肝转移相关microRNA miR-199b通过SIRT1/KISS1调节结直肠癌细胞侵袭转移的机制研究

肝转移是影响结直肠癌预后的重要因素。前期通过miRNA微阵列芯技术、生物信息学预测等筛选鉴定出miR-199b为结直肠癌肝转移的相关miRNA，并可能通过SIRT1/KISS1通路调节结肠癌细胞转移，但其分子机制尚不清楚。本课题拟用免疫组化、原位杂交法探讨结直肠癌组织及肝转移灶中miR-199b及靶基因SIRT1表达与临床病理指标和预后的关系；用活细胞成像侵袭实验、划痕实验等研究miR-199b通过调节SIRT1对结肠癌细胞侵袭迁移能力的影响；通过双荧光素酶、凝胶迁移电泳（EMSA)、定点突变、免疫共沉淀、乙酰化及磷酸化水平鉴定等探索miR-199b通过SIRT1调节KISS1表达影响结直肠癌侵袭转移的分子机制；最后用裸鼠转移实验在动物体内对miR-199b调节结肠癌细胞侵袭转移的机制进行验证。miR-199b/SIRT1/KISS1/信号通路的研究有可能为结直肠癌肝转移治疗提供新靶点。

Liver metastasis is one of the most important factors affecting the prognosis of colorectal cancer. Our preliminary data showed that not only miR-199 b is the tissue-specific microRNA of liver metastasis from colorectal cancer, but also miR-199b might regulate the liver metastasis through SIRT1/KISS1 signal passway. However, its the mechanism is unclear. In this study, in situ hybridization and immunohistochemical techniques were used to study the expression of miR-199 b and SIRT1 in colorecgtal cancer tissues and metastatic tissues as well as the relationship between their expressions and the clinical pathological parameters; invasion assay under liver cell imaging system and wound healing assay were used to detect the invasion and shift of colon cancer cells regulated by miR-199b; dual luciferase report system,electrophoretic mobility shift assay and site-directed mutagenesis, co-immunoprecipitation and assay for acetylization and phosphorylation were used to study the the mechanism of miR-199b regualting the invasion and metastasis through SIRT1/KISS1 pathway; carcinoma oncogeneis model and metastasis assay in nude mice were used to verify the mechanism of miR-199b affecting the invasion and metastasis through miR-199b. The research of miR-199b/SIRT1/KISS1 pathway might contribute the new target to the treatmentof liver metastais from colorecgtal cancer.