天津医药

天津医药 ›› 2022, Vol. 50 ›› Issue (3): 259-264.doi: 10.11958/20211109

• 实验研究 • 上一篇    下一篇

LncRNA PVT1通过竞争miR-149-5p上调CHI3L1并影响变应性鼻炎进展的研究

姜玉秋，唐桥斐，闫智永，张爽   

  1. 沈阳医学院附属第二医院耳鼻喉科（邮编110002）
  • 收稿日期:2021-05-12 修回日期:2021-10-27 出版日期:2022-03-15 发布日期:2022-03-15
  • 基金资助:
    沈阳市科技计划项目（20-205-4-091）

LncRNA PVT1 upregulates CHI3L1 and affects the progression of allergic rhinitis by competition with miR-149-5p

JIANG Yuqiu, TANG Qiaofei, YAN Zhiyong, ZHANG Shuang   

  1. Department of Otorhinolaryngology, the Second Hospital of Shenyang Medical College, Shenyang 110002, China
  • Received:2021-05-12 Revised:2021-10-27 Published:2022-03-15 Online:2022-03-15

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摘要： 目的　探讨长链非编码RNA（LncRNA）PVT1对变应性鼻炎（AR）进展的影响及其相关作用机制。方法　24只BALB/c小鼠，其中6只作为对照组，余18只采用卵白蛋白和氢氧化铝建立AR模型并采用随机数字表法均分为模型组、AR+NC组（尾静脉注射空载体慢病毒）、AR+shPVT1组（尾静脉注射LncRNA PVT1干扰载体慢病毒），每组6只。采用行为学评分评估各组小鼠过敏症状；HE染色观察各组小鼠鼻黏膜组织病理学改变；实时荧光定量PCR检测各组小鼠鼻黏膜组织中LncRNA PVT1、miR-149-5p和几丁质酶-3样蛋白1（CHI3L1）mRNA的表达；Western blot检测CHI3L1蛋白表达；ELISA分析各组小鼠血清IgE、白细胞介素（IL）-5、肿瘤坏死因子（TNF）-α水平。构建LncRNA PVT1和CHI3L1的野生型（WT）和突变型（MT）荧光素酶报告质粒，单独或同时与miR-149-5p mimic共转染293T细胞，采用双荧光素酶实验分析并计算各组细胞荧光素酶相对活性。结果　与模型组相比，AR+shPVT1组小鼠行为学评分降低；血清中IgE、IL-5和TNF-α水平降低；鼻黏膜组织中LncRNA PVT1 mRNA表达水平降低，miR-149-5p mRNA表达水平升高，CHI3L1 mRNA及蛋白水平均降低，鼻黏膜组织炎症反应减轻。双荧光素酶实验证实LncRNA PVT1通过竞争性结合miR-149-5p，上调miR-149-5p靶基因CHI3L1的表达。结论　在AR模型小鼠中异常高表达的LncRNA PVT1通过发挥竞争性内源RNA作用抑制miR-149-5p表达而上调CHI3L1的表达，引发小鼠鼻黏膜组织的炎性症状并促进过敏反应。

关键词: 鼻炎, 过敏性；RNA, 长链非编码；小鼠, 近交BALB C；壳多糖酶3样蛋白质1；LncRNA PVT1；miR-149-5p

Abstract: Objective　To investigate the effect of long non coding RNA (LncRNA) PVT1 on allergic rhinitis (AR) and its related mechanism. Methods　A total of 24 BALB/c mice were used in this study, of which 6 mice were in the normal group, and the remaining 18 mice were treated with ovalbumin (OVA) and Al (OH)3 to establish the AR model. Eighteen mice were randomly divided into the model group, the AR+NC group (empty vector lentivirus injected via tail vein) and the AR+shPVT1 group (shPVT1 lentivirus injected via tail vein). The behavioral score was used to investigate allergic symptoms of the mice in each group. HE staining was used to analyze the pathological changes of nasal mucosa in mice. qPCR was used to detect LncRNA PVT1, miR-149-5p and chitinase-3-like protein 1 (CHI3L1) relative mRNA expression. The protein expression of CHI3L1 was detected by Western blot assay. The serum levels of IgE, IL-5 and TNF-α were analyzed by ELISA. Luciferase reporter vector of LncRNA PVT1 and CHI3L1 of wild type (WT) and mutant type (MT) were constructed and transfected into 293T cells with miR-149-5p mimic. The relative luciferase activity was analyzed by double luciferase assay. Results　Compared with the model group, the behavior score was significantly decreased， serum levesls of IgE, IL-5 and TNF-α were reduced, the relative mRNA or protein expression of LncRNA PVT1 and CHI3L1 were significantly decreased, the relative mRNA of miR-149-5p was increased and the inflammatory response of the nasal mucosa was reduced in the AR+shPVT1 group. Dual luciferase assay confirmed that LncRNA PVT1 up-regulated the expression of miR-149-5p target gene CHI3L1 through competitive binding miR-149-5p. Conclusion　LncRNA PVT1, which is abnormally highly expressed in AR model mice, can inhibit the expression of miR-149-5p and enhance the expression of CHI3L1 by exerting its ceRNA function, thus promote inflammatory symptoms of nasal mucosa tissue and allergic reactions in mice.

Key words: rhinitis, allergic, RNA, long noncoding, mice, inbred BALB C, chitinase-3-like protein 1, LncRNA PVT1, miR-149-5p