TRIM22 介导的 DENV-2 感染 HUVECs 后，通过激活 AMPK/ERK/mTOR 信号通路诱导自噬的机制。

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

背景：登革病毒 2 型（DENV-2）用于感染原代人脐静脉内皮细胞（HUVEC），以检测三肽基含 22 （TRIM22）介导的 DENV-2 感染后激活的腺苷一磷酸激活的蛋白激酶（AMPK）/细胞外信号调节激酶（ERK）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路诱导的自噬，以进一步揭示 DENV-2 感染的潜在发病机制。

方法：使用定量实时聚合酶链反应（qRT-PCR）筛选 TRIM22 的潜在干扰靶点，并确定敲低效率。使用 CCK8 测定法确定 TRIM22 敲低对 HUVEC 增殖的影响。在 TRIM22 敲低后，使用透射电子显微镜（TEM）确定 HUVEC 自噬体的超微结构，并通过 qRT-PCR 测量 HUVEC 自噬和 AMPK 通路相关基因的表达。此外，通过 Western blot 分析测定 HUVEC 自噬和 AMPK 通路相关蛋白的表达水平。通过流式细胞术（FCM）评估细胞周期和细胞凋亡，并通过 TEM 观察 HUVEC 自噬体的结构。

结果：Western blot 结果表明，DENV-2 感染后 36 小时 TRIM22 蛋白表达水平显著增加，与蛋白质组学预测一致。CCK8 测定显示，TRIM22 敲低后 HUVEC 增殖减少（P < 0.001）。TEM 结果表明，TRIM22 敲低后 HUVEC 自溶体增加，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 和 mTOR 基因的表达水平降低（P < 0.01）；然而，AMPK 基因的表达（P < 0.05）和 P62 基因的表达（P < 0.001）增加。FCM 显示，TRIM22 敲低后，HUVEC 进入 G2 期的百分比增加（P < 0.001），同时细胞凋亡增加。加入自噬抑制剂后，TRIM22 过表达对 DENV-2 感染和 AMPK 通路诱导的 HUVEC 自噬的影响降低。

结论：在 HUVEC 中，TRIM22 蛋白正向调节自噬，并且可能通过 AMPK/ERK/mTOR 信号通路影响自噬。TRIM22 介导的 DENV-2 感染 HUVEC 后，通过激活 AMPK/ERK/mTOR 信号通路诱导自噬。