Construction and Identification of Eukaryotic Expression Vector of pEGFP-N3-TFPI-2_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

 WANGJian ,  WANGXiao-dong , TANGGuan-jie , ZHAOJia

Department of General Surgery, The First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, Liaoning Province, China;

Corresponding author：

WANGXiao-dong, Email: lmup2011@sina.com

Keywords：

Tissue factor pathway inhibitor-2; Eukaryotic expression vector of pEGFP-N3; Double digestion; Sequence analysis; Western blot method

DOI：

10.7507/1007-9424.20140072

Video：

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Objective To construct eukaryotic expression vector of pEGFP-N3-TFPI-2, and to provide the base of studying the function of TFPI-2 gene. Methods Extraction of total RNA from placental tissue was extracted at first, and then reverse transcriptase synthesis of cDNA was carried out. The cDNA fragment of TFPI-2 gene which was obtained by real time PCR (RT-PCR) was inserted into eukaryotic expression vector of pEGFP-N3. After double digestion with XhoⅠand KpnⅠ, the recombinant vector of pEGFP-N3-TFPI-2 was identified in 1% agarose gel electrophoresis and was tested by the sequence analysis. Then, the recombinant vector of pEGFP-N3-TFPI-2 (transfection group) and vector of pEGFP-N3 (blank control group) were transfected into Top10 competent cells with Lipofectamine^TM 2000, but no transfection-related treatment was performed in cells of untransfection group. Western blot method was used to test the expression of TFPI-2 protein in cells of 3 groups. Results The purity of total RNA which were analysis by agarose gel electrophoresis and spectrophotometry were fit for PCR. After coding of TFPI-2 gene fragment and eukaryotic expression vector of pEGFP-N3, the recombinant plasmid of pEGFP-N3-TFPI-2 were got double digestion with XhoⅠand KpnⅠ, and was identified in 1% agarose gel electrophoresis, of which showing that there were 2 specific amplification of strips at 708 bp and 4 700 bp. Result of sequence analysis confirmed that the size of recombinant vector was consistent with the theoretical value. Results of Western blot showed that the expression of TFPI-2 protein in transfection group (0.657 3±0.032 5) was higher than those of blank control group (0.301 7±0.028 7) and untransfection group (0.314 3±0.026 6), P < 0.01. Conclusions The eukaryotic expression vector of pEGFP-N3-TFPI-2 has been constructed successfully, which laiding the foundation for the analysis about function of TFPI-2 gene.

Citation： WANGJian, WANGXiao-dong, TANGGuan-jie, ZHAOJia. Construction and Identification of Eukaryotic Expression Vector of pEGFP-N3-TFPI-2. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2014, 21(3): 300-304. doi: 10.7507/1007-9424.20140072 Copy

