miRNA profiling of sacral chordoma and nucleus pulposus cells

Next-generation RNA sequencing (NGS) was used for the combinatorial analysis of mRNA-miRNA gene expression profiles in sacral chordoma and nucleus pulposus samples, and to predict miRNA-mRNA regulatory networks with altered activity in chordoma. We identified a gene set including key regulators of the Hippo pathway, which is targeted by differently expressed miRNAs, and validated their altered expression by RT-qPCR. These newly identified miRNA/RNA interactions are predicted to have a role in the self-renewal process of chordoma stem cells, which might sustain the high rate of recurrence for this tumor. Overall design: 8 chordoma (CH) and 8 nucleus pulposus (NP) tissue samples were collected from surgically treated Caucasian patients. The chordoma samples were frozen in lN2 immediately after removal and stored at -80 ºC until processing. NP tissues were surgically removed from patients diagnosed with degenerative disc disease (Pfirrmann grade III), undergoing lumbar intervertebral fusion surgery. We established primary NP cell cultures from the NP tissue samples, applying the explantation method without any enzymatic digestions.本研究采用下一代RNA测序（Next-generation RNA sequencing, NGS）对骶骨脊索瘤与髓核样本中的mRNA-miRNA基因表达谱进行联合分析，并预测脊索瘤中活性异常的miRNA-mRNA调控网络。本研究鉴定出一套包含Hippo通路关键调控因子的基因集，该基因集受差异表达miRNA的靶向调控，并通过逆转录实时定量聚合酶链反应（Reverse Transcription Quantitative Polymerase Chain Reaction, RT-qPCR）验证了其表达异常。本研究预测，这些新鉴定出的miRNA/RNA互作关系参与了脊索瘤干细胞的自我更新过程，这或可解释该肿瘤的高复发率。实验整体设计：从接受手术治疗的高加索裔患者中采集了8例脊索瘤（CH）与8例髓核（NP）组织样本。脊索瘤样本在切除后立即置于液氮（lN2）中冷冻，并保存于-80 ℃直至实验处理。髓核组织均采集自确诊为退行性椎间盘疾病（Pfirrmann分级Ⅲ级）且接受腰椎椎间融合手术的患者。本研究采用无需酶消化的外植体培养法，从髓核组织样本中建立了原代髓核细胞培养体系。