miR-506通过BMP-4逆转结直肠癌干细胞干性研究

Colorectal cancer stem cells (CRC-SC) are closely associated with proliferation, metastasis and chemotherapy resistance of colorectal cancer (CRC), reversing CRC-SC stemness is an important way to treat CRC. Our previous research newly discovered that miR-506 function as stemness regulator, which can reverse CRC-SC stemness by BMP-4, and it was also found that miR-506 regulating BMP-4 was achieved by targeting EZH2. The project should be further studied on the basis of the above findings: Firstly, verifying that miR-506 function as stemness regulator in the CRC-SC. Secondly, verifying that miR-506 regulates CRC-SC stemness through BMP-4, and verifyies that miR-506 regulates BMP-4 through EZH2, and studing the mechanism ragarding EZH2 regulating BMP-4 from aspects of DNA and histone methylation. Thirdly, we constructed nude mice transplanted tumor model to verifying that miR-506 could reverse CRC-SC stemness in vivo and study its mechanism. Fourthly, the expression of related molecules in CRC tissue were detected and the correlation of these molecules were analyzed. This project is expected to explore the role and mechanism of miRNA-506 in CRC-SC stemness reversal and provide new ideas and strategies for the treatment of CRC.

结直肠癌干细胞（CRC-SC）与结直肠癌（CRC）增殖转移和化疗耐药密切相关，逆转CRC-SC干性是治疗CRC重要途径。本项目组前期研究新发现miR-506具有干性调控作用，能够通过BMP-4逆转CRC-SC干性，并发现miR-506对BMP-4调控是通过靶向EZH2实现。项目组在上述发现基础上拟深入研究：一是在CRC-SC中验证miR-506具有干性调控功能。二是验证miR-506通过BMP-4调控CRC-SC干性，同时验证miR-506通过EZH2调控BMP-4，并从DNA和组蛋白甲基化角度研究EZH2调控BMP-4机制。三是构建裸鼠移植瘤模型，验证miR-506在体内能够逆转CRC-SC干性及机制。四是检测CRC组织中相关分子表达水平，并进行相关性分析。本项目期望探明miRNA-506在CRC-SC干性逆转中的作用和机制，为治疗CRC提供新思路、新靶点。

背景：以往研究发现miR-506在结直肠癌中具有抑癌基因的特性。能够抑制CRC的增殖和转移。我们拟进一步研究miR-506对结直肠癌（CRC）干性的作用及其机制。.研究内容：采用成球实验，WB等检查miR-506对成球和干性标志分子表达的影响。采用流式检测明确miR-506对CRC中干细胞比例的影响。限制性稀释成瘤实验研究miR-506对CRC细胞皮下成瘤率的影响。流式细胞仪分选ALDH+的CRC细胞。qRT-PCR检查ALDH+和ALDH-细胞中miR-506和干性标志分子的水平。CCK-8及皮下成瘤体内实验研究miR-506对化疗敏感性作用。RNA-seq分析miR-506的下游基因。GSEA分析对差异表达基因进行富集分析。qR-PCR、WB、CCK-8实验检查miR-506是否通过BMP-4发挥调控作用。qR-PCR、WB、双荧光素酶报告基因检查miR-506能否靶向EZH2。MS-PCR、ChIP-qPCR、WB等检查EZH2能否通过DNA甲基化和H3K27me3调控BMP-4。IHC、WB检查化疗敏感和耐药组织中miR-506、BMP-4、干性标志分子表达水平，并分析相关性。.重要结果：体外实验证实miR-506能够抑制CRC细胞成球、干性标志分子表达，减少ALDH+细胞比例。体内实验证实过表达miR-506能够抑制CRC细胞体外成瘤能力。ALDH+细胞较ALDH+细胞具有更强的成球能力和干性标志分子表达水平，同时miR-506表达水平更低。过表达miR-506能够增强CRC对5-Fu和伊立替康的化疗敏感性。RNA-seq证实BMP-4是miR-506下游基因。GSEA分析发现miR-506下游差异基因富集于干性调控通路。在miR-506过表达细胞中沉默BMP4，CRC干性表型被逆转。EZH2是miRNA-506的下游靶基因。EZH2能负向调控BMP-4启动子甲基化水平。同时EZH2能调控BMP-4的启动子H3K27Me3水平，其中EZH2和BMP-4在启动子区的结合位点是3区和4区。临床标本检查发现，miR-506和BMP-4在耐药组织中表达进一步降低，其表达水平和干性标志分子的表达水平呈负相关。.科学意义：本研究为从非编码RNA角度阐释了CRC干性的调控机制，为CRC的诊断和治疗提供了重要靶点。