miR-29a参与胰岛素信号通路调控乳腺癌生长侵袭的机制研究

Type 2 diabetes and obesity may be resulted in hyperinsulinemia in patients. Insulin activation of PI-3K and Ras-MAPK pathway can promote the growth and progress of breast cancer. Studies have shown that miR-29a involved in the insulin signaling pathway of diabetes, and miR-29a were up-regulated in invasive breast cancer cells. Our preliminary cell experiments found, the expression of miR-29a in human breast cancer cells increased along with adding insulin cultured. This project aims to investigate the role of miR-29a in the process of insulin stimulating breast cancer growth and invasiveness and explore the regulatory mechanism of miR-29a involved in insulin signaling pathway, as well as the potential of miR-29a acting as a biological target to treat breast cancer. .We establish the cell model of insulin promoting breast cancer cell growth and proliferation. The expression of miR-29a was analyzed in insulin group and untreated group, which is detected by QRT-PCR and Northern- blot methods. We observe the changes of cell proliferation and invasion after adding miR-29a inhibitor in insulin group and mimics miR-29a in untreated group. This experiment wants to prove the important role of miR-29a in the process of insulin promoting breast cancer cell growth and proliferation.. After establishment of breast cancer metastasis animal models, including MKR+/+ mouse and the same species of non-MKR+/+ mouse, we will interfere in miR-29a targets by injecting miR-29a inhibitor from tail vein for MKR+/+ mouse and mimics miR-29a for non-MKR+/+ mouse . Then the changes of tumor formation rate and distant metastasis rate will be observed in mouse model. The purpose of this part is to illustrate the potential vivo treatment of miR-29a in breast cancer.. We analyze the activation and the key protein expression of PI-3K pathway and Ras-MAPK pathway by RT-PCR and western blot methods, and also by DNA microarry technology ,under the conditions of insulin culture and miR-29a interference. The results of these experiments may show the upstream promoter gene and the downstream target gene of miR-29a, which can ensure the position of miR-29a in the insulin signaling pathway. . In addition, the expression of miR-29a and the key protein expression of PI-3K pathway and Ras-MAPK pathway was detected in 100 cases of breast cancer patients with type 2 diabetes mellitus and 100 cases without type 2 diabetes mellitus. The results will further prove the clinical value of miR-29a in breast cancer.

2型糖尿病和高胰岛素血症会促进乳腺癌发生、进展，其可能机制是激活PI-3K和Ras-MAPK两条信号途径。研究显示，miR-29a参与糖尿病的胰岛素信号通路，且miR-29a在侵袭性乳腺癌细胞表达上调。我们的前期细胞实验发现，加入胰岛素培养乳腺癌MDA-MB231细胞，miR-29a的表达上调。.本项目通过建立胰岛素促进乳腺癌细胞生长的细胞模型和高胰岛素血症的MKR+/+小鼠乳腺癌转移模型，以及临床检测合并糖尿病乳腺癌与非糖尿病乳腺癌患者miR-29a的表达，明确miR-29a在胰岛素促进乳腺癌生长和侵袭的重要作用，并通过设计针对miR-29a靶点的细胞和动物体内干预实验，检测PI-3K途径和Ras-MAPK 途径的激活及表达情况，判断miR-29a的上游启动基因和下游靶向基因，发现miR-29a参与胰岛素信号通路的调控机制，以及miR-29a作为生物靶点治疗乳腺癌的潜力。

糖尿病和肿瘤是近30多年来发病率上升最快的两种疾病，越来越多的证据表明糖尿病、高胰岛素血症和乳腺癌存在复杂关系，而它们复杂关系背后的分子机制也是近年来的研究热点。miR-29a是miR-29家族的重要成员，在2型糖尿病患者血清和高胰岛素高葡萄糖条件下培养3T3-L1脂肪细胞中均表达上调，在乳腺癌患者血清和组织中miR-29a表达也上调，并可通过抑制TTP的表达来调控乳腺癌细胞的EMT和转移。本项目主要开展了三个方面的研究内容：（1）首先，我们通过建立细胞模型及临床标本检测发现，相对普通培养的乳腺癌细胞，在胰岛素培养的乳腺癌细胞中miR-29a表达分别上调了1.325±0.132和1.275±0.113倍，相对糖耐量正常的乳腺癌患者，合并糖尿病和糖耐量异常的患者miRNA-29a表达上调，初步探明了miR-29a在胰岛素促进乳腺癌生长和侵袭的重要作用。（2）为了探索miR-29a参与胰岛素信号通路调控乳腺癌生长侵袭的分子机制，我们进行了针对miR-29a的干扰实验，并分析其对胰岛素信号通路关键分子的影响。我们的研究发现，p-ERK、CDC42、P85α均为miR-29a的靶基因，但miR-29a主要通过ERK磷酸化活化促进细胞生长增殖，IGF-1R可能是miR-29a上游靶基因。Transwell侵袭实验发现，胰岛素和mimics miR-29a能够促进MCF-7细胞的侵袭，而inhibitor miR-29a则能够下调胰岛素所致的MCF-7细胞侵袭能力增加。（3）miR-29a治疗合并糖尿病的乳腺癌的生物靶点作用。我们在高糖、高胰岛素培养下MCF-7 细胞转染anti-miR-29a病毒载体后，细胞的增殖活力和侵袭能力下降；采用STZ小剂量腹腔注射法在雌性裸鼠建立糖尿病乳腺癌的小鼠模型，糖尿病小鼠接种anti-miR-29a病毒载体转染的MCF-7 细胞，移植瘤成瘤能力下降，抑瘤率达47.13％。通过以上研究，我们初步探讨miR-29a参与胰岛素信号通路调控乳腺癌生长侵袭的分子机制，明确miR-29a在胰岛素促进乳腺癌生长和侵袭的重要作用；同时通过细胞和动物体内实验，初步揭示糖尿病和高胰岛素血症促进乳腺癌发生、发展以及由此导致乳腺癌患者预后差的内在原因，探索了miR-29a作为乳腺癌生物治疗靶点应用前景，为合并糖尿病和高胰岛素血症的乳腺癌患者治疗寻找一个新的突破。