title: A novel in vitro model for cancer stem cell culture using ectopically expressed piwil2 stable cell line.
abstract: OBJECTIVE
  Piwil2, a member of Ago/Piwi gene family containing Piwi and PAZ domains, has been shown to be ectopically expressed in different cancer cells, especially its remarkable expression in cancer stem cells (CSCs), and is also known to be essential for germ line stem cell self-renewal in various organisms. The hypothesis that CSC may hold the key to the central problem of clinical oncology and tumor relapse leads to more anticancer treatment studies. Due to emerging controversies and extreme difficulties in studying of CSC, like the cells using in vivo models, more attempts have expended to establish different in vitro models. However, the progress was slow owing to the problems associated with establishing proper CSC cultures in vitro. To overcome these difficulties, we prompted to establish a novel stable cell line over-expressing Piwil2 to develop a potential proper in vitro CSC model.
  
  
  METHODS
  In this experimental study, mouse embryonic fibroblasts (MEFs) were isolated and electroporated with a construct containing Piwil2 cDNA under the control of the cytomegalovirus promoter (CMV). Stable transfectants were selected, and the established MEF-Piwil2 cell line was characterized and designated as CSC-like cells using molecular markers. Functional assays, including proliferation, migration, and invasion assays were performed using characterized CSC like cells in serum-free medium. Additionally, MEF-Piwil2 cell density and viability were measured by direct and indirect methods in normoxic and hypoxic conditions.
  
  
  RESULTS
  The results of reverse transcriptase-polymerase chain reaction (RT-PCR), western blot, and immunocytochemistry revealed an overexpression for Piwil2 in the transfected Piwil2 cells both in the RNA and protein levels. Furthermore, analysis of the kinetic and stoichiometric parameters demonstrated that the specific growth rate and the yield of lactate per glucose were significantly higher in the MEF-Piwil2 group compared to the MEF cells (ANOVA, p< 0.05). Also, analysis of functional assays including migration and invasion assays demonstrated a significantly higher number of migrated and invaded cells in the MEF-Piwil2 compared to that of the MEF cells (ANOVA, p< 0.05). The MEF-Piwil2 cells tolerated hypoxia mimetic conditions (CoCl2 ) with more than 95% viability.
  
  
  CONCLUSIONS
  According to the molecular and functional studies, it has been realized that Piwil2 plays a key role(s) in tumor initiation, progression and metastasis. Therefore, Piwil2 can be used not only as a common biomarker for tumor, but also as a target for the development of new anticancer drug. Finally, the main outcome of our study was the establishment of a novel CSC-like in vitro model which is expected to be utilized in understanding the complex roles played by CSC in tumor maintenance, metastasis, therapy resistance or cancer relapse.
authors.affiliation: 1. Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Shahali
authors.forename: Maryam
authors.initials: M
authors.lastname: Kabir-Salmani
authors.forename: Karim
authors.initials: K
authors.lastname: Nayernia
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Soleimanpour-Lichaei
authors.forename: Mohammad
authors.initials: M
authors.lastname: Vasei
authors.forename: Seyed Javad
authors.initials: SJ
authors.lastname: Mowla
authors.forename: Ehsan
authors.initials: E
authors.lastname: Ranaie
authors.forename: Mehdi
authors.initials: M
authors.lastname: Shakibaie
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Modaresi
keywords: Cancer; Ectopic; Piwil2; Self-Renewal; Stem cells
references.citation: Hum Mol Genet. 2006 Jan 15;15(2):201-11
references.pmid: 16377660
references.citation: Int J Biol Sci. 2011 Apr 26;7(5):517-35
references.pmid: 21552419
references.citation: Autophagy. 2012 Jun;8(6):979-81
references.pmid: 22714142
references.citation: PLoS One. 2011;6(11):e27154
references.pmid: 22110608
references.citation: Mol Reprod Dev. 2006 Feb;73(2):173-9
references.pmid: 16261612
references.citation: Cell. 2007 Feb 23;128(4):683-92
references.pmid: 17320506
references.citation: Semin Radiat Oncol. 2004 Jul;14(3):198-206
references.pmid: 15254862
references.citation: Urol Oncol. 2009 May-Jun;27(3):301-3
references.pmid: 19414117
references.citation: J Clin Invest. 2011 Jan;121(1):384-95
references.pmid: 21157036
references.citation: J Pathol. 2012 Dec;228(4):434-6
references.pmid: 22951989
references.citation: Cancer Res. 2007 Feb 1;67(3):1030-7
references.pmid: 17283135
references.citation: J Cell Mol Med. 2008 Jan-Feb;12(1):67-96
references.pmid: 18053092
references.citation: J Cell Biol. 2012 Aug 6;198(3):281-93
references.pmid: 22869594
references.citation: Cancer Res. 2003 Sep 15;63(18):5821-8
references.pmid: 14522905
references.citation: BMC Cell Biol. 2011 Aug 09;12:32
references.pmid: 21827650
references.citation: Proc Natl Acad Sci U S A. 2011 Jan 25;108(4):1397-402
references.pmid: 21220315
references.citation: Anticancer Agents Med Chem. 2008 Apr;8(3):305-12
references.pmid: 18393789
references.citation: Cancer Res. 2010 Jun 1;70(11):4569-79
references.pmid: 20460541
references.citation: Cell Death Differ. 2008 Mar;15(3):504-14
references.pmid: 18049477
references.citation: Cancer Cell. 2010 Nov 16;18(5):510-23
references.pmid: 21075313
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 9;105(36):13427-32
references.pmid: 18765800
references.citation: Cancer Res. 2009 Feb 15;69(4):1302-13
references.pmid: 19190339
references.citation: Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2482-7
references.pmid: 8637900
references.citation: PLoS One. 2010 Oct 20;5(10):e13406
references.pmid: 20975993
references.citation: Nat Methods. 2007 Apr;4(4):359-65
references.pmid: 17396127
references.citation: Cancer Res. 2006 Oct 1;66(19):9339-44
references.pmid: 16990346