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转录因子HBP1(HMG-box containing protein 1,HBP1)属HMG家族,是1个含有513个氨基酸残基的多肽,可与RB蛋白结合,抑制许多癌基因的表达,从而抑制细胞的增殖.本工作构建了HBP1慢病毒表达载体,病毒包装后感染骨肉瘤细胞U2OS细胞.Western印迹结果显示,感染细胞cyclin D1、c-Myc蛋白质水平降低,而p53蛋白质水平增加;Real-time PCR检测cyclin D1、c-Myc及p53 mRNA水平与蛋白质检测结果一致.HBP1表达载体(pEFBOS-HBP1)和报告基因载体(含cyclin D1、c-Myc或p53 promoter)共转染U2OS细胞后的荧光素酶分析发现,HBP1可抑制cyclinD1、c-Myc启动子转录激活,促进p53启动子转录激活,并且这种抑制或激活作用具有HBP1剂量依赖性.以细胞代龄群体倍增值PD(population doubling)为指标测试细胞生长结合软琼脂集落形成实验证明,HBP1慢病毒感染的U2OS细胞生长速度减缓、集落形成能力下降.上述结果提示,HBP1可能通过调控细胞增殖相关基因的表达抑制骨肉瘤细胞的增殖.
The transcription factor HBP1 belongs to the HMG family and contains a polypeptide of 513 amino acid residues, which binds to the RB protein and inhibits the expression of many oncogenes, thus inhibiting cell proliferation. The HBP1 lentivirus expression vector was constructed and infected with U2OS cells after packaging with the virus.Western blotting showed that the protein levels of cyclin D1 and c-Myc were decreased and p53 protein levels were increased in infected cells. The expression of cyclin D1, c Myc and p53 mRNA levels were consistent with the results of protein detection.Luciferase analysis of U2OS cells co-transfected with the pBPBOS-HBP1 and reporter vectors (including cyclin D1, c-Myc or p53 promoter) found that HBP1 Which can inhibit the transcriptional activation of cyclinD1 and c-Myc promoters and promote the transcriptional activation of p53 promoter, and this inhibition or activation has a dose-dependent manner in HBP1. Cell growth binding was tested by using population doubling PD (population doubling) Soft agar colony formation experiments showed that HBP1 lentivirus-infected U2OS cells slowed down and colony formation ability decreased.The above results suggest that HBP1 may be through the regulation of cell proliferation phase Off gene expression inhibits osteosarcoma cell proliferation.