气郁质外泌体miR-6749-5p促进乳腺癌细胞生长转移的分子机制研究

Breast cancer is the most common malignant tumor in women. The Traditional Chinese Medicine theory of “physique is relating to disease” provides a new thought and approach on the prevention and treatment of breast cancer. Our previous studies have found that qi-stagnation constitution in the biased constitution of TCM exhibits the highest risk in breast cancer-prone, but its mechanism remains unclear. Preliminary experimental results have demonstrated that the serum of qi-stagnation people and its exosomes can significantly accelerate the growth and migration of breast cancer cells. Furthermore the similar effects were also shown in those cancer cells intervened by miR-6749-5p which is up-regulated expression in in serum exosome of qi-stagnation people.Hereby, we speculate that exosome miR-6749-5p may promote cell proliferation exceedingly by targeting genes and relative signal pathways, thus mediating breast cancer morbidity by constitution of qi-stagnation. In this project, experiments in vivo and vitro are used to explore the effect mechanism of serum exosome miR-6749-5p of qi-stagnation constitution in the development of breast cancer by techniques and methods, such as gene transfection, RNA interference, Dual Luciferase Reporter, etc. Firstly, we will verify the effect of serum exosome of qi-stagnation constitution in the growth and metastasis of breast cancer cells; Secondly, to make sure whether the promotion role of serum exosome of qi-stagnation constitution in the occurrence and development of breast cancer is mediated by miR-6749-5p; Finally, to confirm the target genes of miR-6749-5p and its key signal pathways, then illuminate the molecular mechanism of qi-stagnation constitution in breast cancer-prone.

乳腺癌是女性发病率最高的恶性肿瘤，中医“体病相关”理论为乳腺癌防治提供了新的思路和途径。我们前期研究发现气郁质罹患乳腺癌的危险度最高，但是其机制尚不清楚。预实验显示：气郁质血浆及其外泌体均可明显促进乳腺癌细胞的生长，而且气郁质外泌体中miR-6749-5p亦能够显著诱导乳腺癌细胞的生长和迁移。据此，推测外泌体miR-6749-5p可能通过靶基因及其信号通路，导致细胞过度增殖，从而介导气郁质促进乳腺癌发病。本项目拟采用体外结合体内实验，围绕气郁质外泌体miR-6749-5p展开研究，应用基因转染、RNA干扰、利用双荧光素酶报告基因等技术和方法，首先验证气郁质血浆外泌体对乳腺癌细胞生长转移的影响，其次明确气郁质血浆外泌体是否介导miR-6749-5p促进乳腺癌的发生发展，最后确定miR-6749-5p的靶基因及其关键信号通路，阐明气郁质促进乳腺癌发病的分子机制，对于乳腺癌的防治具有重要意义。

乳腺癌作为最常见的肿瘤，严重威胁女性的生命健康。不仅是我国医疗卫生的巨大负担，同时也是全世界公共卫生事业的巨大挑战，因此制定有效的疾病筛查策略，针对不同风险人群进行特异性筛查成为防治乳腺癌的发展方向。本课题通过Meta分析汇总了乳腺癌患者中医体质的分布情况，发现气郁质是乳腺癌患者的常见体质类型，气郁体质增加了罹患乳腺癌的风险，为乳腺癌高危人群的筛选提供了重要线索，而且调理气郁质可能是防治乳腺癌的重要途径。利用乳腺癌细胞MDA-MB-231、MCF-7，给予气郁质外泌体及其miR-6749-5p处理，动态观测细胞增殖活力、细胞周期、细胞迁移和侵袭能力等指标变化，明确气郁质血浆外泌体通过介导miR-6749-5p促进体外培养乳腺癌细胞的生长、迁移和侵袭能力；进一步利用裸鼠异位移植瘤和斑马鱼体内实验验证气郁质外泌体及miR-6749-5p促进乳腺癌细胞体内增殖迁移。最后通过靶基因预测网站预测结合双荧光素酶报告基因验证筛选miR-6749-5p靶基因，明确miR-6749-5p调控的关键靶基因及其关键信号通路，为中医“体病相关”理论提供了依据。