Construction and activity identification of luciferase reporter containing human <i>CTGF</i> gene promoter_West China Medical Journal

Authors：

ZHU Guonian ¹ , ZHONG Lichun ² , WU Sisi ¹ , TIAN Xin ² ,  TANG Xiaoju ²

1. Research Core Facility, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
2. Department of Respiratory and Critical Care Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding author：

TANG Xiaoju, Email: tangxiaoju888@hotmail.com

Keywords：

CTGF gene; Promoter; Luciferase; Reporter gene; Pulmonary fibrosis

DOI：

10.7507/1002-0179.201909159

Video：

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Objective To construct a luciferase reporter fusion containing the human connective tissue growth factor (CTGF) gene promoter.Methods The promoter region of the human CTGF gene (-835/+214) was amplified by polymerase chain reaction (PCR) using specially-designed primers, and subsequently cloned into the pGL3.0-Basic vector. Following screening and verification by single colony PCR, double digestion, and sequencing, the resulting pGL3.0-Basic-CTGF was used to transfect the human embryonic kidney cells 293T, human bronchial epithelial cells HBE and human lung epithelial cells A549, and its function in each cell line was determined by luciferase assay.Results Sequence alignment showed 99.5% identity, suggesting successful construction of the pGL3.0-Basic-CTGF reporter fusion. Promoter activities were detected 48 hours after transfection of pGL3.0-Basic-CTGF into the 293T, HBE, and A549 cells, and the promoter activities were 2.416, 0.027, and 0.121, respectively (P<0.01). Moreover, the luciferase activity in the A549 cells was statistically higher than that in the HBE cells (P<0.01).Conclusions The human pGL3.0-Basic-CTGF luciferase reporter fusion has been successfully constructed. The construct exhibits promoter activity in the bronchial epithelial cells HBE and the lung epithelial cells A549, and can therefore serve as a useful tool for future research in transcriptional regulation.

Citation： ZHU Guonian, ZHONG Lichun, WU Sisi, TIAN Xin, TANG Xiaoju. Construction and activity identification of luciferase reporter containing human CTGF gene promoter. West China Medical Journal, 2020, 35(1): 22-27. doi: 10.7507/1002-0179.201909159 Copy

1.	刘张红, 程锦国, 林文博, 等. 肾病Ⅰ号方对肾间质纤维化大鼠 TGF-β1 和 CTGF 表达的影响. 浙江中医药大学学报, 2019, 43(11): 1258-1265.
2.	Brigstock DR. The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev, 1999, 20(2): 189-206.
3.	符红娜, 孙兆瑞, 葛鑫, 等. 结缔组织生长因子在百草枯致肺纤维化中的作用. 中华急诊医学杂志, 2018, 27(11): 1198-1204.
4.	全国卫管协会转化医学产业分会. 特发性肺纤维化的新药物和靶点之二: Pamrevlumab和CTGF. (2017-12-15)[2019-09-01]. http://dy.163.com/v2/article/detail/D5MI21AA0514BKOA.html.
5.	胡昇庠, 王崇高. CTGF 在胃癌中的表达及其促进血管生成的机制. 局解手术学杂志, 2011, 20(6): 612-615.
6.	俞建顺, 蔡丹莉, 严茂祥, 等. MNNG 诱导的胃癌前病变大鼠胃黏膜 CTGF 及 ET-1 表达的变化. 浙江临床医学, 2019, 21(2): 184-186.
7.	Gibson DJ, Pi L, Sriram S, et al. Conditional knockout of CTGF affects corneal wound healing. Invest Ophthalmol Vis Sci, 2014, 55(4): 2062-2070.
8.	张曼曼, 王恒, 赵逵. CYR61 与结肠癌发生发展的关系. 山东医药, 2013, 20: 95-96.
9.	Babic AM, Chen CC, Lau IF. Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol, 1999, 19(4): 2958-2966.
10.	Grotendorst GR, Okochi H, Hayashi N. A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor gene. Cell Growth Differ, 1996, 7(4): 469-480.
11.	杨莉, 李晓玫, 王海燕. 结缔组织生长因子在关木通相关肾小管间质肾病肾间质纤维化中的作用. 中华肾脏病杂志, 2003, 19(4): 19-24.
12.	朱祎, 唐英, 何立群. 大黄酚对单侧输尿管梗阻模型大鼠肾组织 CTGF、α-SMA 和 FN 的影响. 中南药学, 2014, 12(7): 631-634.
13.	胡锐, 陈振兵, 贾中尉, 等. 结缔组织生长因子抗体抑制慢性卡压后神经纤维化的研究. 中华实验外科杂志, 2012, 29(11): 2259-2262.
14.	赵敏, 徐安莉, 周艳艳, 等. 温补肾阳法对肾阳虚肺纤维化大鼠肺内 CTGF 表达的影响. 中国老年学杂志, 2015, 35(2): 421-424.
15.	Lipson KE, Wong C, Teng Y, et al. CTGF is a central mediator of tissue remodeling and fibrosis and its inhibition can reverse the process of fibrosis. Fibrogenesis Tissue Repair, 2012, 5(Suppl 1): S24.
16.	Solberg N, Krauss S. Luciferase assay to study the activity of a cloned promoter DNA fragment. Methods Mol Biol, 2013, 977: 65-78.

