毛蕊异黄酮通过干预miRNA-375/ERα正反馈环路抗ERα(+)乳腺癌作用的研究

Breast cancer is the first most common cancer in women worldwide, and as the second leading cause of cancer death in women. Two thirds of breast cancers share the common feature of being dependent on the presence and interaction of estrogen with the nuclear estrogen receptor α (ERα) protein. Previous studies have demonstrated that ERα expressed in endothelial cells mediates angiogenesis through both classic genomic, and rapid non-genomic, mechanisms. MicroRNAs (miRNA) are endogenous small noncoding RNAs of 19 to 25 nucleotides, which are involved in post-transcriptional control of gene expression. Some study revealed that miR-375 was overexpressed specifically in ERα-positive breast cancer cells. Pedro de Souza Rocha Simonini identified miR-375 as the first miRNA with the capacity to enhance ERα signaling in breast cells. Their findings define a forward feedback pathway in control of ERα expression and miRNA target prediction identified RASD1 as a potential miR-375 target. Calycosin is a main active component of the herb Radix Astragali, and is considered as a phytoestrogen. Our previous studies suggested that low concentrations of calycosin(<16 μM) had stimulatory effects on the proliferation of ERα-positive cells in vitro and in vivo, and found that the stimulatory effects might related to the level of ERα and the activation of extracellular signal-regulated kinase1/2(ERK1/2) MAPK. However, we found calycosin at high concentrations(>25 μM) exhibited inhibitory effects on the proliferation of MCF-7 cells. It was suggested that apoptosis induced by calycosin on human breast cancer cells was related to ERα-Ras-p38MAPK pathway. Furthermore, our previous studies confirmed that calycosin downregulated the level of miR-375 and upregulated the level of RASD1 protein. Based on the above information, we hypothesize that the positive feedback loop of miRNA-375/ERα and RASD1 may also be involved in the antiproliferative effect induced by calycosin in ERα-positive cells. By using cell counting, MTT assay, flow cytometry, luciferase activity assay, Western blot, qPCR, Xenograft tumor growth, immunohistochemistry, we will identify an upregulated or downregulated miRNA-375 in ERα-positive breast cancer cells that was able to interfere with ERα-MAPK, Akt signaling pathways through the regulation of its target, RASD1 in vitro and in vivo. Furthermore, we focus on the effect of calycosin on the positive feedback loop of miRNA-375/ERα and level of RASD1. Accordingly, further research should be carried out to elucidate the relationship among calycosin, RASD1, MAPK/Akt signaling pathways and the positive feedback loop of miRNA-375/ERα, which may provide experimental foundation for calycosin's future clinical use for ERα-positive breast cancer.

乳腺癌是女性最常见的恶性肿瘤，ERα(+)乳腺癌在临床上占2/3。miRNA是单链短序列小RNA，对下游的靶基因发挥调控作用，有研究提出miR-375与ERα构成正反馈环路，下调RASD1表达水平，促进乳腺癌细胞增殖，但上下游通路研究及体内实验均未进行。课题组研究表明不同浓度毛蕊异黄酮分别通过ERα-ERK1/2 MAPK及ERα-Ras-p38 MAPK通路影响细胞增殖或凋亡。预实验结果显示高浓度毛蕊异黄酮可下调MCF-7细胞miRNA-375水平，上调RASD1蛋白表达。据此，我们推测毛蕊异黄酮通过干预miR-375/ERα的正反馈环路，影响RASD1表达来发挥作用。本项目拟通过细胞生物学、分子生物学、动物模型等技术，观察miR-375、ERα、RASD1在体内外的信号级联，重点论证毛蕊异黄酮对正反馈环路的作用及MAPK/Akt通路蛋白磷酸化的影响，为ERα阳性乳腺癌的防治提供新思路。

乳腺癌是女性最常见的恶性肿瘤，并且在导致女性死亡的癌症中排第二位。ERα (+)乳腺癌在临床上占2/3。miRNA 是单链短序列小RNA，对下游的靶基因发挥调控作用。有研究提出miR-375 与ERα 构成正反馈环路，下调RASD1表达水平。课题组研究表明不同浓度毛蕊异黄酮分别通过ERα -ERK1/2 MAPK及ERα -Ras-p38 MAPK通路影响细胞增殖或凋亡。本项目通过细胞生物学、分子生物学、动物模型等技术，观察miR-375、ERα 、RASD1、IGF-1R及PARP-1在体内外的信号级联，重点论证毛蕊异黄酮对正反馈环路的作用及对MAPK/Akt通路蛋白磷酸化的影响。本课题实验结果显示毛蕊异黄酮可下调乳腺癌细胞miRNA-375 水平，上调RASD1 蛋白表达，影响MAPK、Akt、IGF-1R及PARP-1信号通路蛋白磷酸化水平，从而达到抑制乳腺癌细胞增殖，诱导凋亡的作用，而在体内实验中药物也能抑制荷瘤裸鼠肿瘤生长，其机制与体外实验相同。另一方面，异黄酮类植物雌激素对ERβ阳性人结直肠癌细胞及前列腺癌的增殖也具有抑制作用，并能够诱导细胞凋亡，药物的作用机制与ERβ/miR-17，ERβ/MiR-95及IGF-1R, PI3K/Akt信号通路有关。本项目的实验数据可为众多的异黄酮类植物雌激素抗激素相关性肿瘤及促肿瘤生长提供新的思路与方向，以期为不同药物寻找各自特异性的作用靶点。