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.	劉張紅, 程錦國, 林文博, 等. 腎病Ⅰ號方對腎間質纖維化大鼠 TGF-β1 和 CTGF 表達的影響. 浙江中醫藥大學學報, 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.	符紅娜, 孫兆瑞, 葛鑫, 等. 結締組織生長因子在百草枯致肺纖維化中的作用. 中華急診醫學雜志, 2018, 27(11): 1198-1204.
4.	全國衛管協會轉化醫學產業分會. 特發性肺纖維化的新藥物和靶點之二: 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 在胃癌中的表達及其促進血管生成的機制. 局解手術學雜志, 2011, 20(6): 612-615.
6.	俞建順, 蔡丹莉, 嚴茂祥, 等. MNNG 誘導的胃癌前病變大鼠胃黏膜 CTGF 及 ET-1 表達的變化. 浙江臨床醫學, 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.	張曼曼, 王恒, 趙逵. CYR61 與結腸癌發生發展的關系. 山東醫藥, 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.	楊莉, 李曉玫, 王海燕. 結締組織生長因子在關木通相關腎小管間質腎病腎間質纖維化中的作用. 中華腎臟病雜志, 2003, 19(4): 19-24.
12.	朱祎, 唐英, 何立群. 大黃酚對單側輸尿管梗阻模型大鼠腎組織 CTGF、α-SMA 和 FN 的影響. 中南藥學, 2014, 12(7): 631-634.
13.	胡銳, 陳振兵, 賈中尉, 等. 結締組織生長因子抗體抑制慢性卡壓后神經纖維化的研究. 中華實驗外科雜志, 2012, 29(11): 2259-2262.
14.	趙敏, 徐安莉, 周艷艷, 等. 溫補腎陽法對腎陽虛肺纖維化大鼠肺內 CTGF 表達的影響. 中國老年學雜志, 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. 劉張紅, 程錦國, 林文博, 等. 腎病Ⅰ號方對腎間質纖維化大鼠 TGF-β1 和 CTGF 表達的影響. 浙江中醫藥大學學報, 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. 符紅娜, 孫兆瑞, 葛鑫, 等. 結締組織生長因子在百草枯致肺纖維化中的作用. 中華急診醫學雜志, 2018, 27(11): 1198-1204.
4. 全國衛管協會轉化醫學產業分會. 特發性肺纖維化的新藥物和靶點之二: 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 在胃癌中的表達及其促進血管生成的機制. 局解手術學雜志, 2011, 20(6): 612-615.
6. 俞建順, 蔡丹莉, 嚴茂祥, 等. MNNG 誘導的胃癌前病變大鼠胃黏膜 CTGF 及 ET-1 表達的變化. 浙江臨床醫學, 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. 張曼曼, 王恒, 趙逵. CYR61 與結腸癌發生發展的關系. 山東醫藥, 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. 楊莉, 李曉玫, 王海燕. 結締組織生長因子在關木通相關腎小管間質腎病腎間質纖維化中的作用. 中華腎臟病雜志, 2003, 19(4): 19-24.
12. 朱祎, 唐英, 何立群. 大黃酚對單側輸尿管梗阻模型大鼠腎組織 CTGF、α-SMA 和 FN 的影響. 中南藥學, 2014, 12(7): 631-634.
13. 胡銳, 陳振兵, 賈中尉, 等. 結締組織生長因子抗體抑制慢性卡壓后神經纖維化的研究. 中華實驗外科雜志, 2012, 29(11): 2259-2262.
14. 趙敏, 徐安莉, 周艷艷, 等. 溫補腎陽法對腎陽虛肺纖維化大鼠肺內 CTGF 表達的影響. 中國老年學雜志, 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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