Construction and Identification of Dual Target-Regulated Lentiviral Vector of Colorectal Cancer Suppressor Gene CDX2_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

 ZHENGJian-bao ¹ , HESai ¹ ,  SUNXue-jun ¹ , RENYan-fei ¹ , CHENNan-zheng ¹ , ZHANGShi-yun ¹ , LIUDong ¹ , ZHANGLi ² , WANGHui ³

1. Department of General Surgery, The First Affiliated Hospital of Medical College Xi'an Jiaotong University, Xi'an 710061, Shannxi Province, China;
2. Department of General Surgery, The Second Affiliated Hospital of Medical College Xi'an Jiaotong University, Xi'an 710004, Shannxi Province, China;
3. Department of Anesthesiology, Shaanxi Provincial People's Hospital, Xi'an 710068, Shannxi Province, China;

Corresponding author：

SUNXue-jun, Email: sunxy@mail.xjtu.edu.cn

Keywords：

Hypoxia; Telomerase; Lentiviral vector; Gene therapy; Colorectal cancer

DOI：

10.7507/1007-9424.20140102

Video：

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Objective To build a lentiviral expression vector regulated by two targets 5 copies of HREs and hTERTp, express the target gene CDX2, and to test the activity of hTERT promoter by using LoVo cells for transfection. Methods After the primer sets were designed, the hTERT promoter was cloned by PCR amplification from the genome of colon cancer. The CEA promoter was removed from the original vector pLEGFP-5HRE-CEAp by double digestion and PCR method, and then the hTERTp was introduced into the vector to construct the recombinant plasmid pLEGFP-5HRE-hTERTp. 5HRE-hTERTp was obtained by PCR, while the CMV promoter was removed from the original vector pLVX-EGFP-3FLAG by double digestion and PCR method, and then the 5HRE-hTERTp was introduced into the vector to construct the recombinant plasmid pLVX-5HRE-hTERTp-EGFP-3FLAG. The CDX2 was cloned by PCR amplification from GV230-CDX2-EGFP, and the EGFP was removed from the vector pLVX-5HRE-hTERTp-EGFP-3FLAG by double digestion, and then the CDX2 was introduced into the vector to construct the recombinant plasmid pLVX-5HRE-hTERTp-CDX2-3FLAG. LoVo cells ex vivo was transiently transfected by pLVX-5HRE-hTERTp-EGFP-3FLAG to evaluate the activity of hTERTp by detecting the expression of green fluorescence protein EGFP. Results PCR and sequencing analyzing showed that pLEGFP-5HRE-hTERTp, pLVX-5HRE-hTERTp-EGFP-3FLAG, and pLVX-5HRE-hTERTp-CDX2-3FLAG were sequenced correctly and the same as our designed. pLVX-5HRE-hTERTp-EGFP-3FLAG was successfully transfected into LoVo cells ex vivo and expressed green fluorescence protein EGFP, which showed that hTERTp was activated and promoted the expression of downstream gene. Conclusion The lentiviral expression vector, pLVX-5HREhTERTp-EGFP-3FLAG and pLVX-5HRE-hTERTp-CDX2-3FLAG are successfully constructed, which lays the foundation of further research. But the function of dual-target regulation needs further proof.

Citation： ZHENGJian-bao, HESai, SUNXue-jun, RENYan-fei, CHENNan-zheng, ZHANGShi-yun, LIUDong, ZHANGLi, WANGHui. Construction and Identification of Dual Target-Regulated Lentiviral Vector of Colorectal Cancer Suppressor Gene CDX2. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2014, 21(4): 414-419. doi: 10.7507/1007-9424.20140102 Copy

