HMG盒转录因子1(HBP1)参与H_2O_2诱导的细胞衰老过程

Transcriptional Factor HBP1 Participates in H_2O_2-induced Premature Senescence

为探讨HMG盒转录因子1(HBP1)在过氧化氢(H2O2)诱导的细胞衰老中所起的作用,通过慢病毒感染得到稳定表达HBP1的MDA-MB-231细胞,以H2O2处理细胞.采用Western免疫印迹杂交试验和实时PCR检测HBP1、p16和细胞周期蛋白D1(cyclinD1)表达水平的变化.用荧光免疫试验检测H2O2对HBP1表达的影响,以及HBP1在H2O2的诱导下对于p16和细胞周期蛋白D1启动子的影响.用细胞增殖试验检测H2O2对于细胞增殖的影响.用基因敲减实验和衰老相关β半乳糖苷酶(SA-β-Gal)染色检测在H2O2诱导的细胞衰老中HBP1所起的作用.Western和免疫荧光实验结果显示,细胞经H2O2处理后,HBP1表达增高的同时促进了p16的表达,降低了细胞周期蛋白D1的表达.细胞增殖实验结果显示,H2O2显著抑制了细胞的增殖.基因敲减实验和SA-β-Gal染色实验说明,H2O2可诱导HBP1表达正常的MDA-MB-231细胞衰老,而HBP1的敲减则抑制了H2O2诱导的细胞衰老过程.本研究结果提示,在H2O2诱导的衰老中,HBP1的表达显著增加,并通过促进衰老相关基因p16的表达和抑制生长因子cyclinD1的表达来阻碍细胞增殖,促进细胞衰老.HBP1在H2O2诱导的细胞衰老过程中起着重要作用,H2O2诱导的细胞衰老必须在HBP1存在的情况下才能发生.

MDA-MB-231 cells were transfected with HBP1（HMG-box transcription factor 1） through lentivirus vector to investigate the impacts of HBP1 on cell senescence induced by hydrogen peroxide（H2O2）.Western blotting and real-time PCR were used to detect the HBP1,p16,and cyclinD1 protein and mRNA levels in HBP1-expressing MDA-MB-231 cells with or without H2O2 treatments.Immunofluorescence assay was used to determine the HBP1 expression in response to different doses of H2O2.The effect of HBP1 on p16 or cyclin D1 promoter was evaluated.Cell proliferation assays were also performed with MDA-MB-231 cells treated with H2O2.HBP1 knockdown on H2O2-induced premature senescence in MDA-MB-231 cells was examined by β-galactosidase（SA-p-Gal） staining.The results showed that protein levels of HBP1 and p16 increased dramatically,whereas cyclinD1 decreased after H2O2 treatments.H2O2 enhanced HBP1 activation on p16 and cyclin D1 promoters,and inhibited the proliferation of MDA-MB-231 cells.H2O2 induced premature senescence with the knockdown of HBP1 with shRNA.In summary,transcriptional factor HBP1 was suggested to participate in H2O2-induced premature senescence,and possibly through regulating the expression of p16 and cyclin D1.