title: MicroRNA-1 inhibits proliferation of hepatocarcinoma cells by targeting endothelin-1.
abstract: OBJECTIVE
  MicroRNA-1 (miR-1) has been demonstrated as a tumor-suppressive miRNA, which shows a down-regulated pattern in several human malignancies including hepatocellular carcinoma (HCC). However, the pathophysiologic roles of miR-1 and their mechanisms in HCC tumorigenesis are still not totally elucidated.
  
  
  METHODS
  Pre-miR-1 was cloned into pSuper plasmid to overexpress the miR-1 in hepatoma cells. Real-time PCR and Western blot were applied to detect miR-1, ET-1 mRNA and protein levels respectively. Dual luciferase reporter assay was conducted to investigate the binding site of miR-1 on 3'UTR of ET-1 mRNA. Proliferation of hepatoma cells was evaluated by MTT assay.
  
  
  RESULTS
  We observed that over-expression of miR-1 by miRNA-expressing plasmid transfection in HepG2 and Hep3B cells significantly reduced the proliferation of these cells. To explore the mechanism, we examined the potential target genes of miR-1 by bioinformatics. A potent mitogen, Endothelin-1 (ET-1), attracted our attention. Elevated expression of ET-1 but reduced miR-1 level was detected both in human liver cancer tissues and in hepatoma cell lines using Western Blot and miRNA real-time PCR respectively. By the over-expression and inhibition of miR-1 in HepG2 and Hep3B, we confirmed that miR-1 negatively regulated ET-1 expression in hepatoma cells. A luciferase reporter assay showed that miR-1 regulation was established by pairing to a complementary binding site within the ET-1 3'UTR. Finally, attenuated proliferation of hepatoma cells by over-expression of miR-1 could be partially restored by exogenous ET-1 treatment.
  
  
  CONCLUSIONS
  Our findings demonstrate that miR-1 could inhibit ET-1 expression to attenuate the proliferation of hepatoma cells.
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Li
authors.affiliation: Department of Pharmacology and School of Pharmacy, Second Military Medical University, Shanghai, China.
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Cheng
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wei
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Su
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Peng
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Gao
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Tan
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Zhenguo
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China.
authors.forename: Zhongchun
authors.initials: Z
authors.lastname: Qi
authors.affiliation: Department of Pharmacology and School of Pharmacy, Second Military Medical University, Shanghai, China. Electronic address: ruiyaocheng@yahoo.com.
authors.forename: Yaocheng
authors.initials: Y
authors.lastname: Rui
authors.affiliation: Department of Oncology, Chengdu Military General Hospital, Chengdu, Si Chuan Province, China. Electronic address: zhangtao269@126.com.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
mesh_terms: D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000503:physiopathology; D049109:Cell Proliferation / Q000187:drug effects*; D019332:Endothelin-1 / Q000037:antagonists & inhibitors* / Q000502:physiology; D056945:Hep G2 Cells / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000503:physiopathology; D008156:Luciferases; D035683:MicroRNAs / Q000627:therapeutic use*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D019332:Endothelin-1; C544547:MIRN1 microRNA, human; D035683:MicroRNAs; D008156:Luciferases