Development of new types of retrovirus vectors that modulate epigenetical regulation.

在本项目中，我们设计、制备和改进了几种逆转录病毒/慢病毒载体，这些载体携带有效的转录单位来表达短发夹(Sh)RNA、miRNA以及新开发的抑制特定miRNA(TUD RNA)的RNA诱骗分子。利用这些载体系统，我们通过集中研究SWI/SNF染色质重塑复合体来研究癌症的表观遗传学。我们注意到，在BRM表达缺陷的人肿瘤细胞系中，外源转导到其中的MLV和HIV载体的表达随机地迅速沉默。我们最终证明了BRM型SWI/SNF复合体对于逆转录/慢病毒的稳定表达是必不可少的。BRM进一步被证明具有抗癌潜力，因为外源BRM的引入降低了BRM表达缺陷的细胞系的致癌潜力。在所有被检测的细胞系中，都存在有功能的brm基因并被活跃地转录；brm的表达在t…处被抑制。更多的是转录后水平。我们假设BRM是某些miRNAs的靶标，并筛选了几个候选的miRNA，最后表明miR-199a靶向BRM mRNA。有趣的是，所有BRM缺陷的癌细胞株都有高水平的miR-199a，而在BRM表达的细胞中，miR-199a表达很少，我们进一步证明这些不同的表达模式是通过转录因子Egr1在BRM和miR-199a-5p/-3p之间形成的双负反馈调节的结果。此外，我们还证明了miR-21及其靶标NFIB(负转录调节因子)也在癌细胞系中形成了强大的反馈调节。我们还证明了人类安慰剂蛋白作为连接SWI/SNF复合体和RelB/p52的适配蛋白的功能。我们进一步表明，针对Req的shRNA的引入强烈地抑制了锚定非依赖性的生长，但在单层培养中根本不影响肿瘤细胞系的生长，例如PANC-1，在其中非典型的NFκB途径被结构性激活。较少

In this project, we have designed, prepared and improved several retrovirus/lentivirus vectors that carry efficient transcriptional units for the expression of short hairpin (sh) RNA, miRNA, and the newly developed RNA decoy molecule that inhibits specific miRNA (TuD RNA). Using these vector systems, we have studied cancer epigenetics by concentrating on SWI/SNF chromatin remodeling complex.We have noticed that in human tumor cell lines that are deficient in Brm expression, expression of MLV and HIV vectors that were exogenously transduced into them are rapidly silenced stochastically. We finally demonstrated that Brm-type SWI/SNF complex is essential for the stable expression of retro/lentivirus.Brm is further shown to have antioncogenic potential because exogenous introduction of Brm reduces oncogenic potential of cell lines deficient in Brm expression. In all these cell lines examined, the functional Brm gene is present and is actively transcribed; Brm expression was suppressed at t … More he post-transcriptional level. We hypothesized that Brm is targeted by certain miRNAs and screened several miRNA candidates and finally have shown that miR-199a target Brm mRNA. Interestingly, all the cancer cell lines deficient in Brm have high levels of miR-199a, whereas in Brm expressing cells, miR-199a was marginally expressed.We further show these distinct expression patterns are resulted from double-negativefeedback regulation formed between Brm and miR-199a-5p/-3p via a transcription factor Egr1. We additionally have shown that miR-21 and its target NFIB, a negative transcriptional regulator, also forms a robust feedback regulation in cancer cell lines.We have also shown human requiem protein functions as an adaptor protein that links SWI/SNF complex and RelB/p52. We further showed that introduction of shRNA against REQ strongly suppresses anchourage-independent growth, but does not affect growth in monolayer culture at all in tumor cell lines such as Panc-1, in which non-canonical NFκB pathway is constitutively activated. Less