Identification of deregulated pathways, key regulators, and novel miRNA-mRNA interactions in HPV-mediated transformation. [CGH cell lines-Agilent]. Identification of deregulated pathways, key regulators, and novel miRNA-mRNA interactions in HPV-mediated transformation. [CGH cell lines-Agilent]

Next to a persistent infection with high-risk human papillomavirus (HPV), molecular changes are required for the development of cervical cancer. To identify which molecular alterations drive carcinogenesis, we performed a comprehensive and longitudinal molecular characterization of HPV-transformed keratinocyte cell lines. Comparative genomic hybridization, mRNA, and miRNA expression analysis of four HPV-containing keratinocyte cell lines at eight different time points was performed. Data was analyzed using unsupervised hierarchical clustering, integrated longitudinal expression analysis, and pathway enrichment analysis. Biological relevance of identified key regulatory genes was evaluated in vitro and dual-luciferase assays were used to confirm predicted miRNA-mRNA interactions. We show that the acquisition of anchorage independence of HPV-containing keratinocyte cell lines is particularly associated with copy number alterations. Approximately one third of differentially expressed mRNAs and miRNAs was directly attributable to copy number alterations. Focal adhesion, TGF-beta signaling, and mTOR signaling pathways were enriched among these genes. PITX2 was identified as key regulator of TGF-beta signaling and inhibited cell growth in vitro, most likely by inducing cell cycle arrest and apoptosis. Predicted miRNA-mRNA interactions miR-221-3p_BRWD3, miR-221-3p_FOS, and miR-138-5p_PLXNB2 were confirmed in vitro. Integrated longitudinal analysis of our HPV-induced carcinogenesis model pinpointed relevant interconnected molecular changes and crucial signaling pathways in HPV-mediated transformation. Overall design: Chromosomal profiles of 8 sequential passages of 4 HPV-transformed human foreskin primary keratinocyte cell lines were analyzed using comparative genomic hybridization (CGH) microarrays (SurePrint G3 human CGH microarray 4x180K; Agilent).

除高危型人乳头瘤病毒（high-risk human papillomavirus, HPV）持续感染外，宫颈癌的发生还需要分子层面的改变。为明确驱动癌变进程的分子变异类型，我们对HPV转化的角质形成细胞系开展了全面的纵向分子特征分析。我们针对4株携带HPV的角质形成细胞系，在8个不同时间点分别开展了比较基因组杂交（comparative genomic hybridization, CGH）、mRNA及miRNA表达谱分析。数据分析采用无监督层级聚类、整合纵向表达分析及通路富集分析方法完成。我们通过体外实验评估了所鉴定关键调控基因的生物学相关性，并借助双荧光素酶实验验证了预测的miRNA-mRNA互作关系。研究结果显示，携带HPV的角质形成细胞系获得锚定非依赖性生长的特性，与拷贝数变异的关联尤为显著。约三分之一的差异表达mRNA与miRNA，其表达变化可直接归因于拷贝数变异。上述基因显著富集于黏着斑、转化生长因子-β（transforming growth factor-beta, TGF-β）信号通路及哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）信号通路。我们鉴定出PITX2作为TGF-β信号通路的关键调控因子，其可通过诱导细胞周期阻滞与细胞凋亡在体外抑制细胞增殖。此外，体外实验成功验证了miR-221-3p_BRWD3、miR-221-3p_FOS及miR-138-5p_PLXNB2这三组预测的miRNA-mRNA互作关系。基于本研究构建的HPV诱导癌变模型的整合纵向分析，明确了HPV介导细胞转化过程中相关的互作分子改变与关键信号通路。总体实验设计：我们采用比较基因组杂交微阵列（SurePrint G3人CGH微阵列4x180K；安捷伦（Agilent）），对4株HPV转化的人包皮原代角质形成细胞系的8个连续传代样本的染色体谱进行分析。