1. 刘张红, 程锦国, 林文博, 等. 肾病Ⅰ号方对肾间质纤维化大鼠 TGF-β1 和 CTGF 表达的影响. 浙江中医药大学学报, 2019, 43(11): 1258-1265.
2. Brigstock DR. The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev, 1999, 20(2): 189-206.
3. 符红娜, 孙兆瑞, 葛鑫, 等. 结缔组织生长因子在百草枯致肺纤维化中的作用. 中华急诊医学杂志, 2018, 27(11): 1198-1204.
4. 全国卫管协会转化医学产业分会. 特发性肺纤维化的新药物和靶点之二: Pamrevlumab和CTGF. (2017-12-15)[2019-09-01]. http://dy.163.com/v2/article/detail/D5MI21AA0514BKOA.html.
5. 胡昇庠, 王崇高. CTGF 在胃癌中的表达及其促进血管生成的机制. 局解手术学杂志, 2011, 20(6): 612-615.
6. 俞建顺, 蔡丹莉, 严茂祥, 等. MNNG 诱导的胃癌前病变大鼠胃黏膜 CTGF 及 ET-1 表达的变化. 浙江临床医学, 2019, 21(2): 184-186.
7. Gibson DJ, Pi L, Sriram S, et al. Conditional knockout of CTGF affects corneal wound healing. Invest Ophthalmol Vis Sci, 2014, 55(4): 2062-2070.
8. 张曼曼, 王恒, 赵逵. CYR61 与结肠癌发生发展的关系. 山东医药, 2013, 20: 95-96.
9. Babic AM, Chen CC, Lau IF. Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol, 1999, 19(4): 2958-2966.
10. Grotendorst GR, Okochi H, Hayashi N. A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor gene. Cell Growth Differ, 1996, 7(4): 469-480.
11. 杨莉, 李晓玫, 王海燕. 结缔组织生长因子在关木通相关肾小管间质肾病肾间质纤维化中的作用. 中华肾脏病杂志, 2003, 19(4): 19-24.
12. 朱祎, 唐英, 何立群. 大黄酚对单侧输尿管梗阻模型大鼠肾组织 CTGF、α-SMA 和 FN 的影响. 中南药学, 2014, 12(7): 631-634.
13. 胡锐, 陈振兵, 贾中尉, 等. 结缔组织生长因子抗体抑制慢性卡压后神经纤维化的研究. 中华实验外科杂志, 2012, 29(11): 2259-2262.
14. 赵敏, 徐安莉, 周艳艳, 等. 温补肾阳法对肾阳虚肺纤维化大鼠肺内 CTGF 表达的影响. 中国老年学杂志, 2015, 35(2): 421-424.
15. Lipson KE, Wong C, Teng Y, et al. CTGF is a central mediator of tissue remodeling and fibrosis and its inhibition can reverse the process of fibrosis. Fibrogenesis Tissue Repair, 2012, 5(Suppl 1): S24.
16. Solberg N, Krauss S. Luciferase assay to study the activity of a cloned promoter DNA fragment. Methods Mol Biol, 2013, 977: 65-78.

West China Medical Journal

Construction and activity identification of luciferase reporter containing human CTGF gene promoter

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