Mechanism of autophagy induced by activation of the AMPK/ERK/mTOR signaling pathway after TRIM22-mediated DENV-2 infection of HUVECs

Abstract

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.

Keywords: AMPK/ERK/mTOR signaling pathway; Autophagy; DENV-2; TRIM22.

Fig. 1

A The protein expression of TRIM22 in DENV-2-infected HUVECs was detected in western blot analysis; B The quantitative analysis of TRIM22 expression. ***P < 0.001

Fig. 2

Screening of effective interference targets by qRT-PCR. A Amplication plot, Letters A–P represent different samples; B Melt curve plot of H-GAPDH; C Melt curve plot of H-TRIM22; D The knockdown efficiency of sh-TRIM22-1, sh-TRIM22-2 and sh-TRIM22-3 was detected by RT-qPCR. shTRIM22-1, shTRIM22-2, and shTRIM22-3 were used to target TRIM22. *P < 0.05, **P < 0.01

Fig. 3

Knockdown efficiency detection. A Amplication plot, Letters A–P represent different samples; B Melt curve plot of H-GAPDH; C Melt curve plot of H-TRIM22; D The mRNA expression of TRIM22 after TRIM22 knockdown was detected by RT-qPCR; E, F The protein expression of TRIM22 after TRIM22 gene knockdown was detected by western blot analysis. *P < 0.05, ***P < 0.001

Fig. 4

Lentivirus infection of HUVEC (200×). A, B The GFP fluorescence was evaluated in shCtrl or shTRIM22-infected HUVECs

Fig. 5

The effect of shTRIM22 on cell proliferation as detected by CCK-8 assay. ***P < 0.001

Fig. 6

A, B The ultrastructure of HUVEC autophagy observed by TEM in shCtrl and shTRIM22 groups. Green arrow: autophagosome, red arrow: autolysosome

Fig. 7

Expression of HUVEC autophagy and AMPK pathway-related genes after TRIM22 knockdown. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

Expression of autophagy-related proteins. A The effect of TRIM22 knockdown on the expressions of autophagy-related proteins in HUVECs as detected by western blot analysis; B–F The quantitative expressions analysis of autophagy-related proteins. **P < 0.01, ***P < 0.001

Fig. 9

Expression of AMPK pathway-related proteins. A The effect of TRIM22 knockdown on the expressions of AMPK pathway-related proteins in HUVECs as detected by western blot analysis; B–E The quantitative expressions analysis of AMPK pathway-related proteins. **P < 0.01, ***P < 0.001

Fig. 10

Detection of cell cycle. A The effect of TRIM22 knockdown on the cell cycle of DENV-2-infected HUVECs, the abscissa is the fluorescence intensity of PI, which represents the DNA content of the cells, and the ordinate is the number of cells; B the histogram analysis of each phase of the cell cycle. *P < 0.05, ***P < 0.001

Fig. 11

Effect of TRIM22 knockdown on HUVEC apoptosis following DENV-2 infection. ShCtrl, negative control group; DENV-2 + shCtrl, negative control group for DENV-2 infection; DENV-2 + vshTRIM22, DENV-2 infected HUVECs with TRIM22 knockdown

Fig. 12

The ultrastructure of DENV-2-infected HUVECs after TRIM22 knockdown as observed by TEM. A Control group infected with DENV-2; B DENV-2-infected HUVEC group with TRIM22 knockdown. Black arrow autophagosome; red arrow autolysosome

Fig. 13

Effect of TRIM22 knockdown on autophagy-related factors. A The effect of TRIM22 knockdown on autophagy-related factors in DENV-2-infected HUVECs as detected by western blot analysis; B–G The quantitative expressions analysis autophagy-related proteins. shCtrl group: DENV-2 + shCtrl group; shTRIM22 group: DENV-2 + shTRIM22 group; AICAR group: DENV-2 + shCtrl + AICAR group; shTRIM22 + AICAR group: DENV-2 + TRIM22 + AICAR group. *represents comparison with the shCtrl group; *P < 0.05, **P < 0.01, and ***P < 0.001. #represents comparison with the shTRIM22 + AICAR group; #P < 0.05, ##P < 0.01, and ###P < 0.001

Fig. 14

Effects of TRIM22 knockdown on expression of AMPK/ERK/mTOR pathway-related proteins. A The effect of TRIM22 knockdown on the expressions of AMPK/ERK/mTOR pathway proteins in DENV-2-infected HUVECs as detected by western blot analysis; B–E The quantitative expressions analysis of AMPK pathway-related proteins. *represents comparison with the shCtrl group; *P < 0.05, **P < 0.01, and ***P < 0.001. #represents comparison with the shTRIM22 + AICAR group; #P < 0.05, ##P < 0.01, and ###P < 0.001

Fig. 15

Establishment of a TRIM22 overexpression plasmid. A Results of agarose gel electrophoresis of the restriction enzyme-digested vector; 1 LV-007 Vector linearized by Nhe I and Age I double digestion, 2 LV-007 Vector (vector plasmid without digestion); B Lv-007 vector map, and Nhe I and Age I enzyme digestion. The component order is CMV-MCS-EF1-copGFP-T2A-puromycin; The fluorescent tags is copGFP. C Electrophoresis of the TRIM22 PCR products. A specific band at ~ 1497 bp, which was consistent with the expected size, was noted. D 1–4 positive transformants of the TRIM22 overexpression plasmid; 5 negative control (no-load self-connecting control group). The negative control was used to show no false positives in the amplification process using empty vectors without the target gene as templates. The PCR product size of the negative transformant was 312 bp; 6 Negative control (ddH2O). E False positives result from the contamination of external nucleic acid in the exclusion system

Fig. 16

Overexpression efficiency detection. A Amplication plot, Letters A–P represent different samples; B Melt curve plot of H-GAPDH; C Melt curve plot of H-TRIM22; D The mRNA expression of TRIM22 after TRIM22 overexpression was detected by RT-qPCR. **P < 0.01

Fig. 17

Effect of TRIM22 overexpression on HUVEC autophagy-related factors. A The effect of TRIM22 overexpression on autophagy-related factors in DENV-2-infected HUVECs as detected by western blot analysis; B–F The quantitative expressions analysis autophagy-related proteins. TRIM22-OE: TRIM22 overexpression group; Dorsomorphin: AMPK inhibitor. Empty vector: DENV-2 + Empty vector group; TRIM22-OE: DENV-2 + TRIM22-OE group; 3-MA: DENV-2 + Empty vector + Dorsomorphin; Dorsomorphin + TRIM22-OE: DENV-2 + TRIM22-0E + Dorsomorphin group. *represents comparison with the Empty vector group; *P < 0.05, **P < 0.01, and ***P < 0.001. #represents comparison with the TRIM22-OE + Dorsomorphin group; #P < 0.05, ##P < 0.01, and ###P < 0.001

Fig. 18

Effects of TRIM22 overexpression on AMPK/ERK/mTOR signaling pathway-related proteins. A The effect of TRIM22 overexpression on the expressions of AMPK/ERK/mTOR pathway proteins in DENV-2-infected HUVECs as detected by western blot analysis; B–E The quantitative expressions analysis of AMPK pathway-related proteins. *represents comparison with the Empty vector group; *P < 0.05, **P < 0.01, and ***P < 0.001. #represents comparison with the TRIM22-OE + Dorsomorphin group, #P < 0.05, ##P < 0.01, ###P < 0.001