Apoptosis induced by short hairpin RNA-mediated STAT6 gene silencing in human colon cancer cells

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Author:
ZHANG Ming-sheng()
ZHOU Yun-feng()
ZHANG Wen-jie()
ZHANG Xiao-lian()
PAN Qin()
JI Xue-mei()
LUO Zhi-guo()
WU Jian-ping()
Journal Title:
CHINESE MEDICAL JOURNAL
Issue:
Volume 119, Issue 10, 2006
DOI:
Key Word:
RNA interference;short hairpin RNA;signal transducer and activator of transcription 6;tumor;apoptosis

Abstract: Background The relationship between signal transduction and tumors has become one of the foci in cancer research. Signal transducer and activator of the transcription 6 (STAT6) signaling pathway is found to be activated in some cancer cells. But the function of the pathway in cancer cells is unknown. This study was undertaken to investigate the effect of the Stat6 signaling pathway on apoptosis in human colon cancer cells (HT-29 cells) and the possible mechanism of Stat6 by RNA interference techniques.Methods Four eukaryotic expression plasmid vectors of short hairpin RNA (shRNA) specific for the STAT6gene were designed and generated by molecular biological technology. The plasmid vectors were transfected into HT-29 cells by cation liposomes to block the Stat6 signaling pathway. The expressions of STAT6 mRNA and phosph-Stat6 protein were detected by the reverse transcriptase polymerase chain reaction (RT-PCR) method and flow cytometry respectively to screen the most effective shRNA at 72 hours after transfection. The apoptosis condition of the cells in which the expression of the STAT6 gene had been interfered was analyzed by flow cytometry and confocal microscopy. Both mRNA and protein expression of B cell lymphoma-2 (Bcl-2) and Bax were detected by RT-PCR and western blotting.Results Two effective eukaryotic expression plasmid vectors of shRNA specific for the STAT6 gene were generated successfully. One can reduce the expression of the STAT6 gene by 82.4% and the other by 56.8%(P<0.01). The apoptotic rate of colon cancer cells in which STAT6 gene expression had been interfered was significantly higher than that in controlled colon cancer cells (P<0.01). In the cells in which the Stat6 signaling pathway was blocked, the levels of mRNA and protein Bcl-2 were significantly decreased, whereas those of Bax were significantly increased (P<0.01).Conclusions The Stat6 signaling pathway can inhibit apoptosis in human colon cancer cells. The subsequent disorder of Bcl-2/Bax expression may play an important part in that process. The STAT6 gene may serve as a potential target in cancer therapy.

