CCM3 down-regulating the lead acetate-induced migration of human umbilical vein endothelial cells / 中国职业医学

ABSTRACT

OBJECTIVE: To investigate the effects of knockout cerebral cavernous malformation(CCM) virulence gene CCM3 on the migration induced by lead acetate in immortalized human umbilical vein endothelial cells(HUVECs) and to explore the possible mechanism of endoplasmic reticulum stress(ERS). METHODS: CCM3 wildtype(CCM3-WT) and CCM3 knockout(CCM3-KO) HUVECs were used as experimental cells. a) CCM3-WT and CCM3-KO HUVECs were treated with lead acetate at 0,10,50 and 200 μmol/L for 24 hours. The migration of these cells was observed by woundhealing assay. b) CCM3-WT and CCM3-KO HUVECs were treated with lead acetate at 0,10,50 and 200 μmol/L for 24 hours,and at 50 μmol/L for 0,6,12,24 and 48 hours,and the mRNA expression of genes of unfolded protein response pathway were detected by quantitative real-time polymerase chain reaction; the protein expression of glucose-regulated protein 78(GRP78) was detected by Western blotting. c) CCM3-WT and CCM3-KO HUVECs were divided into lead exposure group and tauroursodeoxycholic acid(TUDCA) group. The former was treated with 50 μmol/L lead acetate for 24 hours,and the latter was pre-treated with ERS inhibitor TUDCA,followed by 50 μmol/L lead acetate. The migration of these cells was observed by wound-healing assay. RESULTS: a) The migration of CCM3-WT and CCM3-KO cells decreased and showed a dose-effect relationship with the increase of lead acetate concentration(P < 0. 05). b) The mRNA relative expression of the GRP78,protein kinase-like endoplasmic reticulum kinase(PERK),transcription activator 4(ATF4) and CCAAT enhancer binding homologous protein(CHOP) in CCM3-KO cells treated with 10,50 and 200 μmol/L lead acetate were higher than that in CCM3-WT cells at the same doses,except for the GRP78 in CCM3-KO cells treated with10 μmol/L lead acetate(P < 0. 05). The mRNA expression of PERK and CHOP in CCM3-KO cells increased in a timeeffect relationship with the increase of lead-exposure time(P < 0. 05). The mRNA relative expression of the four genes in CCM3-KO cells were higher than those in CCM3-WT cells at 48 hours(P < 0. 05). When cells were treated with 50μmol/L lead acetate,the protein expression of GRP78 in CCM3-KO cells was higher than that in CCM3-WT cells(P <0. 05),and the protein expression of GRP78 in CCM3-KO cells increased in a time-effect relationship with the increase of lead-exposure time(P < 0. 05). c) The cell migration of TUDCA group was lower than that of lead-exposure group(P <0. 05). CONCLUSION: Lead acetate may activate ERS by activating the PERK-ATF4-CHOP signaling pathway,thereby reducing the migration of HUVECs. CCM3 gene has a protective effect on cell migration.