尿路上皮细胞癌(UCC)是美国第二常见的泌尿生殖系统恶性疾病,吸烟是UCC发展的主要已知危险因素。接触致癌物,例如烟草烟雾中含有的那些,已知会直接或间接损伤DNA,导致突变,UCC中的染色体缺失事件和表观遗传改变。分子研究表明,9号染色体的改变和P53,RAS,RB和PTEN突变是UCC中最常见的事件之一。最近的研究表明,持续的烟草致癌物暴露驱动并增强了UCC中表观遗传改变细胞的选择,主要是侵入性的疾病。然而,暴露于烟草烟雾后导致UCC的分子事件序列尚不清楚.为了阐明烟草暴露后导致UCC肿瘤发生和进展的分子事件,我们建立了吸烟诱导的UCC的体外细胞模型。将SV-40永生化的正常HUC1人膀胱上皮细胞连续暴露于0.1%香烟烟雾提取物(CSE),直到发生转化。用CSE治疗4个月(mo)后,观察到非恶性尿路上皮细胞的形态改变和细胞增殖增加。通过软琼脂测定评估非锚定生长,在CSE处理6个月后,在尿路上皮细胞中观察到迁移和侵袭潜力的增加。通过进行PI3K-AKT途径特异性的PCRmRNA表达阵列,我们发现HUC1细胞CSE暴露6个月后,26个基因上调,22个基因下调。在改变的基因中,PTEN,FOXO1,MAPK1和PDK1在转化细胞中下调,而AKT1,AKT2,HRAS,RAC1上调。然后通过RT-PCR和蛋白质印迹分析进行验证。此外,全基因组甲基化分析显示MCAM,与未暴露于CSE的细胞相比,DCC和HIC1在CSE处理的尿路上皮细胞中高度甲基化。使用定量甲基化特异性PCR(QMSP)验证了这些基因的甲基化状态,证实与未处理的对照相比,CSE处理的尿路上皮细胞的甲基化增加。因此,我们的研究结果表明,烟草特征可能来自独特的遗传和表观遗传改变模式,并且可以使用吸烟诱导癌症发展的体外细胞模型进行鉴定.
Urothelial cell carcinoma (UCC) is the second most common genitourinary malignant disease in the USA, and tobacco smoking is the major known risk factor for UCC development. Exposure to carcinogens, such as those contained in tobacco smoke, is known to directly or indirectly damage DNA, causing mutations, chromosomal deletion events and epigenetic alterations in UCC. Molecular studies have shown that chromosome 9 alterations and P53, RAS, RB and PTEN mutations are among the most frequent events in UCC. Recent studies suggested that continuous tobacco carcinogen exposure drives and enhances the selection of epigenetically altered cells in UCC, predominantly in the invasive form of the disease. However, the sequence of molecular events that leads to UCC after exposure to tobacco smoke is not well understood. To elucidate molecular events that lead to UCC oncogenesis and progression after tobacco exposure, we developed an in vitro cellular model for smoking-induced UCC. SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. Morphological alterations and increased cell proliferation of non-malignant urothelial cells were observed after 4 months (mo) of treatment with CSE. Anchorage-independent growth assessed by soft agar assay and increase in the migratory and invasive potential was observed in urothelial cells after 6 mo of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found that 26 genes were upregulated and 22 genes were downregulated after 6 mo of CSE exposure of HUC1 cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were downregulated in the transformed cells, while AKT1, AKT2, HRAS, RAC1 were upregulated. Validation by RT-PCR and western blot analysis was then performed. Furthermore, genome-wide methylation analysis revealed MCAM, DCC and HIC1 are hypermethylated in CSE-treated urothelial cells when compared with non-CSE exposed cells. The methylation status of these genes was validated using quantitative methylation-specific PCR (QMSP), confirming an increase in methylation of CSE-treated urothelial cells compared to untreated controls. Therefore, our findings suggest that a tobacco signature could emerge from distinctive patterns of genetic and epigenetic alterations and can be identified using an in vitro cellular model for the development of smoking-induced cancer.