Essential role of miR-200c in regulating self-renewal of breast cancer stem cells and their counterparts of mammary epithelium.
Essential role of miR-200c in regulating self-renewal of breast cancer stem cells and their counterparts of mammary epithelium.
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miR-200c 在调节乳腺癌干细胞及其乳腺上皮细胞自我更新中的重要作用。
DOI：
10.1186/s12885-015-1655-5
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发表时间：
2015-09-23
期刊：
影响因子：
3.8
通讯作者：
Sun JG
中科院分区：
文献类型：
--
作者：
Feng ZM;Qiu J;Chen XW;Liao RX;Liao XY;Zhang LP;Chen X;Li Y;Chen ZT;Sun JG
乳腺癌干细胞(BCSCs)是乳腺癌的起源，是乳腺癌耐药、复发和转移的根本原因。BCSCs可以从突变的乳腺上皮干细胞(MASCs)分化而来。因此，比较BCSCs和MASCs的分子差异可能有助于阐明乳腺癌发生的机制和基因治疗的靶点。具体地说，需要分析BCSCs和MASCs的不同miRNome数据，以找出关键的miRNAs，并揭示它们在调节BCSCs干性方面的作用。用荧光激活细胞分选法从正常乳腺上皮细胞系MCF10A中分离出MUC1ESA+细胞，并通过克隆形成实验和多向分化实验检测其干细胞特性。比较MASCs、BCSCs和乳腺癌MCF-7细胞的miRNA谱，以获得可能调控乳腺肿瘤发生的候选miRNAs。选择从MASCs到BCSCs再到MCF-7细胞连续上调的miR-200C，以确定其在体内外对BCSCs和MASCs的干性调节作用。在生物信息学的基础上，通过双荧光素酶报告系统、蛋白质印迹和救援实验对miR-200C的靶标进行了验证。在二维克隆形成实验中，MUC1ESA+细胞产生了多种形态的集落，包括腔集落、肌上皮集落和混合集落。MUC1MCFESA+细胞的克隆形成能力(61.5MCFESA+)显著高于非干细胞−-10A细胞(53.5MCFESA+)(53.5MCF3.42%)(P <； )。在三维基质培养中，MUC1ESA+细胞向乳房内生长，呈管状结构。共鉴定出12个相关的miRNAs，其中8个在BCSCs中上调，4个在MASCs中下调。在功能获得和功能丧失实验中，miR-200C足以抑制BCSCs和MASCs在体外的自我更新和体内BCSCs的生长。此外，miR-200C负性调控BCSCs和MASCs中的程序性细胞死亡10(PDCD10)。PDCD10可挽救miR-200C抑制的BCSCs致瘤性。越来越多的证据表明，从MASCs到BCSCs有一个里程碑式的转变。其潜在机制尚不清楚。在本研究中，获得了MASCs和BCSCs之间的miRNA图谱。在MASCs和BCSCs中低表达的miRNA-200C被证实为抑癌基因，并在调节两种干细胞的自我更新中发挥重要作用。这些发现揭示了对乳腺肿瘤发生的新见解。PDCD10是miR-200C的靶基因，也是miR-200C调控BCSCs和MASCs茎化的可能机制。本文的在线版本(doi：10.1186/s12885-0151655-5)包含补充材料，授权用户可以使用。
Breast cancer stem cells (BCSCs) have been reported as the origin of breast cancer and the radical cause of drug resistance, relapse and metastasis in breast cancer. BCSCs could be derived from mutated mammary epithelial stem cells (MaSCs). Therefore, comparing the molecular differences between BCSCs and MaSCs may clarify the mechanism underlying breast carcinogenesis and the targets for gene therapy. Specifically, the distinct miRNome data of BCSCs and MaSCs need to be analyzed to find out the key miRNAs and reveal their roles in regulating the stemness of BCSCs. MUC1−ESA+ cells were isolated from normal mammary epithelial cell line MCF-10A by fluorescence-activated cell sorting (FACS) and tested for stemness by clonogenic assay and multi-potential differentiation experiments. The miRNA profiles of MaSCs, BCSCs and breast cancer MCF-7 cells were compared to obtain the candidate miRNAs that may regulate breast tumorigenesis. An miRNA consecutively upregulated from MaSCs to BCSCs to MCF-7 cells, miR-200c, was chosen to determine its role in regulating the stemness of BCSCs and MaSCs in vitro and in vivo. Based on bioinformatics, the targets of miR-200c were validated by dual-luciferase report system, western blot and rescue experiments. In a 2-D clonogenic assay, MUC1−ESA+ cells gave rise to multiple morphological colonies, including luminal colonies, myoepithelial colonies and mixed colonies. The clonogenic potential of MUC1−ESA+ (61.5 ± 3.87 %) was significantly higher than that of non-stem MCF-10A cells (53.5 ± 3.42 %) (P < 0.05). In a 3-D matrigel culture, MUC1−ESA+ cells grew into mammospheres with duct-like structures. A total of 12 miRNAs of interest were identified, 8 of which were upregulated and 4 downregulated in BCSCs compared with MaSCs. In gain- and lost-of-function assays, miR-200c was sufficient to inhibit the self-renewal of BCSCs and MaSCs in vitro and the growth of BCSCs in vivo. Furthermore, miR-200c negatively regulated programmed cell death 10 (PDCD10) in BCSCs and MaSCs. PDCD10 could rescue the tumorigenesis inhibited by miR-200c in BCSCs. Accumulating evidence shows that there is a milignant transformation from MaSCs into BCSCs. The underlying mechanism remains unclear. In present study, miRNA profiles between MaSCs and BCSCs were obtained. Then miRNA-200c, downregulated in both MaSCs and BCSCs, were verified as anti-oncogene, and played essential role in regulating self-renewal of both kinds of stem-like cells. These findings reveal a novel insights of breast tumorigenesis. PDCD10 is a target gene of miR-200c and also a possible mechanism by which miR-200c plays a role in regulating the stemness of BCSCs and MaSCs. The online version of this article (doi:10.1186/s12885-015-1655-5) contains supplementary material, which is available to authorized users.