title: Simultaneously expressed miR-424 and miR-381 synergistically suppress the proliferation and survival of renal cancer cells---Cdc2 activity is up-regulated by targeting WEE1.
abstract: OBJECTIVE
  MiRNAs are intrinsic RNAs that interfere with protein translation. Few studies on the synergistic effects of miRNAs have been reported. Both miR-424 and miR-381 have been individually reported to be involved in carcinogenesis. They share a common putative target, WEE1, which is described as an inhibitor of G2/M progression. Here, we studied the synergistic effects of miR-424 and miR-381 on renal cancer cells.
  
  
  METHODS
  The viability of 786-O cells was analyzed after transfection with either a combination of miR-424 and miR-381 or each miRNA alone. We investigated cell cycle progression and apoptosis with flow cytometry. To confirm apoptosis and the abrogation of G2/M arrest, we determined the level of pHH3, which is an indicator of mitosis, and caspase-3/7 activity. The expression levels of WEE1, Cdc25, γH2AX, and Cdc2 were manipulated to investigate the roles of these proteins in the miRNA-induced anti-tumor effects. To verify that WEE1 was a direct target of both miR-424 and miR-381, we performed a dual luciferase reporter assay.
  
  
  RESULTS
  We showed that the combination of these miRNAs synergistically inhibited proliferation, abrogated G2/M arrest, and induced apoptosis. This combination led to Cdc2 activation through WEE1 inhibition. This regulation was more effective when cells were treated with both miRNAs than with either miRNA alone, indicating synergy between these miRNAs. WEE1 was verified to be a direct target of each miRNA according to the luciferase reporter assay.
  
  
  CONCLUSIONS
  These data clearly demonstrate that these two miRNAs might synergistically act as novel modulators of tumorigenesis by down-regulating WEE1 expression in renal cell cancer cells.
authors.affiliation: Third Xiang-Ya Hospital of Central South University, Department of Urology, Changsha, Hunan/China.
authors.forename: Binghai
authors.initials: B
authors.lastname: Chen
authors.forename: Lujing
authors.initials: L
authors.lastname: Duan
authors.forename: Guangming
authors.initials: G
authors.lastname: Yin
authors.forename: Jing
authors.initials: J
authors.lastname: Tan
authors.forename: Xianzhen
authors.initials: X
authors.lastname: Jiang
mesh_terms: D015153:Blotting, Western; D016203:CDC2 Protein Kinase; D002292:Carcinoma, Renal Cell / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002468:Cell Physiological Phenomena; D002471:Cell Transformation, Neoplastic; D019926:Cyclin B / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases; D015536:Down-Regulation; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics*; D035683:MicroRNAs / Q000378:metabolism / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D018797:Cell Cycle Proteins; D019926:Cyclin B; C554214:MIRN424 microrna, human; D035683:MicroRNAs; D009687:Nuclear Proteins; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases