RNA-Seq analysis of 4N and 2N RPE1 cells following polyploid induction via cytokinesis failure by siRNA knockdown of Anillin [tpo8]. Homo sapiens

Tetraploidization, or genome doubling, is a prominent event in tumorigenesis, primarily because cell division in polyploid cells is error-prone and produces aneuploid cells. This study investigates changes in gene expression evoked in acute and adapted tetraploid cells and their impact on cell-cycle progression. Acute polyploidy was generated by knockdown of essential regulator of cytokinesis Anillin, which resulted in cytokinesis failure and formation of binucleate cells, or by chemical inhibition of Aurora kinases, causing abnormal mitotic exit with formation of single cells with aberrant nuclear morphology. Transcriptome analysis of these acute tetraploid cells revealed common signatures of activation of the tumor-suppressor protein p53. Suppression of proliferation in these cells was dependent on p53 and its transcriptional target - Cdk inhibitor p21. Rare proliferating tetraploid cells can emerge from acute polyploid populations. Gene expression analysis of single-cell derived, adapted tetraploid clones showed upregulation of several p53 target genes and cyclin D2, the activator of Cdk4/6/2. Overexpression of cyclin D2 in diploid cells strongly potentiated the ability to proliferate with increased DNA content despite the presence of functional p53. These results point out that p53-mediated suppression of proliferation of polyploid cells can be averted by increased levels of oncogenes such as Cyclin D2, elucidating a possible route for tetraploidy-mediated genomic instability in carcinogenesis. Overall design: Three biological replicates of cells treated with siRNA against Anillin or a non-targeting control are FACS sorted into 2N or 4N populations and assessed for gene expression differences via RNA Seq for a total of 12 samples.

四倍体化（tetraploidization）又称基因组加倍，是肿瘤发生过程中的关键事件，其核心原因在于多倍体细胞的细胞分裂极易出错，并会产生非整倍体细胞。本研究探讨了急性及适应性四倍体细胞中诱发的基因表达变化，及其对细胞周期进程的影响。

急性多倍体可通过两种方式构建：一是敲低胞质分裂的关键调控因子Anillin（Anillin），引发胞质分裂失败并形成双核细胞；二是通过化学抑制极光激酶（Aurora kinases），导致异常有丝分裂退出，进而生成核形态异常的单细胞。对这些急性四倍体细胞的转录组分析显示，其存在肿瘤抑制蛋白p53激活的共同分子特征。此类细胞的增殖抑制作用依赖于p53及其转录靶标——细胞周期蛋白依赖性激酶（Cdk）抑制蛋白p21。

少数增殖性四倍体细胞可从急性多倍体细胞群中出现。对单细胞衍生的适应性四倍体细胞克隆进行基因表达分析发现，多个p53靶基因以及细胞周期蛋白D2（cyclin D2，Cdk4/6/2的激活因子）的表达均显著上调。在二倍体细胞中过表达细胞周期蛋白D2，即便存在功能完整的p53，也能大幅增强其以更高DNA含量进行增殖的能力。

上述结果表明，通过提高细胞周期蛋白D2这类癌基因的表达水平，可规避p53介导的多倍体细胞增殖抑制，从而阐明了四倍体化介导癌变过程中基因组不稳定性的潜在途径。

整体实验设计：针对靶向Anillin的小干扰RNA（siRNA）及非靶向对照处理的细胞，各设置三次生物学重复，随后通过荧光激活细胞分选（FACS）将其分为2N或4N细胞群体，再通过RNA测序（RNA Seq）检测基因表达差异，总计包含12个样本。