1.	王蒙, 汤志刚. TFPI-2的生物学作用及其与肿瘤的关系[J].安徽医药, 2012, 16(7):1016-1017.
2.	Xu C, Wang H, He H, et al. Low expression of TFPI-2 associated with poor survival outcome in patients with breast cancer[J]. BMC Cancer, 2013, 13:118.
3.	Kim Y, Stolarska MA, Othmer HG. The role of the microenvironment in tumor growth and invasion[J]. Prog Biophys Mol Biol, 2011, 106(2):353-379.
4.	Wong CM, Ng YL, Lee JM, et al. Tissue factor pathway in-hibi-tor-2 as a frequently silenced tumor suppressor gene in hepa-toce-llular carcinoma[J]. Hepatology, 2007, 45(5):1129-1138.
5.	George J, Gondi CS, Dinh DH, et al. Restoration of tissue factor pathway inhibitor-2 in a human glioblastoma cell line triggers caspase-mediated pathway and apoptosis[J]. Clin Cancer Res, 2007, 13(12):3507-3517.
6.	Takada H, Wakabayashi N, Dohi O, et al. Tissue factor pathway inhibitor 2(TFPI2) is frequently silenced by aberrant promoter hypermethylation in gastric cancer[J]. Cancer Genet Cytogenet, 2010, 197(1):16-24.
7.	Glöckner SC, Dhir M, Yi JM, et al. Methylation of TFPI2 in stool DNA:a potential novel biomarker for the detection of colorectal cancer[J]. Cancer Res, 2009, 69(11):4691-4699.
8.	Tsunoda S, Smith E, De Young NJ, et al. Methylation of CLDN6, FBN2, RBP1, RBP4, TFPI2, and TMEFF2 in esophageal squamous cell carcinoma[J]. Oncol Rep, 2009, 21(4):1067-1073.
9.	Watanabe A, Hoshino D, Hosino D, et al. Critical role of tran-sient activity of MT1-MMP for ECM degradation in invadopodia[J]. PLoS Comput Biol, 2013, 9(5):e1003086.
10.	Taniwaki K, Fukamachi H, Komori K, et al. Stroma-derived matrix metalloproteinase (MMP)-2 promotes membrane type 1-MMP-dependent tumor growth in mice[J]. Cancer Res, 2007, 67(9):4311-4319.
11.	Maquoi E, Assent D, Detilleux J, et al. MT1-MMP protects breast carcinoma cells against typeⅠcollagen-induced apoptosis[J]. Oncogene, 2012, 31(4):480-493.
12.	Nonaka T, Nishibashi K, Itoh Y, et al. Competitive disruption of the tumor-promoting function of membrane type 1 matrix meta-lloproteinase/matrix metalloproteinase-14 in vivo[J]. Mol Cancer Ther, 2005, 4(8):1157-1166.
13.	仲任, 宋善俊.组织因子途径抑制物2诱导人卵巢癌A2780细胞凋亡及其机制研究[J].华中科技大学学报:医学版, 2010, 39(5):635-638.
14.	Gessler F, Voss V, Seifert V, et al. Knockdown of TFPI-2 prom-otes migration and invasion of glioma cells[J]. Neurosci Lett, 2011, 497(1):49-54.
15.	Altadill A, Rodríguez M, González LO, et al. Liver expression of matrix metalloproteases and their inhibitors in hepatocellular carcinoma[J]. Dig Liver Dis, 2009, 41(10):740-748.
16.	Guo H, Lin Y, Zhang H, et al. Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells[J]. BMC Mol Biol, 2007, 8:110.
17.	Hibi K, Goto T, Kitamura YH, et al. Methylation of the TFPI2 gene is frequently detected in advanced gastric carcinoma[J]. Anticancer Res, 2010, 30(10):4131-4133.
18.	Hibi K, Goto T, Shirahata A, et al. Detection of TFPI2 methylation in the serum of gastric cancer patients[J]. Anticancer Res, 2011, 31(11):3835-3838.
19.	Rollin J, Iochmann S, Bléchet C, et al. Expression and methylation status of tissue factor pathway inhibitor-2 gene in non-small-cell lung cancer[J]. Br J Cancer, 2005, 92(4):775-783.
20.	Iochmann S, Bléchet C, Chabot V, et al. Transient RNA silencing of tissue factor pathway inhibitor-2 modulates lung cancer cell invasion[J]. Clin Exp Metastasis, 2009, 26(5):457-467.
21.	郑浩, 汤志刚, 黄强, 等. TFPI-2基因CpG岛甲基化与胰腺癌的关系[J].实用医学杂志, 2011, 27(24):4396-4398.
22.	Tang Z, Geng G, Huang Q, et al. Expression of tissue factor pathway inhibitor 2 in human pancreatic carcinoma and its effect on tumor growth, invasion, and migration in vitro and in vivo[J]. J Surg Res, 2011, 167(1):62-69.
23.	Xu Y, Qin X, Zhou J, et al. Tissue factor pathway inhibitor-2 inhibits the growth and invasion of hepatocellular carcinoma cells and is inactivated in human hepatocellular carcinoma[J]. Oncol Lett, 2011, 2(5):779-783.
24.	Zhang Q, Zhang Y, Wang SZ, et al. Reduced expression of tissue factor pathway inhibitor-2 contributes to apoptosis and angiogenesis in cervical cancer[J]. J Exp Clin Cancer Res, 2012, 31(1):1.
25.	张慧燕, 刘鸣, 孙亚男, 等.组织途径抑制因子-2基因重组腺病毒载体的构建及其抑制喉鳞癌侵袭的研究[J].中国现代医学杂志, 2009, 19(18):2741-2743.

