小分子双链RNA对人类细胞中抑癌基因p21表达的上调作用

Up-regulation of Tumor Suppressor Gene p21^(WAF1/CIP1) in Human Cells by Small Double Strand RNA

目的筛选更多的对小分子双链RNA(dsP21-322)介导抑癌基因p21WAF1/CIP1激活起效应的人类细胞系,并初步研究该过程是否依赖p53的表达。方法选择4种表达不同类型p53蛋白的人类细胞系,p53野生型(p53wild type)的人骨肉瘤细胞系U2-OS、人肾上皮细胞系293T,p53突变型(p53mutant type)的人宫颈癌细胞系HeLa及p53缺失(p53null)的人肺腺癌细胞系NCI-H1299,转染dsControl(阴性对照)或dsP21-322至以上细胞,RT-qPCR和Westernblot分别检测p21、p53mRNA和蛋白表达的变化。结果 dsP21-322的转染未明显影响上述细胞系p53的表达;而在U2-OS、HeLa和293T细胞系中均能激活p21的表达,相比dsControl,分别上调p21mRNA的表达3.97倍、4.94倍和4.64倍,Western blot进一步证明在这3种细胞系中p21蛋白表达水平的增加与p21mRNA水平的上调相一致,且与dsControl组相比,差异均有统计学意义(均P<0.05)。但是在p53缺失的NCI-H1299细胞系中,dsP21-322则不能上调p21的表达。结论 dsP21-322介导p21激活现象存在于人类细胞,而其未能上调p53缺失细胞系中p21的表达是否提示该过程可能依赖p53的表达尚有待进一步研究。

Objective To screen more human cell lines susceptible to dsP21-322-mediated tumor suppressor gene p21 WAF1/CIP1 activation and investigate whether this process is dependent on p53 expression.Methods A small double strand RNA,dsP21-322,targeting the p21 promoter at position-322 relative to the transcription start site was synthesized.A dsControl lacking significant homology to all known human sequences was also synthesized and used as a negative control.Four kinds of human cell lines which expressed different types of p53 protein were chosen,including human osteosarcoma cell line U2-OS（p53 wild type）,human embryonic kidney cell line 293T（p53 wild type）,human cervical cancer cell line HeLa（p53 mutant type）and human lung cancer cell line NCI-H1299（p53 null）.All above cell lines were cultured in vitro and transfected with dsControl or dsP21-322 by Entranster-R.RT-qPCR and Western blot were applied to detect the expression levels of p21 and p53 mRNA and protein,respectively.Results Seventy-two h after transfections,dsP21-322 did not affect the expression of p53 in above four kinds of cell lines,but caused a significant induction in p21 mRNA expression in p53 wild type or p53 mutant cell lines.Compared with dsControl transfections,induction of p21mRNA was 3.97-,4.94-,and 4.64-fold in U2-OS,HeLa and 293T cell lines,respectively.Western blot revealed that the elevated levels of p21 protein were strongly correlated to the increase in p21 mRNA expression in these three cell lines.The p21 protein level in dsP21-322 transfections was significantly higher than in dsControl transfections（P0.05）.However,dsP21-322 was unable to elevate the p21 mRNA and protein levels in p53 null NCI-H1299 cell line.Conclusion Activation of p21 expression by dsP21-322 in human cell lines is a pervasive phenomenon.Furthermore,dsP21-322 failed to elevate the p21 expression in p53 null cell,indicating this process might rely on the expression of p53.