Effects of hypoxia inducible factor-1α on P311 and its influence on the migration of murine epidermal stem cells
10.3760/cma.j.issn.1009-2587.2017.05.007
- VernacularTitle: 缺氧诱导因子1α对P311的作用及其对小鼠表皮干细胞迁移的影响
- Author:
Zhengdong XU
1
;
Haisheng LI
;
Song WANG
;
Weifeng HE
;
Jun WU
;
Gaoxing LUO
Author Information
1. Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, the Third Military Medical University, Chongqing 400038, China
- Publication Type:Journal Article
- Keywords:
Hypoxia-inducible factor 1;
Cell migration assays;
Wound healing;
Epidermal stem cells;
P311
- From:
Chinese Journal of Burns
2017;33(5):287-294
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effects of hypoxia inducible factor-1α (HIF-1α) on P311 and its influence on the migration of murine epidermal stem cells (ESCs) under hypoxia in vitro.
Methods:Two kinds of murine ESCs were isolated and obtained from 15 neonatal wild-type C57BL/6J mice and 5 congeneric source P311 gene knock-out mice, respectively. The first passage of cells were used in the following experiments after morphologic observation and detection of expression of cell surface markers CD71 and CD49f with flow cytometer. (1) After cell scratch assay, according to the random number table (the same dividing method below), ESCs of P311 gene knock-out mice were divided into normoxia group (cells were cultured with complete medium in normoxic carbon dioxide incubator, and the subsequent normoxic treatments were the same) and hypoxia group (cells were cultured in hypoxic carbon dioxide incubator containing 1% oxygen, and the subsequent hypoxic treatments were the same), with 12 inserts in each group. ESCs of wild-type mice were divided into normoxia group, pure hypoxia group, dimethyl sulfoxide (DMSO) control group (2 μL DMSO solvent was added for 1 h of normoxia treatment before hypoxia treatment), HIF-1α inhibitor group (cells were treated with 11 μmol/L HIF-1 inhibitor of 2 μL under normoxia condition for 1 h before hypoxia treatment), HIF-1α stabilizer group (the cells were treated with 2 μmol/L FG-4592 of 2 μL under normoxia condition for 1 h before hypoxia treatment), with 12 inserts in each group. Three inserts of each time point in each group were adopted respectively to measure the residual width of scratch under inverted phase contrast microscope at post scratch hour (PSH) 0 (immediately), 12, 24, and 48. (2) After hypoxia treatment, the protein level of HIF-1α in ESCs of wild-type mice was detected by Western blotting at post hypoxia hour (PHH) 0, 12, 24, and 48. (3) ESCs of wild-type mice were divided into pure hypoxia group, DMSO control group, HIF-1α inhibitor group, and HIF-1α stabilizer group as that of experiment (1) with the same treatment. The mRNA expression of P311 and expression of P311 in ESCs were determined by real-time fluorescent quantitative reverse transcription polymerase chain reaction and immunocytochemical staining, respectively, at PHH 0 (immediately), 12, 24, and 48 (with sample numbers of 12). (4) The second passage of human embryonic kidney 293 (HEK-293) cells were divided into empty vector hypoxia group (cells were cultured under hypoxia condition after being transfected with empty vector plasmid), P311 normoxia group (cells were cultured under normoxia condition after being transfected with P311 reporter gene plasmid), P311 hypoxia group (cells were cultured under hypoxia condition after being transfected with P311 reporter gene plasmid), P311 hypoxia+ HIF-1α inhibitor group (cells which were incubated with HIF-1α inhibitor were cultured under hypoxia condition after being transfected with P311 reporter gene plasmid). The luciferase activity was detected at post culture hour (PCH) 0 and 12, respectively, and then the P311 transcriptional regulatory binding site of HIF-1α and the promoter sequence of P311 were predicted and searched by bioinformatics methods. Data were processed with factorial design variance analysis, one-way analysis of variance, LSD test and Bonferroni correction.
Results:(1) The results of ESCs. The cells showed cobblestone-like pattern and different clonal morphology due to the different cell proliferation potential. The proportion of CD71-CD49f+ cells accounted for about 85%. The identification results indicated that the cells showed strong stem cell properties and high purity. Compared with those in cells of normoxia group of P311 gene knock-out mice, the residual widths of scratch of cells in pure hypoxia group were smaller at PSH 12 and 24 (with P values below 0.05), and those in hypoxia group, normoxia group of wild-type mice, DMSO control group, HIF-1α inhibitor group, and HIF-1α stabilizer group were smaller at PSH 12 (with P values below 0.05). Compared with those in cells of normoxia group of wild-type mice, the residual widths of scratch of cells in hypoxia group, pure hypoxia group, and DMSO control group were smaller at PSH 12 and 24 (with P values below 0.05), and the residual width of scratch of cells in HIF-1α stabilizer group was smaller at PSH 12 (P<0.05). Compared with those of cells in pure hypoxia group, the residual widths of scratch of cells in hypoxia group were wider at PSH 12 and 24 (with P values below 0.05), and the residual width of scratch of cells in HIF-1α inhibitor group was wider at PSH 12 (P<0.05), and those of cells in HIF-1α stabilizer group were smaller at PSH 12 and 24 (with P values below 0.05). There was no obvious difference in the width of scratch in cells among the 7 groups (F=19.02, P>0.05). The protein levels of HIF-1α in ESCs of wild-type mice at PHH 0, 12, 24, and 48 were respectively 1.02±0.05, 2.56±0.09, 1.60±0.17, and 1.17±0.03. Compared with that at PHH 0, the protein level of HIF-1α at PHH 12 was significantly enhanced (P<0.01). It began to decline at PHH 24 but was still higher than that at PHH 0 (P<0.05), and the protein level of HIF-1α at PHH 48 was close to the normoxia level (P>0.05). Compared with those of cells in pure hypoxia group, the mRNA expressions of P311 of cells in HIF-1α inhibitor group were significantly decreased at each time point (with P values below 0.05), and those in HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). Compared with those of cells in HIF-1α inhibitor group, the mRNA expressions of P311 of cells in DMSO control group and HIF-1α stabilizer group were significantly increased at PHH 0, 12, and 24 (with P values below 0.05). Compared with those of cells in pure hypoxia group, the expressions of P311 of cells in HIF-1α inhibitor group were significantly decreased at each time point (with P values below 0.05), while those in HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). Compared with those of cells in HIF-1α inhibitor group, the expressions of P311 of cells in DMSO control group and HIF-1α stabilizer group were significantly increased at PHH 12 and 24 (with P values below 0.05). (2) The results of HEK-293 cells. At PCH 0, there was no significant difference in the luciferase activity among cells of empty vector hypoxia group, P311 normoxia group, P311 hypoxia group, and P311 hypoxia+ HIF-1α inhibitor group (F=13.33, P>0.05). At PCH 12, the luciferase activity of cells in P311 hypoxia group was higher than that in empty vector hypoxia group (P<0.01). The luciferase activity of cells in hypoxia group was higher than that in P311 normoxia group (P<0.05), while that of cells in P311 hypoxia+ HIF-1α inhibitor group was lower than that in P311 hypoxia group (P<0.01).
Conclusions:HIF-1α may increase the migration of murine ESCs through inducing the expression of P311 at the early stage of hypoxia.
