天津医药

天津医药 ›› 2021, Vol. 49 ›› Issue (12): 1233-1239.doi: 10.11958/20210909

• 细胞与分子生物学 •    下一篇

3-O-C12-HSL通过抑制lncRNA CD48-AS和CD48的表达而阻碍Mo-DCs成熟

罗燕芬 1，庄奇真 2，张轩 1，陈茶 1，刘杨 2，颜星星 1，肖倩 1，李有强 3△   

  1. 1广东省中医院检验医学部（邮编501180）；2广州中医药大学第二临床医学院；3南方医科大学附属何贤纪念医院检验科
  • 收稿日期:2021-04-19 修回日期:2021-09-18 出版日期:2021-12-15 发布日期:2021-12-27
  • 通讯作者: △通信作者 E-mail：liyouqiang21@126.com E-mail:liyouqiang21@126.com
  • 作者简介:罗燕芬（1987），女，硕士，主管检验技师，主要从事抗感染免疫和临床生化检验方面研究。E-mail：luoyanfenabc@126.com
  • 基金资助:
    国家自然科学基金资助项目（81601736）；广东省中医药局中医药科研项目（20192027）

3-O-C12-HSL hampers the maturation of Mo-DCs by inhibiting the expression of lncRNA CD48-AS and CD48

LUO Yan-fen1, ZHUANG Qi-zhen2, ZHANG Xuan1, CHEN Cha1, LIU Yang2, YAN Xing-xing1, XIAO Qian1, LI You-qiang3△   

  1. 1 Department of Laboratory Medicine, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 501180, China; 2 the Second Clinical Medical College of Guangzhou University of Traditional Chinese Medicine; 3 Department of Laboratory Medicine, He Xian Memorial Hospital, Southern Medical University △Corresponding Author E-mail: liyouqiang21@126.com
  • Received:2021-04-19 Revised:2021-09-18 Published:2021-12-15 Online:2021-12-27

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摘要： 目的 筛选并阐明长链非编码RNA（lncRNA）CD48-AS与其反义互补分子CD48 mRNA在N-3-氧代十二 烷酰-L-同型丝氨酸内酯（3-O-C12-HSL）阻碍人单核细胞诱导的树突状细胞（Mo-DCs）成熟过程中发挥作用的机制。 方法 从健康人外周血中分离Mo-DCs，将未成熟的Mo-DCs细胞分为阴性对照组（0.1% DMSO）、脂多糖（LPS）阳性 对照组（100 μg/L的LPS）和实验组（100 μg/L LPS+40 μmol/L 3-O-C12-HSL）进行lncRNA芯片检测。筛选lncRNA表 达谱中差异表达 5 倍以上的自然反义 lncRNA 进行聚类分析，从中筛选差异具有统计学意义的自然反义 lncRNA CD48-AS。未成熟的 Mo-DCs 分为阴性对照组、LPS 阳性对照组以及 LPS+C5、C10、C25 实验组（分别加入 5、10、 25 μmol/L的3-O-C12-HSL）。利用荧光定量PCR检测lncRNA CD48-AS和反义分子CD48 mRNA在实验体系中的表 达水平；通过生物信息学分析lncRNA CD48-AS与CD48反义互补区及其蛋白编码功能；通过核糖核酸酶保护实验 （RPA）验证lncRNA CD48-AS是否与CD48形成二聚体，从而通过影响靶CD48的表达来发挥调控作用。最后检测 lncRNA CD48-AS在细胞内的定位。结果 3-O-C12-HSL处理Mo-DCs后lncRNA表达谱出现了特异性改变。3-OC 12-HSL可下调由LPS诱导的Mo-DCs中lncRNA CD48-AS及其反义靶分子CD48的表达。生物信息学分析lncRNA CD48-AS 不具备蛋白编码功能，为非编码 RNA；lncRNA CD48-AS 与 CD48 形成 RNA 二聚体从而减少 RNA 酶对 CD48 mRNA的降解，使得CD48 mRNA表达增加；lncRNA CD48-AS在Mo-DCs中主要定位在细胞核中，细胞质中表 达量较少。结论 3-O-C12-HSL可通过下调lncRNA CD48-AS，进而影响CD48的表达来阻碍Mo-DCs的成熟。

关键词: RNA, 长链非编码, 树突细胞, 脂多糖类, CD48抗原, 计算生物学, N-3-氧代十二烷酰-L-同型丝氨酸内 酯, 长链非编码RNA CD48-AS

Abstract: Objective To screen and clarify the mechanism of long non-coding RNA CD48-AS (lncRNA CD48-AS) and the antisense complementary molecule CD48 in the process of N-3-oxododecanoyl-L-homoserine lactone (3-O-C12- HSL) hampering the maturation of human monocyte-induced dendritic cells (Mo-DCs). Methods Mo-DCs were extracted from the peripheral blood of healthy individuals, and the immature Mo-DCs were divided into three groups for lncRNA chip analysis: the negative control group (0.1% DMSO), the lipopolysaccharide (LPS) positive control group (100 μg/L LPS) and the treatment group (100 μg/L LPS+40 μmol/L 3-O-C12-HSL). The natural antisense lncRNA expressed more than 5 times in the lncRNA expression profile were screened for Cluster analysis. The natural antisense lncRNA CD48-AS was screened. Immature Mo-DCs were divided into the negative control group, the LPS positive control group, and the 3-O-C12-HSL treatment group (5, 10, 25 μmol/L 3-O-C12-HSL). The expression of lncRNA CD48-AS and antisense molecule CD48 were measured using quantitative PCR in each group. The complementary regions of lncRNA CD48-AS and CD48 and the protein coding function of lncRNA CD48-AS were analyzed through bioinformatics. Whether lncRNA CD48-AS affected the expression of CD48 by forming a dimer with CD48 was verified through the ribonuclease protection experiment (RPA). Preliminary experiments were carried out on the localization of lncRNA CD48-AS in cells. Results The lncRNA expression profile of Mo-DCs showed specific changes after 3-O-C12-HSL treatment. 3-O-C12-HSL down-regulated the expression of lncRNA CD48-AS and its antisense target molecule CD48 induced by LPS. Bioinformatics analysis showed that lncRNA CD48-AS was a non-coding RNA without protein coding function. lncRNA CD48-AS may form RNA dimers with CD48 to reduce the degradation of CD48 by RNase and increase the expression of CD48. lncRNA CD48-AS was mainly located in the nucleus in Mo-DCs, with less expression in cytoplasm. Conclusion 3-O-C12-HSL can inhibit the maturation of Mo-DCs by down-regulating lncRNA CD48-AS and then affecting the expression of CD48.

Key words: RNA, long noncoding, dendritic cells, lipopolysaccharides, CD48 antigen, computational biology, N-3- oxododecanoyl-L-homoserine lactone, lncRNA CD48-AS

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