• 1.華中科技大學同濟醫(yī)學院附屬協(xié)和醫(yī)院胃腸外科（武漢430022）;;
  • 2.華中科技大學同濟醫(yī)學院附屬協(xié)和醫(yī)院藥劑科（武漢430022）;;
  • 3.華中科技大學同濟醫(yī)學院附屬協(xié)和醫(yī)院腔鏡外科（武漢430022）;

目的 構(gòu)建信號傳導及轉(zhuǎn)錄活化因子3（STAT 3）基因短發(fā)夾RNA（shRNA）表達質(zhì)粒，探討STAT 3抑制后對胃癌MKN-45細胞增殖、凋亡和侵襲的影響。
方法 根據(jù)STAT 3 mRNA 編碼序列，設(shè)計RNA干擾靶點，構(gòu)建STAT 3基因的特異性小RNA干擾質(zhì)粒 （psiRNA-H1/STAT 3） ，使用脂質(zhì)體轉(zhuǎn)染MKN-45細胞，實驗分為對照組、psiRNA-H1轉(zhuǎn)染組和psiRNA-H1/STAT 3轉(zhuǎn)染組。通過RT-PCR和Western blot檢測STAT 3特異性小RNA干擾基因?qū)ξ赴┘毎鸖TAT 3 mRNA和蛋白表達的影響； MTT比色法檢測細胞的生長抑制率； 采用流式細胞術(shù)檢測轉(zhuǎn)染后對MKN-45細胞凋亡的影響； 采用Boyden小室侵襲實驗檢測轉(zhuǎn)染后MKN-45細胞侵襲力的變化。
結(jié)果 成功轉(zhuǎn)染重組體的MKN-45細胞中STAT 3 mRNA和蛋白表達明顯下降（ P ＜ 0.05）。psiRNA-H1/STAT 3轉(zhuǎn)染組各時相均明顯抑制MKN-45細胞生長，且MKN-45細胞凋亡率為34.26% ，相比對照組（3.75% ）和psiRNA-H1轉(zhuǎn)染組（4.43% ）明顯升高（ P ＜ 0.01） 。另外，psiRNA-H1/STAT 3轉(zhuǎn)染組MKN-45細胞侵襲力較另外2組明顯減弱（ P ＜ 0.01）。
結(jié)論 成功構(gòu)建了針對STAT 3基因的shRNA表達載體，通過轉(zhuǎn)染MKN-45細胞，在體外能有效抑制人胃癌細胞STAT 3 mRNA和蛋白表達，細胞增殖、侵襲能力減弱并且促進細胞凋亡，為STAT 3基因靶向治療提供一定的實驗依據(jù)。

引用本文： 童強,舒曉剛,盧曉明,肖勇,牛彥鋒,黎維勇,陶凱雄,王國斌. STAT3基因短發(fā)卡RNA表達質(zhì)粒的構(gòu)建及其對胃癌細胞生長和侵襲的抑制作用. 中國普外基礎(chǔ)與臨床雜志, 2009, 16(1): 6-11. doi: 復制

