Abstract
Aim:
To determine the inhibitory effect of the synthetic STAT3 siRNA on the expression of STAT3 gene in human laryngeal cancer cell lines Hep2 and to investigate the effect of STAT3 siRNA on growth and apoptosis in Hep2 cells.
Methods:
A pair of DNA templates coding siRNA against STAT3-mRNA was synthesized to reconstruct plasmid of pSilencer1.0-U6 siRNA-STAT3. Hep2 cells were transfected with RPMI-1640 media (untreated), plasmid (empty), and STAT3 siRNA, respectively. Northern blot and Western blot analysis of STAT3 and pTyr-STAT3 expression in Hep2 cells and Western blot analysis of Bcl-2 expression in the Hep2 cell was performed 72 h after transfection. MTT, flow cytometry, and AO/EB assay were used for determination of cells proliferation and apoptosis in Hep2 cells.
Results:
pTyr-STAT3 was markedly expressed in untreated Hep2 cells and the vector-treated Hep2 cells, whereas pTyr-STAT3 expression was significantly reduced in STAT3 siRNA-transfected Hep2 cells, indicating that STAT3 siRNA inhibited the activity of STAT3. Transfection of Hep2 cells with STAT3 siRNA significantly inhibited STAT3 expression at both mRNA and protein level in Hep2 cells and the inhibition was characterized by time-dependent transfection. Treatment of Hep2 cells with STAT3 siRNA resulted in dose-dependent growth inhibition of Hep2, this significantly increased apoptotic cell rate, and decreased Bcl-2 expression level in Hep2 cells. STAT3 siRNA had an effect on induction of either early or late stage apoptosis.
Conclusion:
This study demonstrates that STAT3 siRNA effectively inhibits STAT3 gene expression in Hep2 cells leading to growth suppression and induction of apoptosis in Hep2 cells. The use of siRNA technique may provide a novel therapeutic approach to treat laryngeal cancer and other malignant tumors expressing constitutively activated STAT3.
Similar content being viewed by others
Article PDF
References
Garcia R, Yu CL, Hudnall A, Catlett R, Nelson KL, Smithgall T, et al. Constitutive activation of STAT3 in fibroblasts transformed by diverse oncoproteins and in breast carcinoma cells. Cell Growth Differ 1997; 8: 1267–76.
Garcia R, Bowman TL, Niu G, Yu H, Minton S, Muro-Cacho CA, et al. Constitutive activation of STAT3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells. Oncogene 2001; 20: 2499–513.
Grandis JR, Drenning SD, Chakraborty A, Zhou MY, Zeng Q, Pitt AS, et al. Requirement of STAT3 but not stat1 activation for epidermal growth factor receptor-mediated cell growth in vitro. J Clin Invest 1998; 102: 1385–92.
Takemoto S, Mulloy JC, Cereseto A, Migone TS, Patel BK, Matsuoka M, et al. Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins. Proc Natl Acad Sci USA 1997; 94: 13897–902.
Gouilleux-Gruart V, Gouilleux F, Desaint C, Claisse JF, Capiod JC, Delobel J, et al. STAT-related transcription factors are constitutively activated in peripheral blood cells from acute leukemia patients. Blood 1996; 87: 1692–7.
Leong PL, Andrews GA, Johnson DE, Dyer KF, Sichuan Xi, Mai JC, et al. Targeted inhibition of STAT3 with a decoy oligonucleotide abrogates head and neck cancer cell growth. Proc Natl Acad Sci USA 2003; 100: 4138–43.
Mora LB, Buettner R, Seigne J, Diaz J, Ahmad N, Garcia R, et al. Constitutive activation of STAT3 in human prostate tumors and cell lines: direct inhibition of STAT3 signaling induces apoptosis of prostate cancer cells. Cancer Res 2002; 62: 6659–66.
Catlett-Falcone R, Landowski TH, Oshiro MM, Turkson J, Levitzki A, Savino R, et al. Constitutive activation of STAT3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity 1999; 10: 105–15.
Caldenhoven E, van Dijk TB, Solari R, Armstrong J, Raaijmakers JA, Lammers JW, et al. STAT3 beta, a splice variant of transcription factor STAT3, is a dominant negative regulator of transcription. J Biol Chem 1996; 271: 13221–7.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC . Potent and specific genetic interference by double-strand RNA in Caenorhabditis elegans. Nature 1998; 391: 806–11.
