MicroRNA expression signatures of CD14-CD45-EpCAM+ cells in human bone marrow for colorectal cancer liver metastasis

To further development of our gene expression approach to biodosimetry, we have employed microRNA microarray expression profiling to identify genes with the potential to distinguish liver metastasis related microRNA. Colorectal cancer patients were administered anesthesia and 20 mL BM was taken from the right and left anterior iliac crests before surgery. Mononucleated cells were collected using a standard Ficoll-Hypaque gradient technique. To enrich for EpCAM+ cells, CD14+ cells were removed from the whole bone marrow using auto MACSTM pro (Milteny Biotec, Bergisch Gladbach, Germany) with anti-CD14 immunomagnetic beads (clone; TÜK4, Milteny Biotec). Next, CD45+ cells were removed by treatment with anti-CD45 immunomagnetic beads (clone; 5B1; Milteny Biotec). The residual CD14−CD45− cells were then incubated with FcR blocking reagent (Milteny Biotec), followed by incubation with anti-EpCAM immunomagnetic beads (clone; HEA-125, Milteny Biotec), and the CD14−CD45−EpCAM+ cells were taken up. Total RNA of these cells we analyzed the microRNA levels of CD14−CD45−EpCAM+ cells obtained from non-metastasis patients (n = 12) and liver metastasis patients (n = 7). Ten-microRNA consensus signature was identified that distinguished between CD14−CD45−EpCAM+ cells from liver metastasis patients and CD14−CD45−EpCAM+ cells from non-liver metastasis patients. MicroRNA expression of CD14-CD45-EpCAM+ cells in human bone marrow was measured. RNA of these cells we analyzed the microRNA levels of CD14−CD45−EpCAM+ cells obtained from non-metastasis patients (n = 12) and liver metastasis patients (n = 7).

为推进我们用于生物剂量测定的基因表达方法的发展，我们采用微小RNA（microRNA）微阵列表达谱分析，以筛选出具备区分与肝转移相关的微小RNA潜力的基因。结直肠癌患者接受麻醉后，于术前从左右髂前嵴采集20 mL骨髓（bone marrow, BM）。采用标准菲科-泛影葡胺（Ficoll-Hypaque）梯度离心法分离单核细胞。为富集上皮细胞黏附分子阳性（EpCAM+）细胞，我们使用全自动MACS™分选仪（auto MACS™ pro，德国美天旎生物技术公司）搭配抗CD14免疫磁珠（克隆号：TÜK4，德国美天旎生物技术公司）去除全骨髓中的CD14阳性（CD14+）细胞；随后使用抗CD45免疫磁珠（克隆号：5B1，德国美天旎生物技术公司）去除CD45阳性（CD45+）细胞。将残留的CD14阴性、CD45阴性细胞与Fc受体封闭试剂（德国美天旎生物技术公司）孵育后，再加入抗EpCAM免疫磁珠（克隆号：HEA-125，德国美天旎生物技术公司）进行孵育，最终获取CD14⁻CD45⁻EpCAM+细胞。我们对取自无转移患者（n=12）与肝转移患者（n=7）的CD14⁻CD45⁻EpCAM+细胞的总RNA进行分析，检测其微小RNA表达水平。最终筛选得到可区分肝转移患者与无转移患者来源的CD14⁻CD45⁻EpCAM+细胞的10种微小RNA共识特征标记。本研究同时检测了人骨髓中CD14⁻CD45⁻EpCAM+细胞的微小RNA表达水平。我们再次对取自无转移患者（n=12）与肝转移患者（n=7）的CD14⁻CD45⁻EpCAM+细胞的总RNA进行微小RNA水平分析。