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14.	Wang W, Jin B, Li W, et al. Targeted antitumor effect induced by hTERT promoter mediated ODC antisense adenovirus[J]. Mol Biol Rep, 2009, 37(7):3239-3247.
15.	Nemunaitis J, Tong AW, Nemunaitis M, et al. A phase I study of telomerase-specific replication competent oncolytic adenovirus (telomelysin) for various solid tumors[J]. Mol Ther, 2010, 18(2):429-434.
16.	Bougel S, Renaud S, Braunschweig R, et al. PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells[J]. J Pathol, 2010, 220(1):87-96.
17.	Li JT, Bian K, Zhang AL, et al. Targeting different types of human meningioma and glioma cells using a novel adenoviral vector expressing GFP-TRAIL fusion protein from hTERT promoter[J]. Cancer Cell Int, 2011, 11(1):35-48.
18.	王宏芳, 刘扬, 李金华, 等.双重靶向条件复制型腺病毒的包装及抗肿瘤作用[J].西安交通大学学报:医学版, 2013, 33(3):308-312.
19.	Zhang P, Tan J, Yang DB, et al. Gene therapy using the human telomerase catalytic subunit gene promoter enables targeting of the therapeutic effects of vesicular stomatitis virus matrix protein against human lung adenocarcinoma[J]. Exp Ther Med, 2012, 4(5):859-864.
20.	Hashimoto Y, Tazawa H, Teraish F, et al. The hTERT promoter enhances the antitumor activity of an oncolytic adenovirus under a hypoxic microenvironment[J]. PLoS One, 2012, 7(6):e39292.
21.	郑见宝, 孙学军, 王炜, 等. HIF-1α与CDX2在结直肠腺癌组织中的表达及意义[J].中国普外基础与临床杂志, 2010, 17(12):1113-1117.
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23.	李守帅, 孙学军, 郑见宝, 等.尾型同源盒基因-2过表达对结肠癌细胞生物学性状的影响[J].西安交通大学学报:医学版, 2011, 32(1):114-118.
24.	Zheng JB, Sun XJ, Wang W, et al. Hypoxia-inducible factor-1αmodulates the down-regulation of the homeodomain protein CDX2 in colorectal cancer[J]. Oncol Rep, 2010, 24(1):97-104.
25.	郑见宝, 孙学军, 王炜, 等.缺氧下调结肠癌细胞株CDX2表达的研究[J].中国普通外科杂志, 2010, 19(4):364-368.

