Willenbrock Saskia, Wagner Siegfried, Reimann-Berg Nicola, Moulay Mohammed, Hewicker-Trautwein Marion, Nolte Ingo, Murua Escobar Hugo
Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany.
Small Animal Clinic, University of Veterinary Medicine Hannover, Hannover, Germany; Institute of Biophysics, Leibniz University Hannover, Hannover, Germany.
PLoS One. 2014 Jun 10;9(6):e98788. doi: 10.1371/journal.pone.0098788. eCollection 2014.
The architectural transcription factor HMGA2 is abundantly expressed during embryonic development. In several malignant neoplasias including prostate cancer, high re-expression of HMGA2 is correlated with malignancy and poor prognosis. The let-7 miRNA family is described to regulate HMGA2 negatively. The balance of let-7 and HMGA2 is discussed to play a major role in tumour aetiology. To further analyse the role of HMGA2 in prostate cancer a stable and highly reproducible in vitro model system is precondition. Herein we established a canine CT1258-EGFP-HMGA2 prostate cancer cell line stably overexpressing HMGA2 linked to EGFP and in addition the reference cell line CT1258-EGFP expressing solely EGFP to exclude EGFP-induced effects. Both recombinant cell lines were characterised by fluorescence microscopy, flow cytometry and immunocytochemistry. The proliferative effect of ectopically overexpressed HMGA2 was determined via BrdU assays. Comparative karyotyping of the derived and the initial CT1258 cell lines was performed to analyse chromosome consistency. The impact of the ectopic HMGA2 expression on its regulator let-7a was analysed by quantitative real-time PCR. Fluorescence microscopy and immunocytochemistry detected successful expression of the EGFP-HMGA2 fusion protein exclusively accumulating in the nucleus. Gene expression analyses confirmed HMGA2 overexpression in CT1258-EGFP-HMGA2 in comparison to CT1258-EGFP and native cells. Significantly higher let-7a expression levels were found in CT1258-EGFP-HMGA2 and CT1258-EGFP. The BrdU assays detected an increased proliferation of CT1258-HMGA2-EGFP cells compared to CT1258-EGFP and native CT1258. The cytogenetic analyses of CT1258-EGFP and CT1258-EGFP-HMGA2 resulted in a comparable hyperdiploid karyotype as described for native CT1258 cells. To further investigate the impact of recombinant overexpressed HMGA2 on CT1258 cells, other selected targets described to underlie HMGA2 regulation were screened in addition. The new fluorescent CT1258-EGFP-HMGA2 cell line is a stable tool enabling in vitro and in vivo analyses of the HMGA2-mediated effects on cells and the development and pathogenesis of prostate cancer.
结构转录因子HMGA2在胚胎发育过程中大量表达。在包括前列腺癌在内的几种恶性肿瘤中，HMGA2的高重新表达与恶性程度和不良预后相关。据描述，let-7 miRNA家族对HMGA2起负调控作用。let-7和HMGA2的平衡被认为在肿瘤病因学中起主要作用。为了进一步分析HMGA2在前列腺癌中的作用，一个稳定且高度可重复的体外模型系统是前提条件。在此，我们建立了一种犬类CT1258-EGFP-HMGA2前列腺癌细胞系，该细胞系稳定过表达与EGFP相连的HMGA2，此外还建立了仅表达EGFP的参照细胞系CT1258-EGFP，以排除EGFP诱导的效应。两种重组细胞系均通过荧光显微镜、流式细胞术和免疫细胞化学进行表征。通过BrdU检测法确定异位过表达的HMGA2的增殖效应。对衍生的CT1258细胞系和初始的CT1258细胞系进行比较核型分析，以分析染色体一致性。通过定量实时PCR分析异位HMGA2表达对其调节因子let-7a的影响。荧光显微镜和免疫细胞化学检测到EGFP-HMGA2融合蛋白成功表达，且仅在细胞核中积累。基因表达分析证实，与CT1258-EGFP和天然细胞相比，CT1258-EGFP-HMGA2中HMGA2过表达。在CT1258-EGFP-HMGA2和CT1258-EGFP中发现let-7a表达水平显著更高。BrdU检测法检测到与CT1258-EGFP和天然CT1258相比，CT1258-HMGA2-EGFP细胞的增殖增加。CT1258-EGFP和CT1258-EGFP-HMGA2的细胞遗传学分析结果显示，其核型为超二倍体，与天然CT1258细胞的核型相似。为了进一步研究重组过表达的HMGA2对CT1258细胞的影响，还筛选了其他被描述为受HMGA2调控的选定靶点。新的荧光CT1258-EGFP-HMGA2细胞系是一个稳定的工具，可用于体外和体内分析HMGA2对细胞的影响以及前列腺癌的发生发展和发病机制。
