盒检测2个组共培养细胞上清中IL-17质量浓度；流式细胞仪检测2个组共培养细胞中Th17细胞百分比。

结果成功建立小鼠EAU模型。实时荧光定量PCR检测结果显示，与正常对照组相比，EAU组小鼠T细胞中lncRNA 5033413D16Rik相对表达量显著降低，差异有统计学意义( t＝－13.332， P<0.001)。与shRNA-NC转染组相比，shRNA-5033413D16Rik转染组T细胞中lncRNA 5033413D16Rik相对表达量显著降低，差异有统计学意义( t＝－6.338， P<0.01)。shRNA-5033413D16Rik转染组共培养细胞中RORγt、IL-17、IL-23R和GM-CSF mRNA相对表达量分别为1.61±0.13、1.51±0.13、1.85±0.33和1.45±0.11，明显高于shRNA-NC转染组的1.00±0.01、1.00±0.01、1.00±0.01和1.00±0.01，差异均有统计学意义( t＝－6.839、－8.221、－4.538、－4.189，均 P<0.05)；ELISA检测结果显示，shRNA-5033413D16Rik转染组共培养细胞上清中IL-17质量浓度为(2 350.39±367.02)pg/ml，明显高于shRNA-NC转染组的(1 513.31±310.37)pg/ml，差异有统计学意义( t＝－3.016， P＝0.039)；流式细胞仪检测结果显示，shRNA-5033413D16Rik转染组共培养细胞中Th17细胞比例为(17.20±0.44)%，明显高于shRNA-NC转染组的(14.10±0.84)%，差异有统计学意义( t＝－3.264， P＝0.031)。

结论lncRNA 5033413D16Rik可以抑制抗原特异性Th17细胞致病性相关基因的表达，负向调控IRBP 1－20特异性Th17细胞反应。

葡萄膜炎;自身免疫性疾病;实验性自身免疫性葡萄膜炎;辅助性T17细胞;lncRNA 5033413D16Rik
ABSTRACT

ObjectiveTo investigate the effect of long noncoding RNA (lncRNA) 5033413D16Rik (lncRNA 5033413D16Rik) on the activity of interphotoreceptor retinoid-binding protein 1-20 (IRBP) 1-20-specific T helper 17 (Th17) cells in experimental autoimmune uveitis (EAU).

MethodsTwelve SPF grade C57BL/6 female mice aged 8 to 10 weeks were selected and divided into EAU group and normal control group using the random number table method, with 6 mice in each group.Mice in the normal control group received no treatment.Mice in the EAU group were immunized with IRBP 1-20 emulsified in complete Freund's adjuvant to induce EAU.On day 13 after immunization, fundus examination and inflammation scoring were performed, and retinal histopathological changes were observed with hematoxylin and eosin staining.T cells were isolated from the spleen and lymph nodes of EAU mice, and the relative expression of lncRNA 5033413D16Rik was detected by real-time fluorescence quantitative PCR (qRT-PCR).In addition, the isolated T cells were divided into short hairpin RNA (shRNA)-5033413D16Rik transfected group and shRNA-negative control (NC) transfected group, and transfected with corresponding shRNAs, respectively.The knockdown efficiency of shRNA-5033413D16Rik was verified by qRT-PCR.T cells in the two groups were co-cultured with bone marrow-derived dendritic cells (BMDCs) under Th17 polarizing conditions.The relative expression of retinoic acid-related orphan receptor (RORγt), interleukin 17 (IL-17), IL-23 receptor (IL-23R), and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA in co-cultured cells were analyzed by qRT-PCR.The IL-17 concentration in co-culture supernatants was assayed by ELISA and percentages of Th17 cells were determined by flow cytometry.The use and care of experimental animals complied with the Regulations for the Administration of Affairs Concerning Experimental Animals promulgated by the State Science and Technology Commission.The study protocol was reviewed and approved by the Experimental Animal Ethics Committee of Tianjin Medical University (No.TJYY2019110117).

ResultsEAU model was established successfully.qRT-PCR analysis showed that the expression of lncRNA 5033413D16Rik was significantly decreased in T cells from EAU mice compared with normal control mice ( t=-13.332, P<0.001).Compared with the shRNA-NC transfected group, the relative expression of lncRNA 5033413D16Rik in T cells of the shRNA-5033413D16Rik transfected group was significantly reduced ( t=-6.338, P<0.01).In co-cultured T cells, the expression levels of RORγt, IL-17, IL-23R, and GM-CSF mRNA in shRNA-5033413D16Rik transfected group were 1.61±0.13, 1.51±0.13, 1.85±0.33 and 1.45±0.11, which were significantly higher than 1.00±0.01, 1.00±0.01, 1.00±0.01 and 1.00±0.01 in shRNA-NC-transfected group ( t=-6.839, -8.221, -4.538, -4.189; all at P<0.05).ELISA analysis revealed that IL-17 concentraion in shRNA-5033413D16Rik transfected group was (2 350.39±367.02)pg/ml, which was significantly higher than (1 513.31±310.37)pg/ml ( t=-3.016, P=0.039).Flow cytometry showed that the percentage of Th17 cells in shRNA-5033413D16Rik transfected group was (17.20±0.44)%, which was higher than (14.10±0.84)% in normal control group ( t=-3.264, P=0.031).

