Research on Regulation of Cell Senescence by MiRNA-34a

YANG Ming-ming, FAN Xue-jiao, DU Wen-ting, REN Peng-liang, LIU Ji

Abstract

To determine miRNA-34a regulated cell senescence indirectly through targeting silent mating-type information regulation 2 homologue 1 (SIRT1) in vitro experiment. Methods A constructed pre-miRNA -34a expression vector and a miRNA-1792 expression vector (not directly against any gene) were transfected into HEK293 and HUVEC cell lines respectively. The expression levels of SIRT1 in each cell groups were detected by RT-PCR and Western blot. The HUVEC cells were divided into different group:transfected with pre-miRNA-34a expression vector (HUVEC-pre-miRNA-34a), transfected with miRNA-1792 expression vector (HUVEC-pre-miRNA-1792), treated HUVEC cell with SIRT1 activator resveratrol (final concentration 1 μmol/L, treatment for 2 h)(HUVEC-Res), and HUVEC cells without any treatment as the control. Comet assay was applied to detect the oxidative damage of above-mentioned cells after H2O2 treatment for 2 h, and beta-galactosidase (SA-β-gal) staining was used to detect the senescence of them in different time points after doxorubicin treatment for 2 h. Results Pre-miRNA-34a expression vector was constructed successfully by sequencing confirmation. RT-PCR and Western blot indicated that the overexpression of miRNA-34a down regulated mRNA and protein level of SIRT1 in HEK293-miRNA-34a and HUVEC-miRNA-34a cell groups (P<0.001). Comet assay revealed that HUVEC-miRNA-34a cell group was the most sensitive to H2O2 treatment, and the DNA damage of HUVEC-Res cell group was the most minor. HUVEC-miRNA-34a cell group displayed higher frequency of SA-β-gal staining than that of other cell groups. Conclusion miRNA-34a regulated cell senescence indirectly through targeting SIRT1.

 

Keywords: miRNA-34, SIRT1, Cell senescence, DNA damage

 

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References


Boehm M, Slack FA. Developmental timing microRNA and its target regulate life span. Science,2005;310(5756); 1954-1957.

Melton C, Judson RL, Blelloch R. Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature,2010;463(7281);621-626.

Griffiths-Jones S, Grocok RJ , Dongen SV, el al. miRNABase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res,2006;34(Database): 140-144.

Megraw M. Sethupathy P, Corda B. el al. miRNAGen; a database for the study of animal microRNA genomic organization and function. Nucleic Acids Res, 2007; 35 (Database): 149-155.

Kim DH, Saetrom P. Sn 0 ve O, el al. MicroRNA-directed transcriptional gene silencing in mammalian cells. Proc Natl Acad Sci U S A,2008; 105(42): 16230-16235.

Kirkwood ТВ. Understanding the odd science of aging. Cell, 2005;120(4):437-447.

Vaupel JW. Biodemography of human ageing. Nature, 2010; 464(7288):536-542.

Eitan R, Kushnir M, Lithwick-yanai G, el al. Tumor microRNA expression patterns associated with resistance to platinum based chemotherapy and survival in ovarian cancer patients. Gynecol Oncol,2009;114(2):253-259.

Boehmer ED, Goral J, Fauce DE. el al. Age-dependent decrease in Toll-like receptor 4-mediated proinflammatory cytokine production and mitogen-activated protein kinase expression. J Leukoc Biol,2004;75(2);342-349.

Chang TC, Wentzel ЕA, Kent OA, el al. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell,2007;26(5);745-752.

He L, He X, Lim LP, el al. A microRNA component of the p53 tumour suppressor network. Nature, 2007; 447 ( 7148 ):


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