PO.PR02.01 · 预防研究
利用 Id1 作为宫颈癌质粒报告系统中的生物标志物
Harnessing Id1 as a Biomarker in a Plasmid Reporter System for Cervical Cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：尽管有 HPV 检测等筛查技术的进步，美国宫颈癌（CC）十年发病率仍保持稳定，提示需要新的 CC 筛查技术来应对停滞不前的发病率。我们提出了一种用于筛查的诊断性质粒，通过癌症特异性分化抑制因子 1（Id1）启动子序列的控制诱导报告酶（分泌型胚胎碱性磷酸酶，SEAP）的表达。所得质粒（pId1-SEAP）用于体外转染 CC 细胞，并根据 Id1 表达表征报告酶 SEAP 的产生。方法与结果：采用 Western Blot 和免疫组化确定 Id1 在细胞模型和人体组织中的表达。在多种条件下进行定时转染，以关联 Id1 和 SEAP 表达。与非癌 3T3 成纤维细胞（1.0±0.0）相比，CC 细胞系（HeLa 3.0±0.13，SiHa 2.9±0.27，CaSki 3.1±0.19，均 P<0.0001）表达的标准化基线 Id1 增加。在 CC 组织的微阵列中，正常样本的 Id1 平均染色值为 3E4±3E4，而早期和晚期癌症的 Id1 平均染色值增加（分别为 3E5±1E5 P<0.0001 和 2E5±1E5 P=0.0002）。与使用 pId1-SEAP 的 3T3 细胞（0.16±0.058）相比，使用 pId1-SEAP 的 HeLa 和 SiHa 细胞系产生的标准化 SEAP 量增加（0.63±0.25 和 0.50±0.10，P<0.05）。Id1 和 SEAP 表达强相关（Pearson r=0.99，P=0.012）。仅将 12,500 个暴露于 pId1-SEAP 质粒的 HeLa 细胞与未处理细胞共培养，即可使 SEAP 值增加（3E4±3E3 P=0.004），与背景水平（1E4±4E2）相比。结论与未来方向：pId1-SEAP 可转染 CC 细胞并按内源性 Id1 表达比例产生 SEAP，证明了其在未来 CC 诊断检测中的潜力。这些数据支持进一步研究以表征 pId1-SEAP 在三维组织模型和体内动物模型中的功能，从而开发一种利用 Id1 表达作为生物标志物、基于家庭的宫颈癌筛查方法。
查看英文原文 English abstract
Introduction: Ten-year cervical cancer (CC) incidence in the U.S. has remained stable despite screening advancements such as HPV testing, suggesting that new CC screening technologies are needed to address stagnant rates. We proposed a diagnostic plasmid for screening that induces expression of a reporter enzyme (secreted embryonic alkaline phosphatase, SEAP) through the control of the cancer-specific inhibitor of differentiation 1 (Id1) promoter sequence. The resulting plasmid (pId1-SEAP) was used to transfect CC cells in vitro and characterize reporter SEAP production based on Id1 expression. Methods and Results: Western Blot and immunohistochemistry were used to establish Id1 expression in cell models and human tissues. Timed transfections in various conditions were used to correlate Id1 and SEAP expression. CC cell lines (HeLa 3.0±0.13, SiHa 2.9±0.27, CaSki 3.1±0.19, all P<0.0001) expressed increased normalized baseline Id1 compared to non-cancer 3T3 fibroblasts (1.0±0.0). In a microarray of CC tissues, normal samples had mean Id1 staining value of 3E4±3E4, while early- and late-stage cancers had increased mean Id1 staining (3E5±1E5 P<0.0001 and 2E5±1E5 P=0.0002, respectively). HeLa and SiHa lines produced increased normalized SEAP amounts with pId1-SEAP (0.63±0.25 and 0.50±0.10, P<0.05) compared to 3T3 cells with pId1-SEAP (0.16±0.058). Id1 and SEAP expression were strongly correlated (Pearson's r=0.99, P=0.012). As few as 12,500 pId1-SEAP plasmid-exposed HeLa cells co-cultured with naïve cells resulted in an increased SEAP value (3E4±3E3 P=0.004) compared to background level (1E4±4E2). Conclusions and Future Directions: pId1-SEAP can transfect CC cells to produce SEAP proportionally to endogenous Id1 expression, demonstrating its potential for future diagnostic tests for CC. These data support further studies to characterize pId1-SEAP function in 3-D tissue models and in vivo animal models, towards the development of a home-based approach to screen for cervical cancer using Id1 expression as a biomarker.
利益披露 Disclosure
A. V. Eli, None..
B. B. Kasten, None..
Y. Hartman, None..
R. C. Arend, None..
J. M. Warram, None.