MicroRNA-125a/b靶向Ras/MAPK信号通路在2型糖尿病与乳腺癌关联中的作用

Type 2 diabetes tends to be closely associated with the pathogenesis and the prognosis of breast cancer, while the mechanism still remains unknown. It is documented in recent studies that miRNA-125a/b and Ras/MAPK singal pathway is correlated with breast cancer. In our previous study, higher pathological grade, more poor effect of chemotherapy and lower survival rate were found in patients suffering from breast cancer complicated with type 2 diabetes. Meanwhile in breast cancer tissues, the expression of IR(insulin receptors) and ERK elevates, yet miRNA-125a/b was down-regulated , and MAPK might be targeted by miRNA-125 a/b. Therefore, we propose that miRNA-125a/b targeted regulating Ras/MAPK singal pathway may play an important role in relationship between type 2 diabetes and breast cancer. Thus, the project is to collect breast cancer tissues, establish breast cancer model of mouse complicated with type 2 diabetes and culture MCF-7 breast cancer cells; detect the expression level of gene and protein of miR-125a/b、Ras/MAPK signal pathway by means of Realtime-PCR and Western-blot, and analyze the further relation between type 2 diabetes and breast cancer via up-regulating or down-regulating the gene expression of miRNA-125a/b. We hope to provide a new idea for prevention and treatment of breast cancer complicated with type 2 diabetes.

2型糖尿病与乳腺癌的发生及预后密切相关，但机制尚不明确。miRNA-125a/b及Ras/MAPK信号通路与乳腺癌相关已在研究中证实。申请人前期研究发现合并2型糖尿病的乳腺癌患者病理分级高、化疗疗效差、生存率低，乳腺癌组织Ras/MAPK通路ERK及IR表达水平升高，miRNA-125a/b表达下调，并发现MAPK可能是miRNA-125a/b的靶基因。因此提出假说：miRNA-125a/b靶向调控Ras/MAPK信号通路可能在2型糖尿病与乳腺癌联系中起重要作用？为验证假说，我们通过乳腺癌病理标本、糖尿病合并乳腺癌小鼠模型及MCF-7细胞，采用Realtime-PCR、Western-blot等方法检测miRNA-125a/b、Ras/MAPK通路基因及蛋白表达水平，并通过上调及下调miRNA-125a/b基因表达，分析糖尿病与乳腺癌之间深层联系，为2型糖尿病合并乳腺癌的防治提供新的思路。

2 型糖尿病与乳腺癌的发生及预后密切相关，但机制尚不明确。microRNAs是一类小非编码RNA,在细胞的增值、凋亡、分化的过程中发挥非常重要的作用。本课题组前期在乳腺癌细胞系MCF-7细胞中发现利用高糖高胰岛素条件培养，可以通过上调IRS-1表达并激活Ras/Raf/MAPK通路从而增强乳腺癌细胞的增值、侵袭能力。利用之前收集的乳腺癌标本及癌旁组织，使用microRNA芯片进行差异化分析，筛查了出了几个表达存在明显差异的miRNAs. 进一步进行表达差异验证，发现miR-125b在乳腺癌组织及细胞系中存在明显的低表达。通过双荧光素酶报告基因验证发现miR-125b靶向作用于RAF-1 3’-UTRs。在乳腺癌中过表达miR-125b 明显抑制乳腺癌细胞系的增值、克隆形成及侵袭能力。并且在动物实验中可抑制移植肿瘤的生长。在乳腺癌细胞中过表达miR-125b可以减少RAF-1的表达和 ERK1/2的活性。另外，在乳腺癌组织和细胞系中我们发现miR-125b靶基因RAF-1的表达水平增高。设计siRNA 干扰RAF-1的表达后可以抑制肿瘤的生长、克隆形成、侵袭能力。提示miR-125靶向调控RAF-1在乳腺癌中发挥重要作用。进一步，我们发现高胰岛素（100nM）对待乳腺癌细胞系MCF-7细胞可以减低miR-125b的表达，且过表达miR-125b可以减轻胰岛素对乳腺癌细胞的效应。提示miR-125b可能在2型糖尿病合并乳腺癌的关联作用中发挥重要的作用。