Identification of a novel regulatory mechanism involved in inhibition of transcription of suvivin mRNA in breast cancer cells via p21cip-mediated regulation

摘要

Purpose: To evaluate the effect of p21 Cip1 on survivin transcription levels in breast carcinoma, and to investigate the potential mechanisms. Methods: Epirubicin, a p21 Cip1 activator, was used to treat MCF7 cells. Under the action of normal biological functions of p53, pEGFP-C2-p21 was transfected into MCF7 cells by lipofectamine and positive clones were screened out with G418. The ex pression levels of p21 cip1 , p53 and survivin mRNA were quantitated by real-time fluorescent polymerase chain reaction (RQ-PCR). MTT assay was utilized to measure cellular viability and proliferation after transfection. Flow cytometry was employed to determine the cell cycle. Hoechst 33342 staining was carried out to assess cell apoptosis. Lastly, several transcription factor sites located at the promoter region of survivin gene, such as, sp1 site, E2F site and p300/CBP, were measured by p21 overex pression using RT-PCR. Results: Following epirubicin treatment, within 24 h, the ex pression levels of endogenous p21 cip1 and p53 were up-regulated, whereas that of survivin was down-regulated. After transfection treatment, p21 inhibited the proliferation of MCF7 cells on days 3 and 4, and MCF7 cells overexpressed p21 mRNA, whereas the level of survivin mRNA in MCF7-p21 groups was markedly down-regulated relative to control group, but overex pression of p21 was not sufficient to cause changes in p53 gene ex pression. The overexpressed p21 resulted in G1/G0 phase arrest based on cell cycle analysis, but apoptosis was not induced. In addition, co-transcription factors E2F-1, sp1 and p300/CBP mRNA levels decreased significantly compared with normal p21 ex pression groups. Conclusion: P21 cip1 may down-regulate the ex pression of survivin gene partially by inhibiting the ex pression level of HAT.