STAT3基因短發卡RNA表達質粒的構建及其對胃癌細胞生長和侵襲的抑制作用_《中國普外基礎與臨床雜志》

作者：

童強 ¹ , 舒曉剛 ¹ , 盧曉明 ¹ , 肖勇 ¹ , 牛彥鋒 ¹ , 黎維勇 ² , 陶凱雄 ³ , 王國斌 ^1,3

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

關鍵詞：

信號傳導及轉錄活化因子3 短發夾RNA 胃癌表達載體

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目的構建信號傳導及轉錄活化因子3（STAT 3）基因短發夾RNA（shRNA）表達質粒，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STAT 3 mRNA 編碼序列，設計RNA干擾靶點，構建STAT 3基因的特異性小RNA干擾質粒（psiRNA-H1/STAT 3），使用脂質體轉染MKN-45細胞，實驗分為對照組、psiRNA-H1轉染組和psiRNA-H1/STAT 3轉染組。通過RT-PCR和Western blot檢測STAT 3特異性小RNA干擾基因對胃癌細胞STAT 3 mRNA和蛋白表達的影響； MTT比色法檢測細胞的生長抑制率；采用流式細胞術檢測轉染后對MKN-45細胞凋亡的影響；采用Boyden小室侵襲實驗檢測轉染后MKN-45細胞侵襲力的變化。
結果成功轉染重組體的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轉染組（4.43% ）明顯升高（ P ＜ 0.01）。另外，psiRNA-H1/STAT 3轉染組MKN-45細胞侵襲力較另外2組明顯減弱（ P ＜ 0.01）。
結論 成功構建了針對STAT 3基因的shRNA表達載體，通過轉染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。

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基因短發卡RNA表達質粒的構建及其對胃癌細胞生長和侵襲的抑制作用. 中國普外基礎與臨床雜志, 2009, 16(1): 6-11. doi: 復制

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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 反義基因對裸鼠胃癌移植瘤細胞凋亡的影響 [Ｊ] . 中國普外基礎與臨床雜志， 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. 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 反義基因對裸鼠胃癌移植瘤細胞凋亡的影響 [Ｊ] . 中國普外基礎與臨床雜志， 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.

《中國普外基礎與臨床雜志》

STAT3基因短發卡RNA表達質粒的構建及其對胃癌細胞生長和侵襲的抑制作用

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《中國普外基礎與臨床雜志》

STAT3基因短發卡RNA表達質粒的構建及其對胃癌細胞生長和侵襲的抑制作用

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

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