1. Bowman T, Garcia R, Turkson J, et al . STATs in oncogenesis [Ｊ] . Oncogene, 2000; 19(21)：2474.
2. Buettner R, Mora LB, Jove R. Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention [Ｊ] . Clin Cancer Res, 2002; 8(4)：945.
3. Clevenger CV. Roles and regulation of stat family transcription factors in human breast cancer [Ｊ] . Am J Pathol, 2004; 165 (5)：1449.
4. Turkson J, Jove R. STAT proteins: novel molecular targets for cancer drug discovery [Ｊ] . Oncogene, 2000; 19(56)：6613.
5. 劉孟剛， 楊俊濤， 劉宏鳴， 等. GA 反義基因?qū)β闶笪赴┮浦擦黾毎蛲龅挠绊?[Ｊ] . 中國普外基礎(chǔ)與臨床雜志， 2008； 15 (1)： 4.
6. Niu G, Wright KL, Huang M, et al . Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis [Ｊ] . Oncogene, 2002; 21(13)：2000.
7. Garcia R, Bowman TL, Niu G, et al . Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells [Ｊ] . Oncogene, 2001; 20(20)：2499.
8. Niu G, Bowman T, Huang M, et al . Roles of activated Src and Stat3 signaling in melanoma tumor cell growth [Ｊ] . Oncogene, 2002; 21(46)：7001.
9. Niu G, Shain KH, Huang M, et al . Overexpression of a dominant-negative signal transducer and activator of transcription 3 variant in tumor cells leads to production of soluble factors that induce apoptosis and cell cycle arrest [Ｊ] . Cancer Res, 2001; 61(8)：3276.
10. Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells [Ｊ] . Proc Natl Acad Sci USA, 2002; 99(9)：6047.
11. Tsareva SA, Moriggl R, Corvinus FM, et al . Signal transducer and activator of transcription 3 activation promotes invasive growth of colon carcinomas through matrix metalloproteinase induction [Ｊ] . Neoplasia, 2007; 9(4)：279.
12. Jung JE, Lee HG, Cho IH, et al . STAT3 is a potential modulator of HIF-1-mediated VEGF expression in human renal carcinoma cells [Ｊ] . FASEB J, 2005; 19(10)：1296.
13. Konnikova L, Kotecki M, Kruger MM, et al . Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells [Ｊ] . BMC Cancer, 2003; 3(2)：23.
14. Hsieh FC, Cheng G, Lin J. Evaluation of potential Stat3-regulated genes in human breast cancer [Ｊ] . Biochem Biophys Res Commun, 2005; 335(2)：292.
15. Kim BH, Yin CH, Guo Q, et al . A small-molecule compound identified through a cell-based screening inhibits JAK/STAT pathway signaling in human cancer cells [Ｊ] . Mol Cancer Ther, 2008; 7(9)：2672.
16. Choi SC, Kim SJ, Choi JH, et al . Fibroblast growth factor-2 and -4 promote the proliferation of bone marrow mesenchymal stem cells by the activation of the PI3K-Akt and ERK1/2 signaling pathways [Ｊ] . Stem Cells Dev, 2008; 17(4)：725.
17. Sheen-Chen SM, Huang CC, Tang RP, et al . Prognostic value of signal transducers and activators of transcription 3 in breast cancer [Ｊ] . Cancer Epidemiol Biomarkers Prev, 2008; 17(9) ∶2286.
18. Brasier AR. Expanding role of cyclin dependent kinases in cytokine inducible gene expression [Ｊ] . Cell Cycle, 2008; 7(17) ∶2661.
19. Niu G, Briggs J, Deng J, et al . Signal transducer and activator of transcription 3 is required for hypoxia-inducib le factor-1alpha RNA expression in both tumor cells and tumor-associated myeloid cells [Ｊ] . Mol Cancer Res, 2008; 6(7)：1099.
20. Fielding CA, McLoughlin RM, McLeod L, et al . IL-6 regulates neutrophil trafficking during acute inflammation via STAT3 [Ｊ] . J Immunol, 2008; 181(3)：2189.
  1. 1. Bowman T, Garcia R, Turkson J, et al . STATs in oncogenesis [Ｊ] . Oncogene, 2000; 19(21)：2474.
  2. 2. Buettner R, Mora LB, Jove R. Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention [Ｊ] . Clin Cancer Res, 2002; 8(4)：945.
  3. 3. Clevenger CV. Roles and regulation of stat family transcription factors in human breast cancer [Ｊ] . Am J Pathol, 2004; 165 (5)：1449.
  4. 4. Turkson J, Jove R. STAT proteins: novel molecular targets for cancer drug discovery [Ｊ] . Oncogene, 2000; 19(56)：6613.
  5. 5. 劉孟剛， 楊俊濤， 劉宏鳴， 等. GA 反義基因?qū)β闶笪赴┮浦擦黾毎蛲龅挠绊?[Ｊ] . 中國普外基礎(chǔ)與臨床雜志， 2008； 15 (1)： 4.
  6. 6. Niu G, Wright KL, Huang M, et al . Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis [Ｊ] . Oncogene, 2002; 21(13)：2000.
  7. 7. Garcia R, Bowman TL, Niu G, et al . Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells [Ｊ] . Oncogene, 2001; 20(20)：2499.
  8. 8. Niu G, Bowman T, Huang M, et al . Roles of activated Src and Stat3 signaling in melanoma tumor cell growth [Ｊ] . Oncogene, 2002; 21(46)：7001.
  9. 9. Niu G, Shain KH, Huang M, et al . Overexpression of a dominant-negative signal transducer and activator of transcription 3 variant in tumor cells leads to production of soluble factors that induce apoptosis and cell cycle arrest [Ｊ] . Cancer Res, 2001; 61(8)：3276.
  10. 10. Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells [Ｊ] . Proc Natl Acad Sci USA, 2002; 99(9)：6047.
  11. 11. Tsareva SA, Moriggl R, Corvinus FM, et al . Signal transducer and activator of transcription 3 activation promotes invasive growth of colon carcinomas through matrix metalloproteinase induction [Ｊ] . Neoplasia, 2007; 9(4)：279.
  12. 12. Jung JE, Lee HG, Cho IH, et al . STAT3 is a potential modulator of HIF-1-mediated VEGF expression in human renal carcinoma cells [Ｊ] . FASEB J, 2005; 19(10)：1296.
  13. 13. Konnikova L, Kotecki M, Kruger MM, et al . Knockdown of STAT3 expression by RNAi induces apoptosis in astrocytoma cells [Ｊ] . BMC Cancer, 2003; 3(2)：23.
  14. 14. Hsieh FC, Cheng G, Lin J. Evaluation of potential Stat3-regulated genes in human breast cancer [Ｊ] . Biochem Biophys Res Commun, 2005; 335(2)：292.
  15. 15. Kim BH, Yin CH, Guo Q, et al . A small-molecule compound identified through a cell-based screening inhibits JAK/STAT pathway signaling in human cancer cells [Ｊ] . Mol Cancer Ther, 2008; 7(9)：2672.
  16. 16. Choi SC, Kim SJ, Choi JH, et al . Fibroblast growth factor-2 and -4 promote the proliferation of bone marrow mesenchymal stem cells by the activation of the PI3K-Akt and ERK1/2 signaling pathways [Ｊ] . Stem Cells Dev, 2008; 17(4)：725.
  17. 17. Sheen-Chen SM, Huang CC, Tang RP, et al . Prognostic value of signal transducers and activators of transcription 3 in breast cancer [Ｊ] . Cancer Epidemiol Biomarkers Prev, 2008; 17(9) ∶2286.
  18. 18. Brasier AR. Expanding role of cyclin dependent kinases in cytokine inducible gene expression [Ｊ] . Cell Cycle, 2008; 7(17) ∶2661.
  19. 19. Niu G, Briggs J, Deng J, et al . Signal transducer and activator of transcription 3 is required for hypoxia-inducib le factor-1alpha RNA expression in both tumor cells and tumor-associated myeloid cells [Ｊ] . Mol Cancer Res, 2008; 6(7)：1099.
  20. 20. Fielding CA, McLoughlin RM, McLeod L, et al . IL-6 regulates neutrophil trafficking during acute inflammation via STAT3 [Ｊ] . J Immunol, 2008; 181(3)：2189.