ConclusionsLncRNA 5033413D16Rik can inhibit the expression of pathogenicity related genes in antigen-specific Th17 cells and negatively regulates IRBP 1-20-specific Th17 cell responses.

Uveitis;Autoimmune diseases;Experimental autoimmune uveitis;Th17 cells;LncRNA 5033413D16Rik
Nian Hong, Email: mocdef.6ab21gnohnain
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引用本文

李学佳,张开朗,陈思思,等. lncRNA 5033413D16Rik对实验性自身免疫性葡萄膜炎Th17细胞活性的抑制作用[J]. 中华实验眼科杂志,2024,42(11)：983-990.

DOI:10.3760/cma.j.cn115989-20230220-00056

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自身免疫性葡萄膜炎是常见的致盲眼病，易反复发作，发病机制目前尚不完全明确。实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis，EAU)是一种由人类视网膜抗原诱导的经典动物模型，有助于深入了解自身免疫性葡萄膜炎的发病机制 [ 1 ]。辅助性T17(T helper 17，Th17)细胞是一类炎性CD4 ＋ T细胞亚群，其主要特征是表达特异性的转录因子维甲酸相关核孤儿受体γt(retinoic acid-related orphan receptor，RORγt)和分泌白细胞介素17(interleukin 17，IL-17) [ 2 ]。研究证实，Th17细胞的过度活化和IL-23/IL-17通路的激活是导致自身免疫性葡萄膜炎发生的重要机制 [ 3 , 4 ]。
长链非编码RNA(long non-coding RNA，lncRNA)是一类长度超过200 nt的非编码RNA，通过染色质重塑、转录调控、翻译调控、RNA剪接、RNA降解以及RNA编辑等方式，在生理调节和各种疾病发生和发展中发挥着重要作用 [ 5 ]。研究表明，lncRNA参与了多种自身免疫性疾病的发生 [ 6 , 7 , 8 ]。例如，lncRNA PVT1与Myc相互作用，通过促进糖酵解增强CD4 ＋ T细胞的增生和功能，进而引起Sjögren综合征样自身免疫反应 [ 8 ]。此外，Zhao等 [ 9 ]研究发现LINC01061通过调控miR-612/BRD4轴引发自身免疫性甲状腺炎。本课题组前期通过分析Behcet病患者和健康志愿者的公开基因表达数据集发现，lncRNA 5033413D16Rik在Behcet病患者CD4 ＋ T细胞中表达下调 [ 10 ]。lncRNA 5033413D16Rik已被证实在不同类型的肿瘤中发挥致癌或抗肿瘤功能，并且具有潜在的免疫调控作用 [ 11 ]。但lncRNA 5033413D16Rik对EAU中光感受器间维生素A类结合蛋白 1－20(interphotoreceptor retinoid-binding protein 1－20，IRBP 1－20)特异性Th17细胞的作用及机制尚不明确。因此，本研究拟利用短发夹RNA(short hairpin RNA，shRNA)敲低T细胞中lncRNA 5033413D16Rik的表达，探讨其对EAU中IRBP 1－20特异性Th17细胞活性的影响及可能机制，以期为自身免疫性葡萄膜炎的治疗提供新的思路。
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吴怡媛，陈思思，杨超，等．白细胞介素-23受体过表达对实验性自身免疫性葡萄膜炎小鼠辅助性T细胞17/调节性T细胞平衡的影响[J]. 中华眼底病杂志， 2022，38(5)∶389-395. DOI： 10.3760/cma.j.cn511434-20210809-00428 .
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Wu YY , Chen SS , Yang C ,et al. Effect of interleukin-23R overexpression on Th17/Treg balance in experimental autoimmune uveitis mice[J]. Chin J Ocul Fundus Dis， 2022，38(5)∶389-395. DOI： 10.3760/cma.j.cn511434-20210809-00428 .
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李学佳：酝酿和设计实验、实施研究、采集数据、统计分析、分析/解释数据、起草文章；张开朗：实施研究、采集数据、统计分析；陈思思：实施研究、采集数据、统计分析、分析/解释数据、对文章的知识性内容作批评性审阅；魏瑞华：酝酿和设计实验、统计分析、研究指导；粘红：酝酿和设计实验、统计分析、研究指导、对文章的知识性内容作批评性审阅及定稿

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天津市视网膜功能与疾病重点实验室自主与开放课题 (2020tjswmm001)
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