HMGA2通过调控miR-205促进结肠癌细胞侵袭转移的作用及机制研究

As a transcriptional factor, HMGA2 plays a very important role in invasion and metastasis of colon cancer. We previously found that overexpression of HMGA2 could promote cell invasion and cause dyregulation of EMT markers in colon cancer. Computer analysis revealed that there were multiple HMGA2 AT binding domains in the promoter of miR-205, and significant reduction in expression and luciferase activity of miR-205 were noted when HMGA2 was overexpressed. Morever, we also found that there was a binding site of miR-205 which was highly conservative existing in the 3'UTR of SMAD4. Based on these findings, we want to explore the function and mechanism of HMGA2 in the regulation of invasion and metastasis via miR-205 in colon cancer. Firstly, a series of models in vitro and in vivo will be set up to explore the role of HMGA2 in colon cancer. Secondly, the direct binding between HMGA2 protein and the promoter of miR-205 will be explored. A lot of technologies will be used, such as ChIP, EMSA, luciferase analysis, point mutation assay, et al. Thirdly, the regulatory mechanism between miR-205 and SMAD4 is to be elucidated. In conclusion, this project will provide theoretic evidence for the novel diagnosis and therapy targeted at HMGA2-miR-205-SMAD4 signaling pathway in colon cancer.

HMGA2是与结肠癌侵袭转移密切相关的重要转录因子。我们前期研究发现高表达HMGA2能使结肠癌细胞侵袭转移能力显著增强，并影响EMT相关分子表达；软件分析表明肿瘤转移关键分子miR-205启动子区域存在多个HMGA2结合位点，luciferase assay和realtime PCR证实HMGA2能使miR-205的转录活性和表达水平显著降低；我们还发现在SMAD4的3'UTR区域存在高度保守的miR-205结合位点。本项目拟在此基础上，通过构建一系列体内外模型进一步阐明HMGA2能够促进结肠癌细胞的侵袭转移；采用ChIP、EMSA、定点突变、蛋白截短实验等方法证明HMGA2能与miR-205启动子区域直接结合并下调其转录，明确两者的结合位点；揭示miR-205参与调控下游SMAD4的分子机制。本课题将为临床以HMGA2-miR-205-SMAD4为基础进行靶向治疗提供实验基础和理论依据。

结直肠癌是严重危害人类生命健康的最常见的恶性肿瘤之一，其发病率在全球约排第3位，中国居第4位。结直肠癌的发病机制仍不完全清楚，它的发生和发展是一个复杂的病理渐变过程，涉及到多个原癌基因的激活和抑癌基因的丢失。HMGA2 (High Mobility Group A2) 作为高迁移率族蛋白A家族中的一员，它的三个AT-hook功能结构域能够与DNA结构中富含A和T的序列结合，继而引起DNA结构的改变，从而调控（促进或者抑制）下游靶基因的转录。首先，我们通过一系列体内和体外实验发现，HMGA2能够促进结肠癌细胞的侵袭和远处转移。其次，我们发现HMGA2通过与miR-205（-2000/+1）、FN1（+175/+196）和IL11（-2147/-2132）的启动子区域直接结合而参与其转录调控过程，IL11通过pSTAT3介导的信号通路而促进结直肠癌细胞的上皮间质转化（EMT）过程。另外，HMGA2还参与Wnt/β-catenin通路，我们发现，HMGA2蛋白能够与WNT1（+79/+482）和Dvl2（-264/+217）的启动子区域直接结合，从而促进结直肠癌细胞对5-FU耐药性的增加和诱导肿瘤细胞干性的发生。除对结直肠癌进行研究之外，我们还对HMGA2在乳腺癌中的作用及分子机制进行了研究。在大样本临床组织样本中，我们发现，HMGA2的表达与乳腺癌的病理分级和预后密切相关。HMGA2表达越高，患者预后越差，尤其在病理分级为II-III级、分化程度越低以及在三阴乳腺癌患者中尤为明显。另外，我们通过来源于公共大数据库的基因富集分析（GSEA）发现，HMGA2的高表达与肿瘤的metaplastic和mesenchymal表型密切相关。在分子机制方面，我们发现HMGA2通过促进p53蛋白Ser15位点的磷酸化而增加乳腺癌细胞对doxorubicin的耐药性。综上所述，本项目揭示了HMGA2在肿瘤发生发展过程中的关键分子机制，为HMGA2在临床上的功能和作用方面提供了巨大的证据支持，为HMGA2将来应用于临床预防、诊断和治疗提供了必要的基础，为结肠癌的发生和发展机制提供新的视角。