Effect of siRNA-mediated α-TAT1 gene silencing on migration behavior of endothelial cells in rats with hepatopulmonary syndrome

Abstract

Objective To investigate the effect of silencing alpha tubulin acetyltransferase 1 (α-TAT1) on migration behavior of endothelial cells induced by hepatopulmonary syndrome (HPS). Methods Online database Tabula Muris was used to analyze the expression of α-TAT1 in various cell subsets in the lungs. Twenty-four male SD rats were randomly divided into control group (Sham group, n=6) and common bile duct ligation group (HPS group, n=18). The rats in HPS group were euthanasized at 2 and 4 weeks after modelling, and then the expression of α-TAT1 in pulmonary vascular endothelial cells was detected by immunofluorescence colocalization. The sera from the Sham and HPS rats were used to stimulate human umbilical vein endothelial cells (HUVECs) for 12 and 24 h, respectively. Then the obtained HUVECs were divided into 4 groups: Sham serum + siRNA NC group, Sham serum + siRNA α-TAT1 group, HPS serum+ siRNA NC group, HPS serum + siRNA α-TAT1 group. The expression levels of α-TAT1 and Ace-α-tubulin in HUVECs were detected by Western blotting. Immunofluorescence assay was applied to observe the levels of polymerized microtubules of α-Tubulin in HUVECs after nocodazole (10 μmol/L) pretreatment to evaluate the stability of microtubule structure. Cell scratch assay combined with cell immunofluorescence assay was employed to observe the nuclear localization of Golgi apparatus and cell migration ability of HUVECs. The angiogenesis ability of HUVECs was tested by in vitro angiogenesis test. Results In vivo and in vitro experiments showed that the expression of α-TAT1 in endothelial cells was significantly increased after HPS inducement. The expression levels of α-TAT1 and Ace-α-tubulin were significantly down-regulated, and the stability of microtubules was weakened in the siRNA α-TAT1 interference group (P < 0.01). In addition, the distribution of GM130 labeled Golgi apparatus in the protrusion of HUVECs was down-regulated in the siRNA α-TAT1 interference group, as well as the migration ability (P < 0.01). And the length of angiogenesis and network level were also significantly declined (P < 0.01). Conclusion Silencing α-TAT1 reduces the migration and angiogenesis of endothelial cells in HPS, which was associated with weakened stabilization of microtubule.

Keywords

• hepatopulmonary syndrome
• endothelial cells
• pathological angiogenesis
• α-tat1
• polarity of migration