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[Research on regulation of cell senescence by miRNA-34a].

OBJECTIVE
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 micromol/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-beta-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-beta-gal staining than that of other cell groups.


CONCLUSION
miRNA-34a regulated cell senescence indirectly through targeting SIRT1.