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Study on the Effect of Human CMV Early Transcriptional Enhancer on Human CEA Promoter
Qiong-Jiang Chen1, Zhen-Yu Jia1*, Ping Chen2, Qun Wei2, Min Zhang1, Jun-Qiang Chen1,Xing Zhang1, Jing-Jia Ye3, Jiang Cao2
1 Zhejiang Academy of Medical Sciences, Hangzhou 310013, China; 2Sir Run Run Shaw Institute of Clinical Medicine, Zhejiang University , Hangzhou 310016, China; 3Clinical Research Center, 2nd Affiliated Hospital of Zhejiang
Abstract: In order to explore the probability of Cytomegalovirus (CMV) early transcriptional enhancer in targeted gene therapy for carcinoembryonic antigen (CEA) positive cancers， CEA promoter (369 bp) and CMV early transcriptional enhancer (531 bp) were cloned by PCR using genomic DNA of human colon carcinoma SW620 cells and pcDNA3.1 (+) plasmid as template， respectively according to literatures. CEA promoter and CMV early transcriptional
enhancer were subcloned into luciferase reporter vector pGL4.10 to obtain pGL4.10-CEA and pGL4.10-CMV-CEA， and co-transfected with transfection-efficiency normalization vector pGL4.74 into CEA-positive human
colon carcinoma LoVo， HT-29， SW620， lung carcinoma A549 cells， breast carcinoma MCF7 cells and CEA-negative colon carcinoma SW480 cells， cervix carcinoma HeLa cells and lung fibroblast LL47 cells. The transcription initiation efficiencies of CEA promoter in these cells were analyzed by Dual Luciferase Assay system and relative luciferase unit (RLU) was used to evaluate the expression efficiency. The results showed that CMV early transcriptional enhancer up-regulated CEA promoter transcriptional activity significantly by 7.46 to 70.16 folds in the above CEA-positive cancer cells， however， it also up-regulated CEA promoter transcriptional activity in CEA-negative Hela and fibroblast cells by 24.01 to 76.40 folds. Therefore， the above CMV enhancer can greatly up-regulate CEA promoter transcriptional activity， but may attenuate the specificity of CEA promoter. The side effect of the nonspecificity of CEA promoter with CMV early transcriptional enhancer should be considered for targeted gene therapy of CEApositive cancer. Combination of other ways such as tissue-specific transcription factor-binding sites in the upstream regulatory region are needed for maintaining the specificity of CEA promoter when utilizing CMV early transcriptional enhancer in targeted gene therapy of CEA-positive cancer.
enhancer were subcloned into luciferase reporter vector pGL4.10 to obtain pGL4.10-CEA and pGL4.10-CMV-CEA， and co-transfected with transfection-efficiency normalization vector pGL4.74 into CEA-positive human
colon carcinoma LoVo， HT-29， SW620， lung carcinoma A549 cells， breast carcinoma MCF7 cells and CEA-negative colon carcinoma SW480 cells， cervix carcinoma HeLa cells and lung fibroblast LL47 cells. The transcription initiation efficiencies of CEA promoter in these cells were analyzed by Dual Luciferase Assay system and relative luciferase unit (RLU) was used to evaluate the expression efficiency. The results showed that CMV early transcriptional enhancer up-regulated CEA promoter transcriptional activity significantly by 7.46 to 70.16 folds in the above CEA-positive cancer cells， however， it also up-regulated CEA promoter transcriptional activity in CEA-negative Hela and fibroblast cells by 24.01 to 76.40 folds. Therefore， the above CMV enhancer can greatly up-regulate CEA promoter transcriptional activity， but may attenuate the specificity of CEA promoter. The side effect of the nonspecificity of CEA promoter with CMV early transcriptional enhancer should be considered for targeted gene therapy of CEApositive cancer. Combination of other ways such as tissue-specific transcription factor-binding sites in the upstream regulatory region are needed for maintaining the specificity of CEA promoter when utilizing CMV early transcriptional enhancer in targeted gene therapy of CEA-positive cancer.
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