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，它们携带用于表达短发夹（sh）RNA、miRNA和新开发的抑制特异性miRNA的RNA诱饵分子（TuD RNA）的有效转录单位。利用这些载体系统，我们已经研究了癌症表观遗传学，主要集中在SWI/SNF染色质重塑复合物上，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载体的表达被迅速地随机沉默。我们最终证明了Brm型SWI/SNF复合物对于逆转录病毒/慢病毒的稳定表达是必需的。Brm的外源性引入降低了Brm表达缺陷的细胞系的致癌潜力，从而进一步证明了Brm具有抗癌潜力。在所有检测的这些细胞系中，功能性Brm基因都存在，并且转录活跃; ...更多信息 转录后水平。我们假设Brm被某些miRNA靶向，并筛选了几种miRNA候选物，最终证明miR-199 a靶向Brm mRNA。有趣的是，所有缺失Brm的肿瘤细胞株都有高水平的miR-199 a表达，而在表达Brm的肿瘤细胞株中，miR-199 a的表达水平很低，这是由于miR-199 a-5 p/-3p与Brm之间通过转录因子Egr 1形成的双负反馈调节所致。此外，我们还发现miR-21及其靶点NFIB（一种负转录调节因子）在癌细胞系中也形成了强有力的反馈调节，我们还发现人类Requiem蛋白作为衔接蛋白连接SWI/SNF复合物和RelB/p52。我们进一步表明，针对p53的shRNA的引入强烈抑制了非依赖于锚定的生长，但在单层培养的肿瘤细胞系如Panc-1中根本不影响生长，其中非经典NFκB途径被组成性激活。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