title: miRNA expression patterns in chemoresistant breast cancer tissues.
abstract: OBJECTIVE
  Breast cancer chemoresistance is a major obstacle to the successful treatment of patients. miRNAs perform critical roles in biological processes, including tumorigenesis and chemoresistance. However, little clinical data are available regarding the relationship between miRNA expression patterns and breast cancer chemoresistance.
  
  
  METHODS
  We created a doxorubicin-resistant MCF-7 (MCF-/Adr) cell line using a pulse-selection method; then verified the resistance of the MCF-7/Adr cell line to doxorubicin by using the methyl thiazolyl tetrazolium (MTT) assay, terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining, and Intracellular doxorubicin accumulation assay. Then, we performed qRT-PCR to detect the expression patterns of 14 selected miRNAs (which are related to breast cancer resistance) in both cell lines. Subsequently, we performed a bioinformatics analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, to determine the putative functions of 13 differentially expressed miRNA-targeted genes. Finally, we tested the expression levels of these 13 miRNAs in 10 chemotherapy non-responder breast cancer tissues and 29 responder tissues. All statistical analyses were performed by a two-tailed Student's t-test, and a P value less than 0.05 was considered statistically significant.
  
  
  RESULTS
  The results of the MTT assay showed that the MCF-7/Adr cell line was significantly more resistant to doxorubicin compared to the MCF-7 cells The results of the TUNEL assay indicated that doxorubicin induced an increase in the number apoptotic cells in the MCF-7 group. Additionally, the accumulation of doxorubicin was higher in MCF-7 cells compared to MCF-7/Adr cells, which was consistent with the MTT and TUNEL results. The qRT-PCR results demonstrated that compared to the parental MCF-7 cell line, miR-200a, miR-141, miR-200c, miR-31, miR-429, and miR-196b were over-expressed, and let-7e, miR-576-3p, miR-125b-1, miR-370, miR-145, miR-765, and miR-760 were significantly down-regulated in MCF-7/Adr cells. The GO analysis results revealed that the predicted target genes of these 14 miRNAs primarily regulated protein binding, zinc ion binding, DNA binding, and transcription factor activity. The KEGG data demonstrated that these target genes are mainly involved in the MAPK signaling pathway, regulation of the actin cytoskeleton, cytokine-cytokine receptor interaction, and other signaling pathways. Compared to the breast cancer tissues from chemotherapy responders, 10 miRNAs were identified to be dysregulated in the chemoresistant breast cancer tissues. Three of these miRNAs were up-regulated (miR-141, miR-200c, and miR-31), and 7 were down-regulated (let-7e, miR-576-3p, miR-125b-1, miR-370, miR-145, miR-765, and miR-760).
  
  
  CONCLUSIONS
  In this study, we identified 10 dysregulated miRNAs in both breast cancer cells and chemoresistant tissues, which might be biomarkers for the prognosis of breast cancer chemoresistance. Our study contributes to a comprehensive understanding of prognostic biomarkers during clinical treatment, and we hypothesize that the miRNA signatures of drug-resistant carcinoma tissues could be useful for developing new strategies for targeted therapies in patients with chemoresistant breast cancer.
authors.affiliation: Yangzhou Maternal and Child Health Hospital, Affiliated with Yangzhou Medical University, 225002 Yangzhou, China.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Lv
authors.affiliation: The Affiliated Jiangyin Hospital of Southeast University Medical College, 214400 Jiangyin, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Xia
authors.affiliation: Nanjing Maternity and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, 210004 Nanjing, China.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Xu
authors.affiliation: Nanjing Maternity and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, 210004 Nanjing, China.
authors.forename: Erhu
authors.initials: E
authors.lastname: Sun
authors.affiliation: Nanjing Maternity and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, 210004 Nanjing, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Ma
authors.affiliation: Nanjing Maternity and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, 210004 Nanjing, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Gao
authors.affiliation: Nanjing Maternity and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, 210004 Nanjing, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: Nanjing Maternity and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, 210004 Nanjing, China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Nanjing Maternity and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, 210004 Nanjing, China.
authors.forename: Fengliang
authors.initials: F
authors.lastname: Wang
authors.affiliation: Nanjing Maternity and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, 210004 Nanjing, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Chen
authors.affiliation: Nanjing Maternity and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, 210004 Nanjing, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhou
authors.affiliation: Nanjing Maternity and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, 210004 Nanjing, China. Electronic address: xhfybj@163.com.
authors.forename: Ziyi
authors.initials: Z
authors.lastname: Fu
authors.affiliation: Nanjing Maternity and Child Health Medical Institute, Affiliated Nanjing Maternal and Child Health Hospital, Nanjing Medical University, 210004 Nanjing, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Xie
mesh_terms: D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000502:physiology*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D061986:MCF-7 Cells; D035683:MicroRNAs / Q000096:biosynthesis*; D011485:Protein Binding / Q000502:physiology
chemical_list: D035683:MicroRNAs; D004317:Doxorubicin
keywords: Breast cancer; Chemoresistance; miRNA