Host transcriptome changes associated with episome loss and selection of keratinocytes containing integrated HPV16

Integration of high-risk human papillomavirus (HRHPV) into the host genome is a key event in cervical neoplastic progression. Integration is associated with deregulated expression of the viral oncogenes E6 and E7 and acquisition of a selective growth advantage for cells containing integrants. Overexpression of the viral transcriptional regulator E2 from heterologous promoters has an inhibitory effect on transcription from integrated HRHPV. We therefore hypothesised that loss of E2-expressing episomes from cells in which integration had previously occurred would be required for such cells to gain a growth advantage. Using the unique W12 model of cervical squamous carcinogenesis, we show that cells containing integrated HPV16 reproducibly emerged during long-term culture when there had been a rapid fall in episome numbers. During the period of emergence it is possible to isolate single-cell clones containing an intracellular mixture of the integrant being selected and episomes at reduced load. Microarray analysis showed that episome loss was closely associated with endogenous activation of antiviral response genes that are also inducible by the type I interferon (IFN) pathway. Taken together, our results indicate that episome loss, associated with induction of antiviral response genes, is a key event in the spontaneous selection of cervical keratinocytes containing integrated HPV16. We conclude that cervical carcinogenesis requires not only HRHPV integration, but also loss of inhibitory episomes. Keywords: Time course, human papillomavirus, episome loss The overall aim was to determine host gene expression changes associated with episome loss in W12 cells undergoing selection for integrated HPV16. We therefore compared gene expression in W12 populations representing different stages in the process of integrant selection. We compared W12p10EPI (episome-only) with clones W12.MRP20 (undergoing selection for the integrant and episome loss) and W12.MRP29 (containing only the selected integrant), which are henceforth referred to as W12.MRP20MIX and W12.MRP29INT. These three conditions were treated as a time-course. Preparation and hybridisation of probes for microarray analysis: Total RNA from W12 samples was used to generate biotin-labelled cRNA for microarray analysis. Two technical replicates were performed for each sample to control for variation in labelling and hybridisation efficiency. Double-stranded cDNA was synthesised using SuperScript (Invitrogen), employing the (dT)24-T7 promoter primer. Biotin-labelled cRNA was then generated by Bioarray in vitro transcription (Enzo), and fragmented by metal-induced hydrolysis. Probe from each replicate was hybridised, washed, stained and scanned by the Medical Research Council Geneservice (Cambridge, UK) using standard Affymetrix procedures. We used GeneChip HG-U133 Plus 2.0 Arrays (Affymetrix). MAS 5.0 transformation of raw data was performed and further statistical analysis carried out using GeneSpring (Agilent Technologies). Real-time RT-PCR of cDNA was performed to validate changes in expression of selected host genes.

高危型人乳头瘤病毒（high-risk human papillomavirus, HRHPV）整合进入宿主基因组是宫颈肿瘤发生进展中的关键事件。整合与病毒癌基因E6和E7的表达失调，以及携带整合体的细胞获得选择性生长优势相关。通过异源启动子过表达病毒转录调控因子E2，可对整合的HRHPV的转录产生抑制作用。因此我们提出假说：在先前已发生病毒整合的细胞中，丢失表达E2的游离型基因组（episome），是此类细胞获得生长优势的必要条件。

本研究采用独特的宫颈鳞状细胞癌发生模型W12开展实验，结果显示，当游离型基因组数量快速下降时，携带整合型HPV16的细胞可在长期培养过程中稳定出现。在该细胞出现的阶段，可分离得到同时包含待筛选整合体和低载量游离型基因组的单细胞克隆。

微阵列分析结果表明，游离型基因组的丢失与I型干扰素（type I interferon, IFN）通路诱导的抗病毒应答基因的内源性激活密切相关。综合来看，本研究结果显示，与抗病毒应答基因诱导相关的游离型基因组丢失，是自发筛选获得携带整合型HPV16的宫颈角质形成细胞的关键事件。我们得出结论：宫颈癌变不仅需要HRHPV整合，还需要丢失具有抑制作用的游离型基因组。

关键词：时间进程（Time course）、人乳头瘤病毒（human papillomavirus）、游离型基因组丢失（episome loss）

本研究的总体目标是明确在W12细胞中，与游离型基因组丢失相关的宿主基因表达变化，该过程伴随着针对整合型HPV16的筛选。因此，我们比较了代表整合体筛选过程不同阶段的W12细胞群体的基因表达水平。我们将仅含游离型基因组的W12p10EPI，与分别处于整合体筛选及游离型基因组丢失阶段的克隆W12.MRP20（后续简称W12.MRP20MIX）、仅携带筛选后整合体的克隆W12.MRP29（后续简称W12.MRP29INT）进行比较。将这三种实验条件视为一个时间进程。

微阵列分析探针的制备与杂交：使用W12样本的总RNA合成生物素标记的cRNA，用于微阵列分析。每个样本设置两次技术重复，以控制标记与杂交效率的差异。采用SuperScript逆转录酶（Invitrogen公司），以(dT)24-T7启动子引物合成双链cDNA。随后通过Bioarray体外转录试剂盒（Enzo公司）生成生物素标记的cRNA，并通过金属诱导水解进行片段化。将每个重复的探针交由英国剑桥医学研究委员会基因服务中心（Medical Research Council Geneservice）按照标准Affymetrix操作流程进行杂交、洗涤、染色与扫描。本研究使用GeneChip HG-U133 Plus 2.0芯片（Affymetrix公司）。对原始数据进行MAS 5.0标准化转换，并采用GeneSpring软件（安捷伦科技公司）开展后续统计分析。通过实时定量逆转录PCR（real-time RT-PCR）验证所选宿主基因的表达变化。