Expression data from SIX1 knocked-down HKc/DR cells

The homeodomain transcription factor SIX1 plays a critical role in embryogenesis, is not expressed in normal adult tissue, but is expressed in many malignancies, including cervical cancer. SIX1 drives the progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward malignancy: HKc/HPV16 express high levels of SIX1 mRNA and protein; overexpression of SIX1 in HKc/HPV16 produces pre-malignant, differentiation-resistant lines (HKc/DR); SIX1 overexpression in HKc/DR induces tumorigenicity. In this paper, we explore the consequences of inhibition of SIX1 expression in premalignant HKc/DR. Only partial inhibition of SIX1 expression could be obtained in HKc/DR by RNA interference. Decreased SIX1 expression (up to 80%) in HKc/DR resulted in slower proliferation, decreased HPV16-E6/E7 mRNA levels, and increased p53 protein levels. Gene expression changes induced in HKc/DR by anti-SIX1 shRNA were indicative of mesenchymal-epithelial transition (EMT) and changes in TGF-beta signaling. We conclude that HPV16-transformed cells depend on SIX1 for survival, continuous HPV16 E6/E7 gene expression and EMT. Three-condition, one-color experiment. HKc/DR cells were transfected with control or one of two different SIX1-targeting siRNA duplexes and gene expression was performed on RNA isolated from them. 4 biological replicates per each experimental group.

同源域转录因子SIX1（homeodomain transcription factor SIX1）在胚胎发生中发挥关键调控作用，在正常成人组织中不表达，但在包括宫颈癌（cervical cancer）在内的多种恶性肿瘤中均有表达。SIX1可促进HPV16永生化人角质形成细胞（HPV16-immortalized human keratinocytes，HKc/HPV16）向恶性表型进展：HKc/HPV16本身可高水平表达SIX1 mRNA与蛋白；在HKc/HPV16中过表达SIX1可构建癌前、分化抵抗的细胞系（HKc/DR）；而在HKc/DR中过表达SIX1则可诱导细胞产生致瘤性。本研究探讨了在癌前HKc/DR细胞中抑制SIX1表达的生物学效应。通过RNA干扰（RNA interference）仅能实现HKc/DR细胞中SIX1的部分表达抑制。当HKc/DR细胞中SIX1表达下调幅度最高达80%时，可观察到细胞增殖速率减慢、HPV16-E6/E7 mRNA水平降低，以及p53蛋白水平升高。抗SIX1短发夹RNA（short hairpin RNA, shRNA）诱导HKc/DR细胞产生的基因表达变化，提示存在间质-上皮转化（mesenchymal-epithelial transition, EMT）以及转化生长因子β（TGF-β）信号通路的改变。本研究得出结论：HPV16转化的细胞依赖SIX1以维持存活、持续表达HPV16 E6/E7基因以及发生间质-上皮转化。本实验为三条件单通道基因表达实验：将HKc/DR细胞转染阴性对照或两种不同的靶向SIX1的小干扰RNA（small interfering RNA, siRNA）双链体，随后从提取的细胞总RNA中开展基因表达谱分析；每个实验组设置4次生物学重复。