miR-1/133a和miR-206/133b基因簇调控钙网蛋白基因表达影响乳腺癌干细胞生物学行为的机制研究

Our previous work verified that miR-1, miR-206, miR-133a and miR-133b, the members of miR-1/133a and miR-206/133b clusters, are all down-regulated significantly in various breast cancer stem cells (BCSCs). The overexpression of miR-1, miR-206, miR-133a and miR-133b respectively in BCSCs suppressed calreticulin expression markedly, resulting in decreased proliferation of BCSCs. Analyzed with bioinformatic softwares, we did not find any binding sites of miR-1, miR-206, miR-133a and miR-133b in the 3'-UTR of CALR. In MDA-MB-231 BCSCs over-expressing the members of miR-1/133a and miR-206/133b clusters, we detected a significant down-regulation of transcription factor EVI-1. Furthermore, with the analysis by bioinformatic softwares, we predicted that the binding sites of miR-1, miR-206, miR-133a and miR-133b might exist in the 3'-UTR of EVI-1, which would suggest that EVI-1 might be one of the downstream target genes of miR-1, miR-206, miR-133a and miR-133b. We also predicted that the promoter region of CALR gene contained several EVI-1 binding sites, which would suggest that EVI-1 might be involved in regulating the expression of CALR. Down-regulating the expression of calreticulin in MDA-MB-231 BCSCs could suppress the proliferation and migration significantly. On the basis of our previous series researches, we proposed the following working hypotheses: the four members of miR-1/133a and miR-206/133b clusters all show a low expression level in BCSCs. The low expressed miR-1, miR-206, miR-133a and miR-133b may up-regulate the expression of calreticulin by negatively regulating the transcription factor EVI-1. The up-regulated calreticulin will affect the biological behavior of BCSCs, including increasing the viability of BCSCs, promoting the epithelial mesenchymal transition of stem cells and enhancing the invasion and migration of BCSCs. One or more domains of calreticulin may play crucial roles in the regulation of migration and invasion of BCSCs. To testify the above hypotheses, this program is designed on the basis of our previous work to verify the detailed target binding sites between miR-1, miR-206, miR-133a, miR-133b and target gene EVI-1. We attempt to investigate whether miR-1, miR-206, miR-133a, miR-133b regulate the transcription and expression of calreticulin via controling EVI-1 gene, and to clarify the regulatory mechanism. Next, we plan to analyze the effects of the alterations of calreticulin expression on BCSCs' biological functions as well as the related molecular mechanisms, and to clarify the crucial domain X of calreticulin which maybe essential in the functions of BCSCs. Finally, we propose to find the best way to inhibit the proliferation and migration of BCSCs through administrating the miR-1, miR-206, miR-133a, miR-133b and domain X alone or in combination, and to observe the antitumor effects on BCSCs xenografts in nude mice. This project will provide a new strategy to improve the therapeutic efficacy of breast cancer.

我们前期工作证明miR-1/133a和miR-206/133b基因簇4个成员在不同的乳腺癌干细胞中低表达，发挥抑癌基因的作用。上述基因簇调节乳腺癌干细胞生物学功能的效果与机制未见报道。通过系列前期研究提出了工作假说：乳腺癌干细胞中低表达的miR-1、miR-206、miR-133a和miR-133b可能通过负性调控转录因子EVI-1的表达，上调钙网蛋白的表达，进而影响乳腺癌干细胞的生物学行为。本项目拟先验证miR-1/133a和miR-206/133b基因簇4个成员与靶基因EVI-1的结合与结合位点；研究miR-1/133a和miR-206/133b基因簇通过调控EVI-1基因影响钙网蛋白的转录和表达，调节乳腺癌干细胞生物学功能的机制；明确钙网蛋白调节乳腺癌干细胞生物学功能的关键结构域X，以及结构域X抗体单独或联合应用对乳腺癌干细胞裸鼠移植瘤的抑瘤效应。研究结果能为乳腺癌的治疗提供新靶点。

我们前期工作证明miR-1/miR-206在乳腺癌组织中低表达，发挥抑癌基因的作用。上述基因簇调节乳腺癌干细胞生物学功能的效果与机制未见报道。通过一系列研究我们发现：乳腺癌干细胞中低表达的miR-1、miR-206可能通过负性调控转录因子EVI-1的表达，上调钙网蛋白的表达，进而影响乳腺癌干细胞的生物学行为。本项目先验证miR-1和miR-206与靶基因EVI-1的结合与结合位点；EVI-1基因又影响钙网蛋白的转录和表达，调节乳腺癌干细胞生物学功能的机制；并且合成钙网蛋白不同结构域以明确钙网蛋白调节乳腺癌干细胞生物学功能的关键结构域X。研究结果能为乳腺癌的治疗提供新靶点。