1. 王蒙, 汤志刚. TFPI-2的生物学作用及其与肿瘤的关系[J].安徽医药, 2012, 16(7):1016-1017.
2. Xu C, Wang H, He H, et al. Low expression of TFPI-2 associated with poor survival outcome in patients with breast cancer[J]. BMC Cancer, 2013, 13:118.
3. Kim Y, Stolarska MA, Othmer HG. The role of the microenvironment in tumor growth and invasion[J]. Prog Biophys Mol Biol, 2011, 106(2):353-379.
4. Wong CM, Ng YL, Lee JM, et al. Tissue factor pathway in-hibi-tor-2 as a frequently silenced tumor suppressor gene in hepa-toce-llular carcinoma[J]. Hepatology, 2007, 45(5):1129-1138.
5. George J, Gondi CS, Dinh DH, et al. Restoration of tissue factor pathway inhibitor-2 in a human glioblastoma cell line triggers caspase-mediated pathway and apoptosis[J]. Clin Cancer Res, 2007, 13(12):3507-3517.
6. Takada H, Wakabayashi N, Dohi O, et al. Tissue factor pathway inhibitor 2(TFPI2) is frequently silenced by aberrant promoter hypermethylation in gastric cancer[J]. Cancer Genet Cytogenet, 2010, 197(1):16-24.
7. Glöckner SC, Dhir M, Yi JM, et al. Methylation of TFPI2 in stool DNA:a potential novel biomarker for the detection of colorectal cancer[J]. Cancer Res, 2009, 69(11):4691-4699.
8. Tsunoda S, Smith E, De Young NJ, et al. Methylation of CLDN6, FBN2, RBP1, RBP4, TFPI2, and TMEFF2 in esophageal squamous cell carcinoma[J]. Oncol Rep, 2009, 21(4):1067-1073.
9. Watanabe A, Hoshino D, Hosino D, et al. Critical role of tran-sient activity of MT1-MMP for ECM degradation in invadopodia[J]. PLoS Comput Biol, 2013, 9(5):e1003086.
10. Taniwaki K, Fukamachi H, Komori K, et al. Stroma-derived matrix metalloproteinase (MMP)-2 promotes membrane type 1-MMP-dependent tumor growth in mice[J]. Cancer Res, 2007, 67(9):4311-4319.
11. Maquoi E, Assent D, Detilleux J, et al. MT1-MMP protects breast carcinoma cells against typeⅠcollagen-induced apoptosis[J]. Oncogene, 2012, 31(4):480-493.
12. Nonaka T, Nishibashi K, Itoh Y, et al. Competitive disruption of the tumor-promoting function of membrane type 1 matrix meta-lloproteinase/matrix metalloproteinase-14 in vivo[J]. Mol Cancer Ther, 2005, 4(8):1157-1166.
13. 仲任, 宋善俊.组织因子途径抑制物2诱导人卵巢癌A2780细胞凋亡及其机制研究[J].华中科技大学学报:医学版, 2010, 39(5):635-638.
14. Gessler F, Voss V, Seifert V, et al. Knockdown of TFPI-2 prom-otes migration and invasion of glioma cells[J]. Neurosci Lett, 2011, 497(1):49-54.
15. Altadill A, Rodríguez M, González LO, et al. Liver expression of matrix metalloproteases and their inhibitors in hepatocellular carcinoma[J]. Dig Liver Dis, 2009, 41(10):740-748.
16. Guo H, Lin Y, Zhang H, et al. Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells[J]. BMC Mol Biol, 2007, 8:110.
17. Hibi K, Goto T, Kitamura YH, et al. Methylation of the TFPI2 gene is frequently detected in advanced gastric carcinoma[J]. Anticancer Res, 2010, 30(10):4131-4133.
18. Hibi K, Goto T, Shirahata A, et al. Detection of TFPI2 methylation in the serum of gastric cancer patients[J]. Anticancer Res, 2011, 31(11):3835-3838.
19. Rollin J, Iochmann S, Bléchet C, et al. Expression and methylation status of tissue factor pathway inhibitor-2 gene in non-small-cell lung cancer[J]. Br J Cancer, 2005, 92(4):775-783.
20. Iochmann S, Bléchet C, Chabot V, et al. Transient RNA silencing of tissue factor pathway inhibitor-2 modulates lung cancer cell invasion[J]. Clin Exp Metastasis, 2009, 26(5):457-467.
21. 郑浩, 汤志刚, 黄强, 等. TFPI-2基因CpG岛甲基化与胰腺癌的关系[J].实用医学杂志, 2011, 27(24):4396-4398.
22. Tang Z, Geng G, Huang Q, et al. Expression of tissue factor pathway inhibitor 2 in human pancreatic carcinoma and its effect on tumor growth, invasion, and migration in vitro and in vivo[J]. J Surg Res, 2011, 167(1):62-69.
23. Xu Y, Qin X, Zhou J, et al. Tissue factor pathway inhibitor-2 inhibits the growth and invasion of hepatocellular carcinoma cells and is inactivated in human hepatocellular carcinoma[J]. Oncol Lett, 2011, 2(5):779-783.
24. Zhang Q, Zhang Y, Wang SZ, et al. Reduced expression of tissue factor pathway inhibitor-2 contributes to apoptosis and angiogenesis in cervical cancer[J]. J Exp Clin Cancer Res, 2012, 31(1):1.
25. 张慧燕, 刘鸣, 孙亚男, 等.组织途径抑制因子-2基因重组腺病毒载体的构建及其抑制喉鳞癌侵袭的研究[J].中国现代医学杂志, 2009, 19(18):2741-2743.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction and Identification of Eukaryotic Expression Vector of pEGFP-N3-TFPI-2

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