  • [1]Gilman AG,Simon MI,Bourne HR,Harris BA,Long R,Ross EM,et al Overview of the alliance for cellular signaling.Nature 2002; 420:703-706.
  • [2]Darnell JE,Kerr IM,Stark GR.Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.Science 1994;264:1415-1421.
  • [3]Ihle JN,Kerr IM.Jaks and Stats in signaling by the cytokine receptor superfamily.Trends Genet 1995; 11:3951-3963.
  • [4]Schindler C,Darnell JE.Transcriptional responses to polypeptide ligands:the Jak-STAT pathway.Annu.Rev Biochem 1995; 64:621-651.
  • [5]Takeda K,Tanaka T,Shi W,Matsumoto M,Minami M,Kashiwamura S,et al.Essential role of Stat6 in IL-4signaling.Nature 1996; 380:627-630.
  • [6]Takeda K,Kamanaka M,Tanaka T,Kishimoto T,Akira S.Impaired IL-13-mediated functions of macrophages in Stat6-deficient mice.J Immunol1996; 157:3220-3222.
  • [7]Kaplan MH,Schindler U,Smiley ST,Grusby MJ.Stat6 is required for mediating responses to IL-4 and for the development if Th2 cells.Immunity 1996; 4:313-319.
  • [8]Shimoda K,van Deursen J,Sangster MY,Sarawar SR,Carson RT,Tripp RA,et al.Lack of IL-4-induced Th2responses and IgE class switching in mice with disrupted Stat6 gene.Nature 1996; 380:630-633.
  • [9]Gooch JL,Christy B,Yee D.STAT6 mediates interleukin-4 growth inhibition in human breast cancer cells.Neoplasia 2002; 4:324-331.
  • [10]Skinnider BF,Elia A J,Gascoyne RD.Signal transduer and activator of transcription 6 is frequently activated in Hodgkin and Reed-Sternberg cells of Hodgkin lymphoma.Blood 2002; 99:618-626.
  • [11]Pisani P,Parkin DM,Ferlay J.Estimates of the worldwide mortality from eighteen major cancers in 1985.Int J Cancer 1993; 54:594-606.
  • [12]Gudkov AV,Zelnick CR,Kazarov AR,Thimmapaya R,Suttle DP,Beck WT,et al.Isolation ofgenetic suppressor elements,inducing resistance to topoisomerase Ⅱ-interactive cytotoxic drugs,from human topoisomerase Ⅱ cDNA.Proc Natl Acad Sci 1993; 90:3231-3235.
  • [13]Collins FS,Green ED,Guttmacher AE,Guyer MS.A vision for the future of genomic research.Nature 2003;422:835-847.
  • [14]Novina CD,Sharp PA.The RNAi revolution.Nature 2004;430:161-164.
  • [15]Matzke M,Matzke A,Kooter MJ.RNA:guiding gene silencing.Science 2001; 293:1980-1983.
  • [16]Fire A,Xu S,Montgomery MK,Kostas SA,Driver SE,Mello CC.Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans.Nature 1998; 391:806-811.
  • [17]Ngo H,Tschudi C,Gull K,Ullu E.Double-stranded RNA induces mRNA degradation in Trypanosoma brucei.Proc Natl Acad Sci USA 1998; 95:14687-14692.
  • [18]Kennerdell JR,Carthew RW.Heritable gene silencing in Drosophila using double-stranded RNA.Nat Biotechnol 2000; 17:896-898.
  • [19]Yu JY,DeRuiter SL,Turner DL.RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.Proc Natl Acad Sci USA 2002;99:6047-6052.
  • [20]Kisielow M,Kleiner S,Nagasawa M,Faisal A,Nagamine Y.Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA.Biochem J 2002; 363 (Pt 1):1-5.
  • [21]Tuschl T.Expanding small RNA interference.Nat Biotechnol 2002; 20:446-448.
  • [22]Agami R.RNAi and related mechanisms and their potential use for therapy.Curr Opin Chem Biol 2002; 6:829-834.
  • [23]Krutzik PO,Nolan GP.Intracellular phospho-protein staining techniques for flow cytometry:monitoring single cell signaling events.Cytometry A 2003; 55:61-70.
  • [24]Krutzik PO,Irish JM,Nolan GP,Perez OD.Analysis of protein phosphorylation and cellular signaling events by flow cytometry:techniques and clinical applications.Clin Immunol 2004; 110:206-221.
  • [25]Fisher DE.Apoptosis in cancer therapy:crossing the threshold.Cell 1994; 78:539-542.
  • [26]Linehan LA,Warren WD,Thompson PA,Grusby M J,Berton MT.Stat6 is required for IL-4-mediated germline Ig gene transcription and switch recombination.J Immunol1998; 161:302-310.
  • [27]Tomkinson A,Duez C,Lahn M,Gelfand EW.Adoptive transfer of T cells induces airway hyperresponsiveness independently of airway eosinophilia but in a signal transducer and activator of transcription 6-dependent manner.J Allergy Clin Immunol 2002; 109:810-816.
  • [28]Ni Z,Lou W,Lee SO.Selective activation of members of the signal transducers and activators of transcription family in prostate carcinoma.J Urol 2002; 167:1859-1862.
  • [29]Suzanne O,Michael JG,Virginia KC.Cutting edge:STAT6-deficient mice have enhanced tumor immunity to primary and metastatic mammary carcinoma.J Immunol 2000; 165:6015-6019.
  • [30]Zhang W J,Koltun WA,Thompson JL,Tilberg AF,Galka E,Poritz LS,et al.Human B lymphoblast cell lines defective of Stat6 signaling produce high levels of proinflammatory cytokines IL-12,TNF-α and IFN-γ.Int J Oncology 2004; 24:447-453.
  • [31]Galka E,Thompson JL,Zhang W J,Poritz LS,Koltun WA.Stat6null phenotype human lymphocytes exhibit increased apoptosis.J Surg Res 2004; 122:14-20.
  • [32]Dorsett Y,Tuschl T.siRNAs:applications in functional genomics and potential as therapeutics.Nat Rev Drug Discov 2004; 3:318-329.
  • [33]Hammond SM,Boettcher S,Caudy AA,Kobayashi R,Hannon GJ.Argonaute 2,a link between genetic and biochemical analyses of RNAi.Science 2001;293:1146-1150.
  • [34]Elbashir SM,Harborth J,Weber K,Tuschl T.Analysis of gene function in somatic mammalian cells using small interfering RNAs.Methods 2002; 26:199-213.
  • [35]Gan HZ,Zhang GZ,Zhao JS,Zhang FC,Bu LS,Yang S J,et al.Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors.Chin Med J 2005; 118:893-902.
  • [36]Miyagishi M,Taira K.U6 promoter driven shRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells.Nat Biotechnol 2002; 20:497-500.
  • [37]Czauderna F,Fechtner M,Aygun H,Arnold W,Klippel A,Giese K,et al.Functional studies of the Pl(3)-kinase signaling pathway employing synthetic and expressed shRNA.Nucleic Acid Res 2003; 31:670-682.
  • [38]Reed JC.Bcl-2 family proteins.Oncogene 1998; 17:3225-3236.
  • [39]Gross A,McDonnell JM,Korsmeyer S.Bcl-2 family members and the mitochondria in apoptosis.Genes Dev 1999; 13:1899-1911.
  • [40]Oltvai ZN,Millman CL,Korsmeyer SJ.Bcl-2heterodimerizes in vivo with a conserved homolog Bax,that accelerates programmed cell death.Cell 1993; 74:609-619.
  • [41]Whitte E.Life,death,the pursuit of apoptosis.Genes Dev 1996; 10:1-15.
  • [42]Levy P,Robin H,Bertrand F,Kornprobst M,Capeau J.Butyrate-treated colonic Caco-2 cells exhibit defective integrin-mediated signaling together with increased apoptosis and differentiation.J Cell Physiol 2003; 197:336-347.
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