LINC00261 Suppresses Epithelial-Mesenchymal Transition and Metastatic Potential of Colorectal Cancer by Down-regulating the MeCP2-ZEB1 Feedback Loop

Accumulating evidence suggests that epithelial-mesenchymal transition (EMT) contributes to metastasis, a major cause of death in patients with colorectal cancer (CRC). MeCP2 (methyl CpG binding protein 2), a key modulator for initiating gene transcription, plays a pivotal role in EMT, but its upstream signaling pathways are poorly understood. We collected 137 colorectal cancer patients’ samples, including tumor tissues (n=137), adjacent normal tissues (n=137) and liver metastases (n=11). Three pairs of colorectal cancer cell lines were sequenced to identify high abundance of lincRNAs in primary tumor tissues. Two independent experiments (electrophoretic mobility shift assay and RNA immunoprecipitation assay) were used to characterize the interaction between LINC00261 and MeCP2. Subsequently, the effects of LINC00261 on phosphorylation of MeCP2 and promotion of the MeCP2-ZEB1 feedback were examined. Consequently long intergenic non-protein coding RNA 261 (LINC00261) was down-regulated in highly metastatic CRC cells and liver metastases from patients with CRC. Down-regulating LINC00261 promoted the expression of EMT markers and increased the invasive and migratory properties of multiple CRC cell lines. Furthermore, knocking down LINC00261 increased colon-derived liver metastasis in an orthotopic CRC model and promoted distant metastatic colonization in a lung metastasis model. Mechanistic studies revealed that LINC00261 interacted with MeCP2 and stabilized it in the nucleus by inhibiting Ser80 phosphorylation Interestingly, ZEB1 could re-modulate MeCP2 expression by binding to the MeCP2 promoter, and they formed a positive feedback loop consequently. In contrast, the lower LINC00261 expression in CRC samples correlated with reduced E-cadherin and membrane β-catenin levels, and was associated with shorter overall survival. Taken together, LINC00261 suppresses EMT and CRC cells’ metastatic potential by negatively regulating the MeCP2-ZEB1 feedback loop; LINC00261’s loss in metastatic tissues may be a potential prognostic marker of aggressive disease. wild type colorectal cancer cells comparing with knockdown colorectal cancer cells

越来越多的证据表明，上皮间质转化（epithelial-mesenchymal transition, EMT）与转移密切相关，而转移是结直肠癌（colorectal cancer, CRC）患者死亡的主要原因。甲基CpG结合蛋白2（methyl CpG binding protein 2, MeCP2）作为启动基因转录的关键调控因子，在EMT中发挥关键作用，但目前对其上游信号通路的了解仍十分有限。本研究收集了137例结直肠癌患者的样本，包括肿瘤组织（n=137）、癌旁正常组织（n=137）以及肝转移灶（n=11）。通过对3对结直肠癌细胞系进行测序，以筛选在原代肿瘤组织中高丰度表达的基因间长链非编码RNA（long intergenic non-protein coding RNA, lincRNA）。采用两项独立实验——电泳迁移率变动分析（electrophoretic mobility shift assay）与RNA免疫沉淀实验（RNA immunoprecipitation assay），验证了LINC00261与MeCP2之间的相互作用。随后，本研究探究了LINC00261对MeCP2磷酸化的调控作用，以及其对MeCP2-ZEB1反馈环路的促进效应。研究结果显示，基因间长链非编码RNA261（long intergenic non-protein coding RNA 261, LINC00261）在高转移性结直肠癌细胞以及结直肠癌患者的肝转移灶中表达下调。下调LINC00261的表达可促进上皮间质转化标志物的表达，并增强多种结直肠癌细胞系的侵袭与迁移能力。此外，在结直肠癌原位模型中敲低LINC00261可增加结肠来源的肝转移灶形成，而在肺转移模型中则可促进远端转移定植。机制研究揭示，LINC00261可与MeCP2相互结合，并通过抑制丝氨酸80（Ser80）位点的磷酸化，在细胞核内稳定该蛋白。有趣的是，ZEB1可通过结合MeCP2启动子区域重新调控其表达，二者由此形成正向反馈环路。与之相反，结直肠癌样本中低水平的LINC00261表达与E-钙粘蛋白（E-cadherin）及膜β-连环蛋白的表达降低相关，且与患者更短的总生存期密切相关。综上，LINC00261可通过负向调控MeCP2-ZEB1正向反馈环路，抑制上皮间质转化及结直肠癌细胞的转移潜能；LINC00261在转移组织中的缺失或许可作为侵袭性结直肠癌的潜在预后标志物。野生型结直肠癌细胞与经LINC00261敲低的结直肠癌细胞