Knockdown of TRAF3IP2 suppresses the expression of VEGFA and the proliferation of keratinocytes and vascular endothelial cells

Abstract

Objective: To investigate the expression level of TRAF3IP2 in psoriasis lesion, and to explore the functional roles of TRAF3IP2 on proliferation, apoptosis, cytokine expression and secretion of both keratinocytes and vascular endothelial cells in vitro.

Methods: The expression of TRAF3IP2 in skin samples of patients with psoriasis was analyzed by immunohistochemistry and qRT-PCR. To identify the effect of TRAF3IP2 knockdown on HaCaT and HUVEC cells, a plasmid vector expressing siRNA targeting TRAF3IP2 mRNA was designed and transfected into cells with Lipofectamine 2000. The levels of cytokines were identified using the ELISA Kits and qRT-PCR. Furthermore, cell proliferation, cell cycle and apoptosis were examined by using MTT, PI and Annexin V-FITC/7AAD assays, respectively. Furthermore, the expression of apoptosis-related proteins (Cleaved-Caspase 3, Caspase 3 and Bax) were detected by western blotting.

Results: TRAF3IP2 was significantly upregulated in psoriasis lesion. TRAF3IP2 knockdown reduced the expression of vascular endothelial growth factor A (VEGFA) and the release of IL-6, and IL-8, but had no effect on IL-23 in both HaCaT and HUVEC cells. In addition, knockdown of TRAF3IP2 significantly inhibited cell proliferation through blocking the cell cycle in the G2/M phase. Moreover, knockdown of TRAF3IP2 increased the expression of Caspase 3, Cleaved-Caspase 3 and Bax, which was supported by the increased apoptosis of both HaCaT and HUVEC cells.

Conclusion: Taken together, these results indicated that TRAF3IP2 might play a contributive role in the pathogenesis of psoriasis and may serve as a new target for the treatment of psoriasis. VEGF related pathways may be involved in the mechanism beneath.

Keywords: Cell biology; Genetics; Molecular biology; Physiology.

Fig. 1

TRAF3IP2 expression was upregulated in psoriatic lesions. (a) Representative Immunohistochemistry staining of healthy human skin and psoriatic human skin and positive expression of TRAF3IP2 in psoriatic dermal vascular endothelial cells. (b) Semi-quantitative analysis of TRAF3IP2 staining results from 29 healthy and 97 psoriatic skin samples. (c) qRT-PCR was used to detect the expression of TRAF3IP2 in healthy and psoriatic skin. **p < 0.01, vs the siNC group.

Fig. 2

Efficiency of TRAF3IP2 knockdown in HaCaT and HUVEC cells. (a) Morphological characterization of HaCaT and HUVEC cells growing in DMEM and RPMI-1640 medium, respectively (x100). The efficiency of TRAF3IP2 knockdown in HaCaT and HUVEC cells was verified using qRT-PCR (b, c) and western blotting (d, e), respectively. Data are shown as mean ± SD. n = 3 independent experiments, *p < 0.05 and **p < 0.01, vs the siNC group. Note: The full, uncropped versions of the blots can be found as supplementary materials.

Fig. 3

TRAF3IP2 knockdown affects the secretion and expression of cytokines in HUVEC and HaCaT cells. Secretion and expression levels of VEGFA, IL-6, IL-8 and IL-23 in HaCaT cells (a, c) and HUVEC cells (b, d) were measured by using ELISA and qRT-PCR, respectively. Data are shown as mean ± SD. n = 3 independent experiments, *p < 0.05, **p < 0.01, vs the siNC group.

Fig. 4

Effect of TRAF3IP2 knockdown on proliferation, cell cycle and apoptosis of HUVEC and HaCaT. (a) MTT assays were performed to assess HaCaT and HUVEC cell viability after treatment with TRAF3IP2 siRNA. It was found that HUVEC and HaCaT cells exhibited a significant decrease in cellular viability after transfection with siTRAF3IP2 compared with cells transfected with control siRNA. (b) Percentage of cells at different periods of cell cycle. It was found that TRAF3IP2 knockdown significantly increased the percentage of cells in G1 period. (c) It was revealed that most of the HUVEC and HaCaT cells fall into the upper left quadrant which is known to represent normal living cells. The upper right quadrant represented HUVEC and HaCaT cells were undergoing apoptosis and it was shown that, after transfected with siTRAF3IP2, a significantly increased in the percentage of HUVEC and HaCaT cells undergone apoptosis. (d) Quantification of the percentage of cells undergoing apoptosis in c. The expression of apoptosis-related proteins (Caspase 3 and Bax) were detected by western blotting (e), and the images were processed and analyzed by ImageJ software (f). Data are shown as mean ± SD. n = 3 independent experiments, *p < 0.05, **p < 0.01, vs the siNC group. Note: The full, uncropped versions of the blots can be found as supplementary materials.