小鼠脑组织及培养神经元Neuro-2a在内质网应激反应时的基因表达谱分析

Gene Expression Profile Analyses of Mouse Brain and Cultured Neuro-2a Cells under Endoplasmic Reticulum Stress

内质网是真核细胞的重要细胞器。某些细胞内外因素如病原体感染等能引起从内质网到胞浆和胞核的信号传导途径活化,即内质网应激反应。但是,目前国内外尚无针对内质网应激反应的基因表达谱分析报道。本研究中,用3种已报道的内质网应激反应诱导剂,包括蛋白质糖基化抑制剂衣霉素(tunicamycin)、内质网Ca^(2+)-ATPases抑制剂毒胡萝卜素(thapsigargin)和乙脑病毒(Japanese encephalitis virus,JEV),分别处理小鼠颅腔和小鼠脑神经瘤细胞(Neuro-2a),试剂处理组与未处理组的第二代RNA测序分析发现,衣霉素、毒胡萝卜素和乙脑病毒在体外和体内均引起分子伴侣基因Hsp70表达上调,诱导内质网应激反应。衣霉素、毒胡萝卜素和乙脑病毒体外处理诱导的内质网应激反应信号通路中,基因差异表达相似性高于体内处理组。乙脑病毒和糖基化抑制剂衣霉素体内外处理,主要诱导内质网应激反应的非折叠蛋白质反应信号通路,引起相关基因Atf4、Bip、Edem和Perk等表达上调。内质网Ca^(2+)-ATPases抑制剂毒胡萝卜素主要诱导内质网超负荷反应,激活NF-κB信号通路。乙脑病毒诱导的内质网应激反应相关差异表达基因数量最多,体外与体内合计有40种。乙脑病毒体内外处理上调的基因包括Bax、Casp12、Atf4、Bip、Edem和Perk等,下调的基因包括Sec23/24、Nef、Svip和Jnk等。糖基化抑制剂衣霉素体内外处理上调基因包括Gadd34、Atf4、Ermani和Bip等,下调基因包括Grp94、Atf6、Sec23/24和Nef等。内质网Ca2+-ATPases抑制剂毒胡萝卜素体内外处理上调的基因包括Sec61、Trap和Ask1等。衣霉素、毒胡萝卜素和乙脑病毒体内外处理也通过内质网应激反应,调控与炎症或凋亡相关的MAPK信号通路和P53信号通路。本研究首次通过使用3种内质网应激反应诱导剂分别处理小鼠和细胞,揭示了体内外内质网应激反应引起的基因表达谱变化,为内质网应激反应相关疾病的治疗提供了新思路。

Endoplasmic retieulum （ER） is a very important organelle in eukaryotic cells. Intracellualr and extracellular stimulus such as virus infection can trigger ER stress response in which signaling transduces from endoplasmic reticulum to cytoplasm and nucleus. However, gene expression profile analyses of endoplasmic reticulum stress response have not been reported. In this study, mouse brain and Neuro-2a cells were treated with endoplasmic reticulum stress response inducer including tunicamycin （protein glycosylation inhibitor）, thapsigargin （ER Ca2＋ -ATPases inhibitor）, and Japanese encephalitis virus （JEV）. Global mRNA expression changes induced by tunicamycin, thapsigargin and JEV were determined by next-generation sequencing technology. Common upregulated gene by tunicamycin, thapsigargin and JEV in vivo and in vitro was Hsp70. Tunicamycin, thapsigargin and JEV induced ER stress response in mouse brain and Neuro-2a ceils. Compared to in vivo treatment, in vitro treatment of three inducers had higher similarity in differently expressed genes. Moreover, JEV and Tunicamycin mainly induced unfolded protein response signaling pathway of ER stress response which up-regulated the expression of genes such as Atf4, Bip, Edem and Perk. Thapsigargin, an ER Ca2--ATPases inhibitor, induced endoplasmic reticulum overload response signaling pathway of ER stress response by activating NF-KB signaling pathway. Of three inducers, JEV in vivo and in vitro caused the most differently expressed genes related to ER stress response （in total 40 genes）. JEV upregulated the expression of genes such as Pdls, Bax, Caspl2, Perk, elF2c- and Traf2 and down-regulated the expression of genes such as Sec23/24, Nef, Grp94, Svip and .Ink. Tunicamycin, an ER protein glycosylation inhibitor, up- regulated the expression of genes such as Gadd34, Atf4, Ermani and Bip and downregulated the expression of genes such as Grp94, Atf6, Sec23/24 and Nef in vivo and in vitro. Thapsigargin upregulated the expression of genes such as Sec61 , Trap and Ask1. Tunicamycin, Thapsigargin and JEV in vivo and in vitro regulated MAPK and P53 signaling pathways involved in inflammation and apoptosis. This study revealed the ER stress-induced gene expression changes in vivo and in vitro by using three ER stress inducer including tunicamycin, thapsigargin and JEV to treat mouse brain and Neuro-2a, which provided a novel insight for the treatment of ER stress response-related diseases.