神经内分泌分化的结肠癌细胞源性外泌体中Lnc-SNRNP35诱导TAMs促进结直肠癌进展的机制研究

Neuroendocrine differentiation (NED) promotes colorectal cancer progression. Previously, our results showed that NED colon cancer cells could recruit tumor associated macrophagocytes(TAMs) through secreting chemokines CXCL11 and CXCL10 acting on the receptor CXCR3. however，this recruit mechanism was poorly known. Further，we found that exosomes derived from NED colon cancer cells could promote the migration of TAMs. Then we found differently expressed LncRNA through RNA sequencing, and Lnc-SNRNP35 in the exosomes was up-regulated. Further experiments confirmed that when Lnc-SNRNP35 was over-expressed in the TAMs, it could up-regulate the CXCR3 levels in TAMs. And then we found that Lnc-SNRNP35 and CXCR3 3 'UTR region exists miR-3908 binding sites at the same time by bio-information analysis. Thus, we supposed that Lnc-SNRNP35 in exosomes derived from NED colon cancer cells acting as ceRNA(competitive RNA) could increase CXCR3 level in TAMs through binding with miR-3908，and promote the infiltration of TAMs. This study would shed light on the roles of NED in colorectal cancer which was urgently needed in clinic.

神经内分泌样分化(NED)可促进结直肠癌的发生发展。我们已证实NED的结肠癌细胞可以通过分泌CXCL10和CXCL11作用于CXCR3受体招募肿瘤相关性巨噬细胞(TAMs)从而促进结直肠癌的进展，然而此招募机制尚不清楚。预实验发现NED结肠癌细胞来源的外泌体明显能够增强TAMs的迁移，将此外泌体进行LncRNA测序后发现Lnc-SNRNP35在NED结肠癌细胞来源的外泌体中表达明显上调，并且可上调TAMs中CXCR3的表达，生物信息分析表明Lnc-SNRNP35和CXCR3的3’UTR区域同时存在miR-3908结合位点。由此我们提出NED结肠癌细胞来源的外泌体通过Lnc-SNRNP35可作为ceRNA（竞争性RNA）结合miR-3908促进TAMs表面CXCR3的表达，从而诱导TAMs浸润的新机制。本研究有望阐明NED促进结直肠癌进展的作用机制，为结直肠癌的诊治提供新靶点。

神经内分泌分化（Neuroendocrine differentiation，NED）是结直肠癌预后不良的一个重要影响因素，但目前对于NED促进结直肠癌进展的作用机制仍未完全阐明。我们的前期研究成果表明，NED的结肠癌细胞能通过分泌趋化因子CXCL10和CXCL11，增加肿瘤组织中肿瘤相关性巨噬细胞（TAMs）的迁移聚集，从而促进结直肠癌的发生、发展，且大量文献证实CXCL10和CXCL11是CXCR3的选择性配体，其构成的内分泌轴可作用于癌组织及癌旁细胞。同时，预实验结果表明NED的结肠癌细胞来源性外泌体（Exosome）能够显著增加TAMs的迁移浸润能力。于是我们通过构建NED的结肠癌细胞株及其对照细胞株，分离、提纯、鉴定其分泌的外泌体后，将提取的外泌体与TAMs体外共培养，通过Transwell实验再次证实了NED结肠癌细胞来源的外泌体能增强TAMs趋化能力，同时通过Western-blot检测到共培养后的TAMs中CXCR3受体的表达上调；再者，通过RNA高通量测序和生物信息学分析对比NED与non-NED结肠癌细胞来源性外泌体时，筛选到若干LncRNA在NED的结肠癌细胞源性外泌体中表达上调，经过在细胞株中的q-PCR验证后发现仅Lnc-HOXB8-1:2在NED的结肠癌细胞源性外泌体中明显高表达；同时，基于基因库预测，在分析可能与Lnc-HOXB8-1:2相互作用的下游miRNA时发现Lnc-HOXB8-1:2上具有hsa-miR-6825-5p潜在结合位点，而且CXCR3上同样也存在hsa-miR-6825-5p的靶点；在TAMs中过表达Lnc-HOXB8-1:2后其CXCR3受体的表达增加，而hsa-miR-6825-5p的表达则下降。上述实验结果表明，神经内分泌分化的结肠癌细胞所分泌的外泌体，其携带的Lnc-HOXB8-1:2可作为ceRNA竞争性结合hsa-miR-6825-5p调控CXCR3受体的表达，进而协同趋化因子CXCL10和CXCL11促进TAMs向肿瘤组织大量迁移浸润，从而推动了结直肠癌的进展。本项目帮助我们首次从外泌体角度分析了神经内分泌分化促进结直肠癌进展的相关作用机制，从而为探索结直肠癌的临床诊疗新靶点提供了一定的理论依据。