Effects of BNIP3 on the migration of human dermal microendothelial cell and its possible molecular mechanism
10.3760/cma.j.cn501120-20200927-00425
- VernacularTitle:低氧条件下B淋巴细胞瘤-2与腺病毒E1B 19 000相互作用蛋白3对人真皮微血管内皮细胞迁移的影响及其机制
- Author:
Junhui ZHANG
;
Qiong ZHANG
;
Jiezhi JIA
;
Hongmei LI
;
Can ZHANG
;
Jiongyu HU
;
Dongxia ZHANG
;
Yuesheng HUANG
- From:
Chinese Journal of Burns
2020;37(1):E019-E019
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effects and mechanism of B-cell lymphoma-2/E1B 19 000 interacting protein 3 (BNIP3) on the migration of human dermal microvascular endothelial cells (HDMECs) under hypoxia.Methods:The experimental research method was applied. (1) HDMECs were divided into normoxic group received routine culture and hypoxic 6, 12, 24 h groups treated with hypoxia under oxygen volume fraction of 2% for corresponding time. Western blotting was used to detect the protein expressions of BNIP3 and microtubule-associated protein 1 light chain 3Ⅱ (LC3Ⅱ) in HDMECs. (2) HDMECs were divided into normoxia+unloaded group, normoxia+BNIP3 knockdown group, hypoxia+unloaded group, and hypoxia+BNIP3 knockdown group which were transfected with unloaded virus or BNIP3 knockdown virus and were subjected normoxic or hypoxic treatment. The BNIP3 protein expression was detected by Western blotting and immunofluorescence staining. The scratch area at 24 h post scratching was detected by scratch test, and the wound healing rate was calculated. The curve distance of cell movement was measured with the living cell workstation, and the speed of movement was calculated within 3 hours. (3) HDMECs were grouped and treated as experiment (2). Western blotting and immunofluorescence staining were performed to detect the protein expression of LC3Ⅱ. The samples were 3 in the above-mentioned experiments. Data were statistically analyzed with one-way analysis of variance and LSD test.Results:(1) Compared with normoxic group, the protein expressions of BNIP3 and LC3Ⅱ of cells in hypoxic 6, 12, 24 h groups were significantly increased (P<0.01). (2) After 6 hours of culture, compared with hypoxia+unloaded group, the BNIP3 expression of cells in hypoxia+BNIP3 knockdown group was significantly decreased (P<0.05). The red fluorescence of BNIP3 expression of cells in normoxia+unloaded group and normoxia+BNIP3 knockdown group was weak, the red fluorescence of cells in hypoxia+unloaded group was strong, and the red fluorescence of cells in hypoxia+BNIP3 knockdown was significantly decreased compared with that in hypoxia+unloaded group. After scratching for 24 hours, the scratch of cells in hypoxia+unloaded group basically healed, while the remaining scratch area in the other three groups were large. The wound healing rates in normoxia+unloaded group, normoxia+BNIP3 knockdown group, hypoxia+unloaded group, and hypoxia+BNIP3 knockdown group were (61±4)%, (58±4)%, (88±4)% and (57±4)%, respectively. There was significant difference in general comparison among these groups (F=14.57, P<0.01). The wound healing rate in hypoxia+unloaded group was significantly higher than that in normoxia+unloaded group (P<0.01) and hypoxia+BNIP3 knockdown group (P<0.05). Within 3 hours of observation, the range of cell movement in hypoxia+unloaded group was significantly larger than that in normoxia+unloaded group, and the range of cell movement in hypoxia+BNIP3 knockdown group was significantly smaller than that in hypoxia+unloaded group. Within 3 hours of observation, the curve movement velocity of cells in hypoxia+unloaded group was significantly higher than that in normoxia+unloaded group and hypoxia+BNIP3 knocdown group (P<0.01). (3) After 6 hours of culture, compared with hypoxia+unloaded group, the BNIP3 protein expression of cells in hypoxia+BNIP3 knockdown group decreased significantly (P<0.05). After 6 hours of culture, the red fluorescence of LC3 expression of cells was weak in normoxia+unloaded group and normoxia+BNIP3 knockdown group, the red fluorescence of LC3 expression of cells was significantly enhanced in hypoxia+unloaded group, and the red fluorescence of LC3 expression of cells was significantly inhibited in hypoxia+BNIP3 knockdown group.Conclusions:BNIP3 can promote the migration and motility of HDMECs under hypoxia, and autophagy may be involved in the regulation migration and motility of HDMECs by BNIP3.
