miR-143-3p和miR-195-5p低表达在结直肠癌肝转移中的作用与调控机制研究

We propose this idea that CRC cells and TAMs jointly promote CRC liver metastases by a different miRNA-target gene pathway based on our previous finding that the expression level of both miR-143-3p and miR-195-5p was reduced more in liver metastatic CRC than that in primary CRC and our recent discovery that re-expression of miR-143-3p and miR-195-5p reversed the invasion ability of CRC cells and liver metastasis potential of CRC through inhibiting the expression of target gene(ASAPS and ITGA6)in CRC cells and inhibiting the expression of target gene( FGF2 and MYB)in TAMs respectively, which was derived from our newly established technology of miRNA re-expression（Oncogene,2013）and liver targeting nanotechnology（Biomaterials,2012).In this proposal, the impact of reduced expression of miRNAs on CRC liver metastasis by target gene proteins will be subjected to further analysis at molecular and tissue levels in combination with in vivo animal experimentations and clinical data analysis. These studies will yield further insight into the mechanism of how reduced expression of miR-143-3p and miR-195-3p would promote the CRC liver metastasis, leading to a new target on which more efficient methods helping prevent hepatic metastasis of CRC can be built.

在课题组前期发现miR-143-3p 和miR-195-5p的表达水平在肝转移结直肠癌（CRC）中较原位CRC中显著下降，及近期运用已建立的miRNA再表达（Oncogene,2013）和肝靶向纳米（Biomaterials, 2012）技术发现miR-143-3p 和miR-195-5p的再表达能分别通过抑制CRC细胞中靶基因蛋白（ASAPS和ITGA6）和抑制肿瘤相关巨噬细胞（TAMs）中靶基因蛋白（FGF2和MYB）抑制肿瘤细胞的侵袭和肝转移瘤的形成，提出CRC细胞和TAMs通过不同miRNA-靶基因途径协同促进CRC肝转移的新构想。本项目拟通过分子生物学、组织水平、动物体内实验和临床资料分析，进一步研究并揭示低表达miRNAs如何借助相关靶基因影响CRC肝转移进程，旨在阐明miR-143-3p和miR-195-3p低表达促进CRC肝转移的分子机制，有望为CRC肝转移防治提供新靶点

【目的】旨在阐明结直肠癌（CRC）借助miRNA及相关靶基因途径影响侵袭和转移进程的分子机制，为CRC肝转移的临床诊断、治疗和预后评估提供新的生物学标志物和靶点。.【方法】1、microRNA芯片检测CRC肝转移相关表达缺失的miRNA并用实时荧光定量PCR进一步验证。2、检测过表达miR-143-3p对CRC增殖能力和迁移能力的影响。3. LoVo细胞株提取培养基上清液，用佛波酯和LoVo上清液一起刺激THP-1细胞转化为M2型巨噬细胞，ELISA和流式细胞术测定特定的细胞因子表达；提取M2巨噬细胞中miR-195-5p用RT-PCR和免疫印迹证实miR-195下调。4. 运用生物信息学技术和网络资源预测miR-143-3p和miR-195-5p的候选靶基因，并通过Western Blot法检测转染miR-模拟物的CRC细胞中各候选靶基因蛋白水平的表达。5、Real-time PCR、Western Blot法分别检测siRNA对Lovo细胞中靶基因（ITGA6和ASAP3）mRNA和蛋白表达水平的抑制效率。6、应用Transwell和MTT检测分别低表达靶基因蛋白对CRC细胞增殖和迁移能力的影响。7、运用组织芯片+免疫组化技术检测各靶基因蛋白在CRC组织和正常结直肠组织中的表达差异并分析与患者的临床病理特征的相关性。.【结果】1、miR-143-3p和miR-195-5p在肝转移CRC中的表达下降为59倍和16倍。2、过表达miR-143-3p能够抑制Lovo细胞的侵袭和迁移能力；miR-195-5p 表达上调的TAM减弱了LoVo的侵袭能力。3、生物信息学技术和网络资源预测出4个miR-143-3p的候选靶基因：ASAP3、MSI2、CRELD和ITGA6，进一步验证出最佳靶基因为ITGA6 和ASAP3，降低ITGA6 和ASAP3的表达能够抑制Lovo细胞的侵袭和迁移能力；筛选出miR-195-5p的3个主要的靶基因分别为CCNE1,ASH-1L和MAGE； western-blot及免疫组化验证得出MAGE在肿瘤中表达量显著下降。4、高表达的ITGA6与患者的TNM分期密切相关(P<0.05)，高表达的ASAP3与患者TNM分期及淋巴结转移密切相关(P<0.05)。.【结论】1、miR-143-3p和miR-195-5p在肝转移CRC中表达水平显著下降,