Construction of shRNA Expression Vectors for STAT3 Gene and Its Inhibitory Effect on The Growth and Invasion of Gastric Cancer Cells_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

TONG Qiang ¹ , SHU Xiaogang ¹ , LU Xiaoming ¹ , XIAO Yong ¹ , NIU Yanfeng ¹ , LI Weiyong ² , TAO Kaixiong ³ , WANG Guobin ^1,3

1.Department of Gastrointestinal Surgery;;
2.Pharmaceutical Preparation Section;;
3. Department of Laparoscope Surgery;;
Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, ChinaCorresponding Author: TONG Qiang, E-mail: etongqiang@yahoo.com.cn;

Corresponding author：

Keywords：

Signal transducer and activator of transcription 3; Short hairpin RNA; Gastric cancer; Expression vector【Foundation item】 Project of Key Scientific and Technological Foundation of Hubei Province（No.2006AA301A05）

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Objective To study the effect of knockdown of signal transducer and activator of transcription 3 (STAT3) expression by short hairpin RNA (shRNA) on proliferation, apoptosis and invasion of human gastric cancer cell line MKN-45 in vitro . Methods Specific shRNA plasmids to STAT3 were constructed, and then transfected into MKN-45 cells by lipofectamine methods. Cells were divided into three groups: control group, psiRNA-H1 transfected group as negative group and psiRNA-H1/STAT3 transfected group. Semi-quantitative RT-PCR and Western blotting were used to detect the expression of STAT3 mRNA and protein, respectively. Proliferation and apoptosis of the transfected cells were observed by methyl thiazolyl tetrazolium (MTT) method and flow cytometry (FCM), respectively. The invasion of the transfected MKN-45 cells was measured by Boyden chamber. Results Compared with the negative control cells, semi-quantitative RT-PCR and Western blotting showed that the expressions of STAT3 mRNA and protein were down-regulated in the psiRNA-H1/STAT3 transfected group ( P ＜ 0.05) . The subcloned recombinant plasmid expressing shRNA effectively inhibited MKN-45 cell growth and proliferation while empty plasmid had no such specific effect. Cell apoptosis rate increased significantly in psiRNA-H1/STAT3 transfected group （ P ＜ 0.01）, and the invasion of MKN-45 cells was efficiently inhabited in psiRNA-H1/STAT3 transfected group as compared with control group and psiRNA-H1 transfected group( P ＜ 0.01).Conclusion Recombinant plasmid psiRNA-H1/STAT3 shRNA significantly inhibits the proliferation and invasion of MKN-45 cells and promotes their apoptosis.

Citation： TONG Qiang,SHU Xiaogang,LU Xiaoming,XIAO Yong,NIU Yanfeng,LI Weiyong,TAO Kaixiong,WANG Guobin. Construction of shRNA Expression Vectors for STAT3 Gene and Its Inhibitory Effect on The Growth and Invasion of Gastric Cancer Cells. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2009, 16(1): 6-11. doi: Copy

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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.	刘孟刚，杨俊涛，刘宏鸣，等. GA 反义基因对裸鼠胃癌移植瘤细胞凋亡的影响 [Ｊ] . 中国普外基础与临床杂志， 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. 刘孟刚，杨俊涛，刘宏鸣，等. GA 反义基因对裸鼠胃癌移植瘤细胞凋亡的影响 [Ｊ] . 中国普外基础与临床杂志， 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.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction of shRNA Expression Vectors for STAT3 Gene and Its Inhibitory Effect on The Growth and Invasion of Gastric Cancer Cells

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