title: Suppressive Effect of Constructed shRNAs against Apollon Induces Apoptosis and Growth Inhibition in the HeLa Cell Line.
abstract: BACKGROUND
  Cervical cancer is the second most common female cancer worldwide. Inhibitors of apoptosis proteins (IAPs) block apoptosis; therefore, therapeutic strategies targeting IAPs have attracted the interest of researchers in recent years. Apollon, a member of IAPs, inhibits apoptosis and cell death. RNA interference is a pathway in which small interfering RNA (siRNA) or shRNA (short hairpin RNA) inactivates the expression of target genes. The purpose of this study was to determine the effect of constructed shRNAs on apoptosis and growth inhibition through the suppression of apollon mRNA in HeLa cell line.
  
  
  METHODS
  Three shRNAs with binding ability to three different target sites of the first region of apollon gene were designed and cloned in pRNAin-H1.2/Neo vector. shRNA plasmids were then transfected in HeLa cells using electroporation. Down-regulation effects of apollon and the viability of HeLa cells were analyzed by RT-PCR, lactate dehydrogenase assay, and MTT assay, respectively. Also, the induction and morphological markers of apoptosis were evaluated by caspase assay and immunocytochemistry method.
  
  
  RESULTS
  The expression of shRNA in HeLa cells caused a significant decrease in the level of apollon mRNA1. In addition, shRNA1 effectively increased the mRNA level of Smac (as the antagonist of apollon), reduced the viability of HeLa cells and exhibited immunocytochemical apoptotic markers in this cell line.
  
  
  CONCLUSIONS
  Apollon gene silencing can induce apoptosis and growth impairment in HeLa cells. In this regard, apollon can be considered a candidate therapeutic target in HeLa cells as a positive human papillomavirus cancer cell line.
authors.affiliation: Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Ewin, Chamran highway, Tehran, Iran.
authors.forename: Saeideh
authors.initials: S
authors.lastname: Milani
authors.affiliation: Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Ewin, Chamran highway, Tehran, Iran.|Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors.forename: Mojgan
authors.initials: M
authors.lastname: Bandehpour
authors.affiliation: Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Hemmat EXP., Tehran, Iran.
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Sharifi
authors.affiliation: Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Ewin, Chamran highway, Tehran, Iran.|Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.|Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran .
authors.forename: Bahram
authors.initials: B
authors.lastname: Kazemi
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics*; D002470:Cell Survival / Q000235:genetics; D005260:Female; D006367:HeLa Cells; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics*; D047908:Intracellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics; D024101:Mitochondrial Proteins / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics* / Q000627:therapeutic use; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000628:therapy
chemical_list: D051017:Apoptosis Regulatory Proteins; C401541:BIRC6 protein, human; C412403:DIABLO protein, human; D051034:Inhibitor of Apoptosis Proteins; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering
keywords: Coronary artery disease; Genetic association study; Iran; Single nucleotide polymorphisms