title: Evidence for transcriptional interference in a dual-luciferase reporter system.
abstract: The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3'untranslated region (3'UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3'UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs. We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups. The results suggested that curcumin promoted the transcription of the luciferase genes located downstream of the simian vacuolating virus 40 (SV40) early enhancer/promoter, but not those located downstream of the human cytomegalovirus (CMV) immediate-early or the herpes simplex virus thymidine kinase (HSV-TK) promoters. These results explain the discrepancies between the two luciferase reporter systems. The current study underscores the importance of taking caution when interpreting the results of dual-luciferase reporter assays and provides strategies to overcome the potential pitfall accompanying dual-luciferase reporter systems.
authors.affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.
authors.forename: Guo-Qing
authors.initials: GQ
authors.lastname: Wu
authors.affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.
authors.forename: Hong-Ying
authors.initials: HY
authors.lastname: Zhou
authors.affiliation: Zhejiang Academy of Traditional Chinese Medicine, Tongde Hospital of Zhejiang Province, 234 Gucui Road, Hangzhou, Zhejiang 310012, China.
authors.forename: Ke-Qun
authors.initials: KQ
authors.lastname: Chai
authors.affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Xue
authors.affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.
authors.forename: Ai-Hong
authors.initials: AH
authors.lastname: Zheng
authors.affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.
authors.forename: Xiu-Ming
authors.initials: XM
authors.lastname: Zhu
authors.affiliation: Department of Biochemistry, Guangzhou University of Chinese Medicine, 232 Waihuang Road East, Guangzhou, Guangdong 510006, China.
authors.forename: Jian-Yong
authors.initials: JY
authors.lastname: Xiao
authors.affiliation: Zhejiang Academy of Traditional Chinese Medicine, Tongde Hospital of Zhejiang Province, 234 Gucui Road, Hangzhou, Zhejiang 310012, China.
authors.forename: Xu-Hua
authors.initials: XH
authors.lastname: Ying
authors.affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.
authors.forename: Fu-Wei
authors.initials: FW
authors.lastname: Wang
authors.affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Rui
authors.affiliation: Cell and Molecular Biology Laboratory, Zhoushan Hospital, Zhoushan, Zhejiang 316000, China.
authors.forename: Li-Yun
authors.initials: LY
authors.lastname: Xu
authors.affiliation: Department of Cardio-Thoracic Surgery, Zhoushan Hospital, Zhoushan, Zhejiang 316000, China.
authors.forename: Yong-Kui
authors.initials: YK
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, 651 Dongfeng Road East, Guangzhou, Guangdong 510060, China.
authors.forename: Yi-Ji
authors.initials: YJ
authors.lastname: Liao
authors.affiliation: State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, 651 Dongfeng Road East, Guangzhou, Guangdong 510060, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Xie
authors.affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.
authors.forename: Li-Qin
authors.initials: LQ
authors.lastname: Lu
authors.affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.
authors.forename: Dong-Sheng
authors.initials: DS
authors.lastname: Huang
mesh_terms: D020413:3' Untranslated Regions / Q000502:physiology*; D045744:Cell Line, Tumor; D000071221:Enhancer of Zeste Homolog 2 Protein; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis*; D063151:Polycomb Repressive Complex 2 / Q000096:biosynthesis* / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D020413:3' Untranslated Regions; D008156:Luciferases; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2