1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013[J]. CA Cancer J Clin, 2013, 63(1):11-30.
2. Laszlo L. Predictive and Prognostric Factors in the complex treatment of patients with colorectal cancer[J]. Magy Onkol, 2010, 54(4):383-394.
3. 中华人民共和国卫生部.中国癌症预防与控制规划纲要(2004~2010)[J].中国肿瘤, 2004, 13(2):65-68.
4. Shayakhmetov DM, Di Paolo NC, Mossman KL. Recognition of virus infection and innate host responses to viral gene therapy vectors[J]. Mol Ther, 2010, 18(8):1422-1429.
5. Chen EQ, Song XQ, Wang YL, et al. Construction of a highly-active, liver-specific transcriptional regulatory element through combination of the albumin promoter andα-fetoprotein enhancer[J]. Plasmid, 2011, 65(2):125-131.
6. Dong K, Wang R, Wang X, et al. Tumor-specific RNAi targeting eIF4E suppresses tumor growth, induces apoptosis and enhances cisplatin cytotoxicity in human breast carcinoma cells[J]. Breast Cancer Res Treat, 2009, 113(3):443-456.
7. Asayama M, Kadowaki S, Yamaguchi K. Issues of molecular targeted therapies in combination with chemotherapy in metastatic colorectal cancer[J]. Nihon Rinsho, 2011, 69(3):464-471.
8. Law AYS, Ching LY, Lai KP, et al. Identification and characterization of the hypoxia-responsive element in human stanniocalcin-1 gene[J]. Mol Cell Endocrinol, 2010, 314(1):118-127.
9. Shi Q, Zhang P, Zhang J, et al. Adenovirus-mediated brain-derived neurotrophic factor expression regulated by hypoxia response element protects brain from injury of transient middle cerebral artery occlusion in mice[J]. Neurosci Lett, 2009, 465(3):220-225.
10. Wang W, Sun XJ, Lu L, et al. Cytotoxicity of lymphocytes activ-ated by superantigen toxic-shock-syndrome toxin-1 against colorectal cancer LoVo cells[J]. Mol Cell Biochem, 2013, 376(1-2):1-9.
11. Marignol L, Foley R, Southgate TD, et al. Hypoxia response element-driven cytosine deaminase/5-fluorocytosine gene therapy system:a highly effective approach to overcome the dynamics of tumour hypoxia and enhance the radiosensitivity of prostate cancer cells in vitro[J]. J Gene Med, 2009, 11(2):169-179.
12. Ruan H, Su H, Hu L, et al. A hypoxia-regulated adeno-associa-ted virus vector for cancer-specific gene therapy[J]. Neoplasia, 2001, 3(3):255-263.
13. 范永卫, 杨赤, 万玉良, 等.人端粒酶逆转录酶启动子介导的靶向性肿瘤基因治疗的研究进展[J].第四军医大学学报, 2009, 30(12):1149-1151.
14. Wang W, Jin B, Li W, et al. Targeted antitumor effect induced by hTERT promoter mediated ODC antisense adenovirus[J]. Mol Biol Rep, 2009, 37(7):3239-3247.
15. Nemunaitis J, Tong AW, Nemunaitis M, et al. A phase I study of telomerase-specific replication competent oncolytic adenovirus (telomelysin) for various solid tumors[J]. Mol Ther, 2010, 18(2):429-434.
16. Bougel S, Renaud S, Braunschweig R, et al. PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells[J]. J Pathol, 2010, 220(1):87-96.
17. Li JT, Bian K, Zhang AL, et al. Targeting different types of human meningioma and glioma cells using a novel adenoviral vector expressing GFP-TRAIL fusion protein from hTERT promoter[J]. Cancer Cell Int, 2011, 11(1):35-48.
18. 王宏芳, 刘扬, 李金华, 等.双重靶向条件复制型腺病毒的包装及抗肿瘤作用[J].西安交通大学学报:医学版, 2013, 33(3):308-312.
19. Zhang P, Tan J, Yang DB, et al. Gene therapy using the human telomerase catalytic subunit gene promoter enables targeting of the therapeutic effects of vesicular stomatitis virus matrix protein against human lung adenocarcinoma[J]. Exp Ther Med, 2012, 4(5):859-864.
20. Hashimoto Y, Tazawa H, Teraish F, et al. The hTERT promoter enhances the antitumor activity of an oncolytic adenovirus under a hypoxic microenvironment[J]. PLoS One, 2012, 7(6):e39292.
21. 郑见宝, 孙学军, 王炜, 等. HIF-1α与CDX2在结直肠腺癌组织中的表达及意义[J].中国普外基础与临床杂志, 2010, 17(12):1113-1117.
22. Zheng JB, Sun XJ, Qi J, et al. Effects of homeodomain protein CDX2 expression on the proliferation and migration of Lovo colon cancer cells[J]. Pathol Oncol Res, 2011, 17(3):743-751.
23. 李守帅, 孙学军, 郑见宝, 等.尾型同源盒基因-2过表达对结肠癌细胞生物学性状的影响[J].西安交通大学学报:医学版, 2011, 32(1):114-118.
24. Zheng JB, Sun XJ, Wang W, et al. Hypoxia-inducible factor-1αmodulates the down-regulation of the homeodomain protein CDX2 in colorectal cancer[J]. Oncol Rep, 2010, 24(1):97-104.
25. 郑见宝, 孙学军, 王炜, 等.缺氧下调结肠癌细胞株CDX2表达的研究[J].中国普通外科杂志, 2010, 19(4):364-368.

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CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction and Identification of Dual Target-Regulated Lentiviral Vector of Colorectal Cancer Suppressor Gene CDX2

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