miR-7下调乳腺癌干细胞亚群的机制研究

The breast cancer stem cells (BCSCs) are one of the main reasons that result in the death of breast cancer patients due to without radical treatment of breast cancer. In our previous study, we found that the miR-7 was low expression in BCSCs detected by miRNA gene chips, and that the miR-7 overexpression significantly inhibited the expression of oncogene SETDB1, and reversed an epithelial-methsenchymal transition in MDA-MB-231 cells and BCSCs by downregulating STAT3 cell signaling. We also found that, from the previous study, the miR-7 overexpression could decrease the sub-population of BCSCs in MDA-MB-231 cell line, however, the mechanisms of leading to down-regulation of sub-population of BCSCs is poorly understood now. . In this study, we will infect the lenti-miR-7 into the MDA-MB-231, MCF-7 and BT549 cell lines as well as the cells isolated from the breast cancer patients sampled for excisional tumor tissues in different clinical stages, and detect the ALDH1+cells and BCSC sub-population by using FCM. Furthermore, we will test the molecular expression of HA, HAS2, CD44, TGF-β1, PI3K, Akt, mTOR, and SAMD2/3/7/ etc in the lenti-miR-7 infected breast cancer cell lines and the breast cancer patients’ samples as well as the breast cancer tissues isolated from the breast cancer bearing NOD/SCID mice that were injected with the BCSCs infected with lenti-miR-7 by using q-PCR, Western blot, breast cancer bearing NOD/SCID mice, and immunohistochemisty assays. The purpose of this study was to inquire into the molecular mechanisms of miR-7 down regulation of the sub-population of BCSCs via decrease of ALDH1A3 expression and inhibition of expression of CD44 and TGF-β1. The study achievement will have implications for therapeutic strategies that aim at targeting treatmet of highly invasive breast cancer by using the method of miR-7 overexpression. It has an important significant that we may solve the tough problem that is how to down regulate sub-population of BCSCs for radical treatment of the breast cancer.

乳腺癌干细胞(BCSC)是导致乳腺癌不能根治而死亡的主要原因之一。本组前期研究发现miR-7在BCSC中低表达，过表达miR-7可通过下调癌基因SETDB1、抑制STAT3信号通路进而减低BCSC上皮间质转换及转移；并发现miR-7还可下调乳腺癌MDA-MB-231细胞系中BCSC亚群，但经何机制发挥该作用尚不清楚。本研究将在乳腺癌MDA-MB-231等细胞系和不同临床分期乳腺癌手术分离的细胞中通过慢病毒过表达miR-7，经流式细胞仪、qPCR、Western blot、荷乳腺癌NOD/SCID鼠及免疫组化等实验，检测ALDH1+细胞、BCSC亚群和HA、CD44、TGFβ1、Akt、mTOR等分子表达；探讨miR-7减低ALDH1A3并抑制CD44和TGFβ1表达而下调BCSC亚群的机制，为应用miR-7下调BCSC亚群靶向治疗乳腺癌提供新方法，对解决如何靶向治疗BCSC的难题有重要意义

一、项目的背景：乳腺癌是女性中最常见的恶性肿瘤之一。因乳腺癌干细胞（BCSC）可使缓解期患者出现复发和转移，成为致死主因。课题组前研究发现，过表达miR-7可显著下调BCSC亚群，但机制不明。本研究基于：因BCSC亚群的多寡与乳腺癌患者的治疗和预后密切相关，阐明miR-7在BCSC中调节作用，可为靶向调控BCSC寻求新的有效靶点。二、主要研究内容（一）：MiR-7下调RELA减低CD44和ESAM表达抑制乳腺癌干细胞亚群的机制研究，利用MDA-MB-231、MCF-7、SK-BR-3及LD四株人乳腺癌细胞系，探讨了miR-7下调BCSC亚群的机制。（二）：miR-7调控ALDH1A3与XIST减低BCSC亚群的机制研究，利用人乳腺癌细胞系及人乳腺癌临床组织标本，探讨了miR-7- ALDH1A3和miR-7/miR-92b- XIST-Slug-ESA轴减少BCSC亚群的调控机制。三、重要结果、关键数据：1.MiR-7下调人乳腺癌细胞系MDA-MB-231，MCF-7，SK-BR-3及LD细胞中的BCSCs亚群数量，降低了RELA，CD44和ESAM表达，并抑制荷人BCSC异种移植瘤在NOD/SCID小鼠生长、血管生成和肿瘤转移。2.临床乳腺癌miR-7低表达且与RELA、CD44和ESAM表达呈负相关;RELA表达与CD44和ESAM两者间呈正相关。3.MiR-7通过靶向核转录因子RELA，降低其对CD44和ESAM转录作用，间接抑制CD44和ESAM表达，从而减低BCSC亚群数及其干性，在乳腺癌细胞和BCSC中发挥抑制效应。4.miR-7对ALDH1A3、miR-7对Slug和ESA分子具有靶向调控作用；miR-7/miR-92b与XIST之间存在相互调节作用，影响BCSC亚群比例。5.ALDH1A3，XIST，Slug和ESA表达与BCSC亚群数呈正相关；miR-7/miR-92b表达与BCSC亚群数呈负相关。6.miR-7能抑制ALDH1A3活性减低BCSC生物标记；miR-7/miR-92b能抑制Slug而下调ESA表达，减低BCSC表面标记，导致BCSC亚群比例减少。四、科学意义：miR-7及RELA、CD44和ESAM以及ALDH1A3以及miR-7/miR-92b调控XIST与Slug可作为靶向BCSC治疗乳腺癌的靶点,为该研究提供了可参考实验数据。