RNA-Seq analysis of 4N and 2N RPE1 cells following polyploid induction via cytokinesis failure or Aurora kinase inhibition [tpo3]

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 either Aurora inhibitor (ZM447439), CytochalasinD, or untreated are FACS sorted into 2N or 4N populations and assessed for gene expression differences via RNA Seq for a total of 18 samples.

四倍体化（Tetraploidization），即基因组加倍（genome doubling），是肿瘤发生过程中的一类重要事件，其核心原因在于多倍体细胞的细胞分裂易出现错误，并会产生非整倍体细胞。本研究探究了急性与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：一是敲低胞质分裂必需调控因子Anillin，这会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，进而形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激活的共同特征。这些细胞的增殖抑制作用依赖于p53及其转录调控靶标——细胞周期蛋白依赖性激酶（Cdk）抑制剂p21。极少数增殖性四倍体细胞可从急性多倍体细胞群中产生。对单细胞来源的适应性四倍体细胞克隆的基因表达分析显示，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——的表达均出现上调。在二倍体细胞中过表达细胞周期蛋白D2，即便存在功能性p53，仍可显著增强细胞以更高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（Cyclin D2）这类致癌基因的表达水平，可规避p53介导的多倍体细胞增殖抑制作用，这阐明了四倍体化介导的基因组不稳定性在致癌过程中的一条潜在通路。实验整体设计：分别经极光激酶抑制剂（ZM447439）、细胞松弛素D（CytochalasinD）处理，或未做处理的细胞，各设置3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，再通过RNA测序（RNA-seq）检测基因表达差异，共计18个样本。