Time resolved gene expression analysis during tamoxifen adaption of MCF-7 cells identifies long non-coding RNAs with prognostic impact

Acquired tamoxifen resistance is a persistent problem for the treatment of estrogen receptor positive, premenopausal breast cancer patients and predictive biomarkers are still elusive. We here analyzed gene expression changes in a cellular model to identify early and late changes upon tamoxifen exposure and thereby novel prognostic biomarkers. Estrogen receptor positive MCF-7 cells were incubated with 4OH-tamoxifen (10 nM) and gene expression analyzed by array hybridization during 12 weeks. Array results were confirmed by nCounter- and qRT-PCR technique. Pathway enrichment analysis revealed that early responses concerned mainly amine synthesis and NRF2-related signaling and evolved into a stable gene expression pattern within 4 weeks characterized by changes in glucuronidation-, estrogen metabolism-, nuclear receptor- and interferon signaling pathways. As a large number of long non coding RNAs was subject to regulation, we investigated 5 of these (linc01213, linc00632 linc0992, LOC101929547 and XR_133213) in more detail. From these, only linc01213 was upregulated but all were less abundant in estrogen-receptor negative cell lines (MDA-MB 231, SKBR-3 and UACC3199). In a web-based survival analysis linc01213 and linc00632 turned out to have prognostic impact. Linc01213 was investigated further by plasmid-mediated over-expression as well as siRNA down-regulation in MCF-7 cells. Nevertheless, this had no effect on proliferation or expression of tamoxifen regulated genes, but migration was increased. In conclusion, the cellular model identified a set of lincRNAs with prognostic relevance for breast cancer. One of these, linc01213 although regulated by 4OH-tamoxifen, is not a central regulator of tamoxifen adaption, but interferes with the regulation of migration.

获得性他莫昔芬耐药是雌激素受体阳性绝经前乳腺癌患者治疗中的持续性难题，目前仍缺乏明确的预测生物标志物。本研究通过细胞模型分析他莫昔芬暴露后的基因表达变化，以期筛选出早期及晚期的异常表达特征，进而发现全新的预后生物标志物。将雌激素受体阳性MCF-7细胞置于4OH-他莫昔芬（4OH-tamoxifen，10 nM）中培养，于12周内通过芯片杂交（array hybridization）技术分析基因表达变化，并采用nCounter和qRT-PCR技术对芯片结果进行验证。通路富集分析（Pathway enrichment analysis）显示，早期应答主要涉及胺类合成及NRF2相关信号通路，并在4周内演变为稳定的基因表达模式，其特征为葡萄糖醛酸化、雌激素代谢、核受体及干扰素信号通路的改变。鉴于存在大量受调控的长链非编码RNA（long non-coding RNA, lncRNA），本研究对其中5种（linc01213、linc00632、linc0992、LOC101929547及XR_133213）进行了深入分析。结果显示，仅linc01213表达上调，且上述5种lncRNA在雌激素受体阴性细胞系（MDA-MB 231、SKBR-3及UACC3199）中的丰度均较低。基于网络的生存分析结果显示，linc01213与linc00632具有预后价值。进一步通过质粒介导的过表达及siRNA干扰下调MCF-7细胞中linc01213的表达，结果显示其对细胞增殖及他莫昔芬调控基因的表达无显著影响，但可增强细胞迁移能力。综上，本细胞模型筛选出了一批与乳腺癌预后相关的长链非编码RNA。其中，linc01213虽受4OH-他莫昔芬调控，但并非他莫昔芬适应性的核心调控因子，而是参与迁移过程的调控。