中华放射医学与防护杂志

杜继聪,刘入菱,程赢,等.TLR2下游辐射防护关键miRNA筛选及miR-21功能验证[J].中华放射医学与防护杂志,2020,40(8):582-589.Du Jicong,Liu Ruling,Cheng Ying,et al.Screening of the key miRNA downstream of TLR2 and validating the function of miR-21 in radioprotection[J].Chin J Radiol Med Prot,2020,40(8):582-589

TLR2下游辐射防护关键miRNA筛选及miR-21功能验证

Screening of the key miRNA downstream of TLR2 and validating the function of miR-21 in radioprotection

投稿时间：2020-04-01

DOI：10.3760/cma.j.issn.0254-5098.2020.08.002

中文关键词: 电离辐射损伤 TLR2 转录组测序 miR-21

英文关键词:Ionizing radiation Damage TLR2 RNA sequence miR-21

基金项目:国家自然科学基金（81872559，81903260）；上海市青年科技英才扬帆计划（19YF1459100）；上海市卫计委科研课题青年项目（20174Y0173）

作者	单位	E-mail
杜继聪	中国人民解放军海军军医大学海军医学系舰船辐射医学防护教研室, 上海 200433
刘入菱	中国人民解放军海军军医大学海军医学系舰船辐射医学防护教研室, 上海 200433
程赢	中国人民解放军海军军医大学海军医学系舰船辐射医学防护教研室, 上海 200433
蔡建明	中国人民解放军海军军医大学海军医学系舰船辐射医学防护教研室, 上海 200433
高福	中国人民解放军海军军医大学海军医学系舰船辐射医学防护教研室, 上海 200433
刘聪	中国人民解放军海军军医大学海军医学系舰船辐射医学防护教研室, 上海 200433	victorliu20102020@163.com

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中文摘要:

目的筛选Toll样受体2（Toll-like receptor 2， TLR2）通路下游与辐射防护调控相关的关键miRNA，并探讨miR-21功能。方法以⁶⁰Co γ射线对野生型（wild type， WT）、TLR2 KO小鼠分别进行6.0、7.0、8.0 Gy全身照射，观察小鼠生存情况；通过转录组测序筛选骨髓细胞内TLR2通路下游关键miRNA，并通过QT-PCR验证其表达。过表达/敲低该miRNA后检测其生物学功能。结果 6.0、7.0、8.0 Gy全身照射后，TLR2 KO小鼠较WT小鼠辐射敏感性增加（χ²=4.490、13.100、7.928，P<0.05），骨髓移植实验证明TLR2 KO小鼠辐射敏感性增加与照射后骨髓细胞损伤有关（χ²=4.291，P<0.05）。转录组测序筛选到差异基因55个（[log2 Fold Change]>0.95，Q<0.05），其中上调基因28个，下调基因27个；定量PCR实验确定TLR2 KO（t=9.420，P<0.01）与MyD88 KO（t=10.700，P<0.01）小鼠骨髓细胞内miR-21表达下调。PAM3CSK4刺激后小鼠骨髓细胞内白介素6（IL-6）（t=13.790，P<0.05）和肿瘤坏死因子α（TNF-α）（t=14.280，P<0.05）表达量上调且依赖于TLR2。miR-21过表达可以促进EL4（t=5.951，P<0.05）和NIH/3T3（t=4.786，P<0.05）辐射后细胞活力，抑制WT小鼠（t=4.842，P<0.05）和TLR2 KO小鼠（t=10.520，P<0.05）BMCs辐射后细胞凋亡。miR-21敲低后降低EL4（t=4.815，P<0.05）和NIH/3T3（t=4.042，P<0.05）辐射后细胞活力。结论 miR-21在TLR2辐射防护进程中具有关键调控作用，其作用机制可能与上调IL-6与TNF-α有关。

英文摘要:

Objective To screen the key miRNA downstream of TLR2 and explore the function of the miR-21. Methods Wild type (WT) and TLR2 KO mice were irradiated with ⁶⁰Co γ-ray to compare their survivals. The downstream miRNAs of TLR2 signaling pathway were screened by RNA sequence in BMCs, and their expressions were verified by QT-PCR. Cell lines with overexpression or knockdown of a miRNA were established to evaluate the function of miRNA. Results The radiosensitivity of TLR2 KO mice was higher than that of TLR2 WT mice(χ²=4.490, 13.100, 7.928, P<0.05). The bone marrow transplantation experiment proved that the increased radiosensitivity of TLR2 KO mice was related to BMCs (χ²=4.291, P<0.05). A total of 55 differentially expressed genes were screened by RNA sequence ([log2 Fold Change]>0.95, Q<0.05), of which 28 were up-regulated and 27 were down-regulated. QT-PCR assay determined that miR-21 was down-regulated in BMCs of TLR2 KO (t=9.420, P<0.01) and MyD88 KO (t=10.700, P<0.01) mice. It was proved by QT-PCR that the expressions of IL-6 (t=13.790, P<0.05) and TNF-α (t=14.280, P<0.05) were increased in a TLR2 dependent manner after PAM3CSK4 stimulation. Overexpression of miR-21 promoted viability of EL4 cells (t=5.951, P<0.05) and NIH/3T3 cells (t=4.786, P<0.05) and reduced BMCs apoptosis in WT (t=4.842, P<0.05) and TLR2 KO (t=10.520, P<0.05) mice after radiation. Inhibition of miR-21 decreased the viability of EL4 cells (t=4.815, P<0.05) and NIH/3T3 cells (t=4.042, P<0.05). Conclusions miR-21 plays a key regulatory role in the process of TLR2 radioprotection, which may be related to the up-regulation of IL-6 and TNF-α.

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