title: Essential role of miR-200c in regulating self-renewal of breast cancer stem cells and their counterparts of mammary epithelium.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, P. R. China. 22346720@qq.com.
authors.forename: Zhong-Ming
authors.initials: ZM
authors.lastname: Feng
authors.affiliation: Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, P. R. China. zlpjyo@126.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Qiu
authors.affiliation: Department of Medical English, College of Basic Medicine, Third Military Medical University, Chongqing, 400038, P. R. China. chenxw12@sina.com.
authors.forename: Xie-Wan
authors.initials: XW
authors.lastname: Chen
authors.affiliation: Department of Medical English, College of Basic Medicine, Third Military Medical University, Chongqing, 400038, P. R. China. liaorx@aliyun.com.
authors.forename: Rong-Xia
authors.initials: RX
authors.lastname: Liao
authors.affiliation: Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, P. R. China. 776325362@qq.com.
authors.forename: Xing-Yun
authors.initials: XY
authors.lastname: Liao
authors.affiliation: Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, P. R. China. 284196729@qq.com.
authors.forename: Lu-Ping
authors.initials: LP
authors.lastname: Zhang
authors.affiliation: Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, P. R. China. 276423923@qq.com.
authors.forename: Xu
authors.initials: X
authors.lastname: Chen
authors.affiliation: Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, P. R. China. 407390154@qq.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, P. R. China. 314475914@qq.com.
authors.forename: Zheng-Tang
authors.initials: ZT
authors.lastname: Chen
authors.affiliation: Cancer Institute of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, P. R. China. sunjg09@aliyun.com.
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Sun
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D000066673:Cell Self Renewal / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results
chemical_list: C533896:MIRN200 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger