Takeuchi Masao, Higashino Atsunori, Takeuchi Kikuko, Hori Yutaro, Koshiba-Takeuchi Kazuko, Makino Hatsune, Monobe Yoko, Kishida Marina, Adachi Jun, Takeuchi Jun, Tomonaga Takeshi, Umezawa Akihiro, Kameoka Yosuke, Akagi Ken-Ichi
Section of Laboratory Equipment, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki-shi, Osaka, Japan.
Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, Kyoto, Japan.
PLoS One. 2015 May 15;10(5):e0126562. doi: 10.1371/journal.pone.0126562. eCollection 2015.
Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN); these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma) and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5), which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1), the cell surface receptor for hedgehog (Hh) signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells) through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional contribution of genes showed by expression profiling to the neoplastic transformation of human fibroblasts and human mesenchymal stem cells (hMSC).
对人类细胞中基因表达变化以及肿瘤转化的综合分析，为了解转化背后的分子机制提供了有价值的信息。为了进一步解决这些问题，我们对用人端粒酶逆转录酶（hTERT）和人乳头瘤病毒16型E6/E7基因永生化的人间充质干细胞系UE6E7T - 3进行了全转录组分析，并将其与这些细胞的转化进程相关联。在培养早期，如先前所述，UE6E7T - 3细胞优先丢失一条13号染色体；此外，肿瘤抑制基因、DNA修复基因和凋亡激活基因均有过表达。在13号染色体丢失后，观察到了驱动渐进性转化的额外非整倍体和基因改变。在此阶段，该细胞系表达癌基因以及与抗凋亡功能、细胞周期进程和染色体不稳定性（CIN）相关的基因；这些促肿瘤发生的变化伴随着肿瘤抑制基因表达的降低。在延长培养后的后期阶段，细胞表现出染色体易位，在裸鼠中获得了不依赖贴壁生长和致瘤性（肉瘤），并表现出编码生长因子和DNA修复基因的表达增加，以及黏附基因的表达降低。特别地，编码一种可能是肉瘤生物标志物的细胞表面蛋白聚糖的磷脂酰肌醇蛋白聚糖-5（GPC5），与转化相关且高表达。patched（Ptc1），即刺猬信号通路（Hh）的细胞表面受体，也显著过表达并与GPC5共定位。敲低GPC5表达可降低细胞增殖，表明它通过Hh信号通路在U3 - DT细胞（源自UE6E7T - 3细胞的转化体）的生长中起关键作用。因此，UE6E7T - 3细胞培养模型是评估通过表达谱显示的基因对人成纤维细胞和人间充质干细胞（hMSC）肿瘤转化的功能贡献的有用工具。