Hammond SM, Bernstein E, Beach D, Hannon GJ . An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 2000; 404: 293–6.
Sharp PA, Zamore PD . Molecular biology: RNA interference. Science 2000; 287: 2431–3.
Wianny F, Zernicka-Goetz M . Specific interference with gene function by double-strand RNA in early mouse development. Nat Cell Biol 2000; 2: 70–5.
Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T . Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411: 494–8.
Grandis JR, Drenning SD, Zeng Q, Watkins SC, Melhem MF, Endo S, et al. Constitutive activation of that stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo. Proc Natl Acad Sci USA 2000; 97: 4227–32.
Lee SO, Lou W, Qureshi KM, Mehraein-Ghomi F, Trump DL, Gao AC . RNA interference targeting Stat3 inhibits growth and induces apoptosis of human cancer cells. Prostate 2004; 64: 303–9.
Konnikova L, Kotecki M, Kruger MM, Cochran BH . Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells. BMC Cancer 2003; 3: 23.
Weber-Nordt RM, Mertelsmann R, Finke J . The JAK-STAT pathway: signal transduction involved in proliferation, differentiation and transformation. Leuk Lymphoma 1998; 28: 459–67.
Takeda K, Clausen BE, Kaisho T, Tsujimura T, Terada N, Forster I, et al. Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of STAT3 in macrophages and neutrophils. Immunity 1999; 10: 39–49.
Kaplan DH, Shankaran V, Dighe AS, Stockert E, Aguet M, Old LJ, et al. Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice. Proc Natl Acad Sci USA 1998; 95: 7556–61.
Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C, et al. STAT3 as an oncogene. Cell 1999; 98: 295–303.
Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N, et al. Targeted disruption of the mouse STAT3 gene leads to early embryonic lethality. Proc Nat1 Acad Sci USA 1997; 94: 3801–4.
Niu G, Heller R, Catlett-Falcone R, Coppola D, Jaroszeski M, Dalton W, et al. Gene therapy with dominant-negative STAT3 suppresses growth of the murine melanoma B16 tumor in vivo. Cancer Res 1999; 59: 5059–63.
Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ . Argonaute 2, a link between genetic and biochemical analyses of RNAi. Science 2001; 293: 1146–50.
Scott S, Higdon R, Beckett L, Shi XB, deVere White RW, Earle JD, et al. Bc1 2 antisense reduces prostate cancer cell survival following irradiation. Cancer Biother Radiopharm 2002; 17: 647–56.
Xia H, Mao QW, Paulson HL, Davidson BL . siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002; 20: 1006–10.
Lewis DL, Hagstrom JE, Loomis AG, Wolff JA, Herweijer H . Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat Gen 2002; 32: 107–8.
Song E, Lee SK, Wang J, Ince N, Ouyan N, Min J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 2003; 9: 347–51.
Contreras JL, Vilatoba M, Eckstein C, Bilbao G, Thompson JA, Eckhoff DE . Caspase-8 and caspase-3 small interfering RNA decreases ischemia/reperfusion injury to the liver in mice. Surgery 2004; 136: 390–400.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Gao, Lf., Xu, Dq., Wen, Lj. et al. Inhibition of STAT3 expression by siRNA suppresses growth and induces apoptosis in laryngeal cancer cells. Acta Pharmacol Sin 26, 377–383 (2005). https://doi.org/10.1111/j.1745-7254.2005.00053.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1745-7254.2005.00053.x
Keywords
This article is cited by
-
SUMOylation and SENP3 regulate STAT3 activation in head and neck cancer
Oncogene (2016)
-
A GMCSF and IL7 fusion cytokine leads to functional thymic‐dependent T‐cell regeneration in age‐associated immune deficiency
Clinical & Translational Immunology (2015)
-
MED19 promotes proliferation and tumorigenesis of lung cancer
Molecular and Cellular Biochemistry (2011)
-
Inhibition of tumor growth and induction of apoptosis in prostate cancer cell lines by overexpression of tissue inhibitor of matrix metalloproteinase-3
Cancer Gene Therapy (2010)
-
Efficient shRNA delivery into B and T lymphoma cells using lentiviral vector-mediated transfer
Journal of Hematopathology (2009)