Mechanism of autophagy induced by activation of the AMPK/ERK/mTOR signaling pathway after TRIM22-mediated DENV-2 infection of HUVECs.
Mechanism of autophagy induced by activation of the AMPK/ERK/mTOR signaling pathway after TRIM22-mediated DENV-2 infection of HUVECs.
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DOI：
10.1186/s12985-022-01932-w
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发表时间：
2022-12-31
期刊：
影响因子：
4.8
通讯作者：
Zuo, Li
中科院分区：
文献类型：
--
作者：
Wu, Ning;Gou, Xiaoqin;Hu, Pan;Chen, Yao;Ji, Jinzhong;Wang, Yuanying;Zuo, Li
采用登登革热病毒2型（DENV-2）感染原代人脐静脉内皮细胞（HUVECs），检测TRIM22介导的DENV-2感染后单磷酸腺苷活化蛋白激酶(AMPK)/细胞外信号调节激酶(ERK)/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路激活诱导的自噬，进一步揭示DENV-2感染的潜在致病机制。采用实时定量聚合酶链反应（qRT-PCR）筛选TRIM22可能的干扰靶点，并测定其敲除效率。采用CCK8法测定TRIM22敲低对HUVEC增殖的影响。TRIM22敲除后，透射电镜（TEM）检测HUVEC自噬体超微结构，qRT-PCR检测HUVEC自噬及AMPK通路相关基因的表达。western blot检测HUVEC自噬及AMPK通路相关蛋白表达水平。流式细胞术观察HUVECs细胞周期和凋亡情况，透射电镜观察HUVECs细胞自噬体结构。Western blot结果显示，DENV-2感染后36 h， TRIM22蛋白表达水平显著升高，这与蛋白质组学预测一致。CCK8检测显示TRIM22敲除后HUVEC的增殖减少（P < 0.001）。TEM结果显示，敲除TRIM22后，HUVEC自溶酶体增加，自噬受到抑制。qRT-PCR结果显示，TRIM22敲低后，抗胸腺细胞球蛋白7 （ATG7）、抗胸腺细胞球蛋白5 （ATG5）、Beclin1、ERK、mTOR基因表达水平降低（P < 0.01）；AMPK基因（P < 0.05）和P62基因（P < 0.001）表达增加。FCM结果显示，TRIM22敲除后，HUVECs处于G2期的比例随细胞凋亡而增加（P < 0.001）。添加自噬抑制剂后，TRIM22过表达对DENV-2感染和AMPK途径诱导的HUVEC自噬的影响减弱。在HUVECs中，TRIM22蛋白正向调节自噬，并可能通过AMPK/ERK/mTOR信号通路影响自噬。在trim22介导的DENV-2感染HUVECs后，AMPK/ERK/mTOR信号通路激活可诱导自噬。在线版本包含补充材料，可在10.1186/s12985-022-01932-w获得。
Dengue virus type 2 (DENV-2) was used to infect primary human umbilical vein endothelial cells (HUVECs) to examine autophagy induced by activation of the adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/mammalian target of rapamycin (mTOR) signaling pathway following tripartite motif-containing 22 (TRIM22)-mediated DENV-2 infection to further reveal the underlying pathogenic mechanism of DENV-2 infection. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to screen putative interference targets of TRIM22 and determine the knockdown efficiency. The effect of TRIM22 knockdown on HUVEC proliferation was determined using the CCK8 assay. Following TRIM22 knockdown, transmission electron microscopy (TEM) was used to determine the ultrastructure of HUVEC autophagosomes and expression of HUVEC autophagy and AMPK pathway-related genes were measured by qRT-PCR. Moreover, HUVEC autophagy and AMPK pathway-related protein expression levels were determined by western blot analysis. Cell cycle and apoptosis were assessed by flow cytometry (FCM) and the autophagosome structure of the HUVECs was observed by TEM. Western blot results indicated that TRIM22 protein expression levels increased significantly 36 h after DENV-2 infection, which was consistent with the proteomics prediction. The CCK8 assay revealed that HUVEC proliferation was reduced following TRIM22 knockdown (P < 0.001). The TEM results indicated that HUVEC autolysosomes increased and autophagy was inhibited after TRIM22 knockdown. The qRT-PCR results revealed that after TRIM22 knockdown, the expression levels of antithymocyte globulin 7 (ATG7), antithymocyte globulin 5 (ATG5), Beclin1, ERK, and mTOR genes decreased (P < 0.01); however, the expression of AMPK genes (P < 0.05) and P62 genes (P < 0.001) increased. FCM revealed that following TRIM22 knockdown, the percentage of HUVECs in the G2 phase increased (P < 0.001) along with cell apoptosis. The effect of TRIM22 overexpression on HUVEC autophagy induced by DENV-2 infection and AMPK pathways decreased after adding an autophagy inhibitor. In HUVECs, TRIM22 protein positively regulates autophagy and may affect autophagy through the AMPK/ERK/mTOR signaling pathway. Autophagy is induced by activation of the AMPK/ERK/mTOR signaling pathway following TRIM22-mediated DENV-2 infection of HUVECs. The online version contains supplementary material available at 10.1186/s12985-022-01932-w.
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影响因子：
11.1
作者：
He C;Klionsky DJ
通讯作者：
Klionsky DJ
影响因子：
21.3
作者：
通讯作者：
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DOI：
10.1016/j.mjafi.2014.09.011
发表时间：
2015-01-01
期刊：
Medical journal, Armed Forces India
影响因子：
--
作者：
Mustafa, M S;Rasotgi, V;Gupta, V
通讯作者：
Gupta, V
影响因子：
30.3
作者：
Heaton NS;Randall G
通讯作者：
Randall G
DOI：
10.1038/nrm3735
发表时间：
2014-02
期刊：
Nature reviews. Molecular cell biology
影响因子：
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作者：
通讯作者：
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