Open Accessp21<sup>WAF1</sup> and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes
Author(s) -
Dan Wang,
Yuzhen Zhang,
Bing Yang,
Fengxiang Zhang,
Ming-Yong Cao,
Cheng Wang,
Minglong Chen
Publication year - 2011
Publication title -
folia histochemica et cytobiologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.384
H-Index - 40
eISSN - 1897-5631
pISSN - 0239-8508
DOI - 10.5603/fhc.2011.0063
Subject(s) - senescence , small hairpin rna , cyclin d1 , hypoxia (environmental) , cell cycle , transfection , gene knockdown , flow cytometry , microbiology and biotechnology , cyclin , biology , chemistry , cell culture , andrology , cell , medicine , oxygen , biochemistry , genetics , organic chemistry
We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatal rat cardiomyocytes. In this research, we investigated the effects of p21(WAF1) (p21) in hypoxia/reoxygenation-induced senescence, using H9c2 cells. A plasmid overexpressing wild type p21(WAF1) and a plasmid expressing small hairpin RNA (shRNA) targeting p21(WAF1) were constructed, and transfected into H9c2 cells to control the p21 expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO(2), balancing the incubator chamber with N(2) for 6 h (hypoxia 6 h), then 21% oxygen for 8 h (reoxygenation 8 h). Cell cycle was examined using flow cytometry. Senescence was assessed using β-galactosidase staining. The expression of p53, p21, p16(INK4a), and cyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution, stronger β-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the opposite results and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silenced cells had a smaller percentage of G1 cells, weaker β-galactosidase activity and lower 16(INK4a) expression, and higher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data implies that p21(WAF1) is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescence process.