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Evidence for transcriptional interference in a dual-luciferase reporter system.

Wu GQ, Wang X, Zhou HY, Chai KQ, Xue Q, Zheng AH, Zhu XM, Xiao JY, Ying XH, Wang FW, Rui T, Xu LY, Zhang YK, Liao YJ, Xie D, Lu LQ, Huang DS - Sci Rep (2015)

Bottom Line: 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.These results explain the discrepancies between the two luciferase reporter systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.

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.

No MeSH data available.


Related in: MedlinePlus

miR-let 7c and miR-101 inhibited EZH2 3′UTR-luciferase activity.(A) The expression of miR-let 7c and miR-101 in A549 cells was significantly elevated after the co-transduction of LV-miR-let 7c and LV-miR-101 (*P < 0.05). (B) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 inhibited EZH2 3′UTR-renilla luciferase activity from p1 (*P < 0.05). (C) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 inhibited EZH2 3′UTR-firefly luciferase activity from p2 (*P < 0.05). (D) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 did not affect the luciferase activity when the luciferase reporter assay was carried out with p1-scram (NS, not statistically significant). (E) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 did not alter luciferase activity when the luciferase reporter assay was carried out with p2-scram and the control vector pRL-TK (NS, not statistically significant). The luciferase activity (hRluc:hluc or hluc:hRluc) in A549 cells co-transduced with LV-miR-let 7c and LV-miR-101 was normalized to 1 relative to the luciferase activity in cells transduced with LV-Control. The data in all of the bar graphs were plotted as the mean ± SEM.
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f5: miR-let 7c and miR-101 inhibited EZH2 3′UTR-luciferase activity.(A) The expression of miR-let 7c and miR-101 in A549 cells was significantly elevated after the co-transduction of LV-miR-let 7c and LV-miR-101 (*P < 0.05). (B) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 inhibited EZH2 3′UTR-renilla luciferase activity from p1 (*P < 0.05). (C) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 inhibited EZH2 3′UTR-firefly luciferase activity from p2 (*P < 0.05). (D) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 did not affect the luciferase activity when the luciferase reporter assay was carried out with p1-scram (NS, not statistically significant). (E) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 did not alter luciferase activity when the luciferase reporter assay was carried out with p2-scram and the control vector pRL-TK (NS, not statistically significant). The luciferase activity (hRluc:hluc or hluc:hRluc) in A549 cells co-transduced with LV-miR-let 7c and LV-miR-101 was normalized to 1 relative to the luciferase activity in cells transduced with LV-Control. The data in all of the bar graphs were plotted as the mean ± SEM.

Mentions: To further evaluate whether miR-let 7c and miR-101 regulate EZH2 3′UTR-luciferase activity in A549 cells, we co-transduced A549 cells with precursor miRNAs (LV-miR-let 7c and LV-miR-101) or control precursor miRNA (LV-Control). As determined by qPCR, the co-transduction of LV-miR-let 7c and LV-miR-101 induced a 7- and 15-fold overexpression of miR-let 7c and miR-101, respectively (Fig. 5A). In agreement with previous reports1234567, the overexpression of miR-let 7c and miR-101 significantly inhibited EZH2 3′UTR-luciferase activity in A549 cells regardless of whether the assay was carried out with p1 or p2 (Fig. 5B,C). Scrambling the EZH2 3′UTR sequence in the luciferase reporter construct p1 or p2 completely abrogated the inhibitory effect of miR-let 7c and miR-101 on luciferase activity (Fig. 5D,E).


Evidence for transcriptional interference in a dual-luciferase reporter system.

Wu GQ, Wang X, Zhou HY, Chai KQ, Xue Q, Zheng AH, Zhu XM, Xiao JY, Ying XH, Wang FW, Rui T, Xu LY, Zhang YK, Liao YJ, Xie D, Lu LQ, Huang DS - Sci Rep (2015)

miR-let 7c and miR-101 inhibited EZH2 3′UTR-luciferase activity.(A) The expression of miR-let 7c and miR-101 in A549 cells was significantly elevated after the co-transduction of LV-miR-let 7c and LV-miR-101 (*P < 0.05). (B) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 inhibited EZH2 3′UTR-renilla luciferase activity from p1 (*P < 0.05). (C) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 inhibited EZH2 3′UTR-firefly luciferase activity from p2 (*P < 0.05). (D) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 did not affect the luciferase activity when the luciferase reporter assay was carried out with p1-scram (NS, not statistically significant). (E) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 did not alter luciferase activity when the luciferase reporter assay was carried out with p2-scram and the control vector pRL-TK (NS, not statistically significant). The luciferase activity (hRluc:hluc or hluc:hRluc) in A549 cells co-transduced with LV-miR-let 7c and LV-miR-101 was normalized to 1 relative to the luciferase activity in cells transduced with LV-Control. The data in all of the bar graphs were plotted as the mean ± SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664949&req=5

f5: miR-let 7c and miR-101 inhibited EZH2 3′UTR-luciferase activity.(A) The expression of miR-let 7c and miR-101 in A549 cells was significantly elevated after the co-transduction of LV-miR-let 7c and LV-miR-101 (*P < 0.05). (B) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 inhibited EZH2 3′UTR-renilla luciferase activity from p1 (*P < 0.05). (C) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 inhibited EZH2 3′UTR-firefly luciferase activity from p2 (*P < 0.05). (D) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 did not affect the luciferase activity when the luciferase reporter assay was carried out with p1-scram (NS, not statistically significant). (E) Compared with LV-Control, LV-miR-let 7c and LV-miR-101 did not alter luciferase activity when the luciferase reporter assay was carried out with p2-scram and the control vector pRL-TK (NS, not statistically significant). The luciferase activity (hRluc:hluc or hluc:hRluc) in A549 cells co-transduced with LV-miR-let 7c and LV-miR-101 was normalized to 1 relative to the luciferase activity in cells transduced with LV-Control. The data in all of the bar graphs were plotted as the mean ± SEM.
Mentions: To further evaluate whether miR-let 7c and miR-101 regulate EZH2 3′UTR-luciferase activity in A549 cells, we co-transduced A549 cells with precursor miRNAs (LV-miR-let 7c and LV-miR-101) or control precursor miRNA (LV-Control). As determined by qPCR, the co-transduction of LV-miR-let 7c and LV-miR-101 induced a 7- and 15-fold overexpression of miR-let 7c and miR-101, respectively (Fig. 5A). In agreement with previous reports1234567, the overexpression of miR-let 7c and miR-101 significantly inhibited EZH2 3′UTR-luciferase activity in A549 cells regardless of whether the assay was carried out with p1 or p2 (Fig. 5B,C). Scrambling the EZH2 3′UTR sequence in the luciferase reporter construct p1 or p2 completely abrogated the inhibitory effect of miR-let 7c and miR-101 on luciferase activity (Fig. 5D,E).

Bottom Line: 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.These results explain the discrepancies between the two luciferase reporter systems.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology &Cancer Biotherapy Center, Zhejiang Provincial People's Hospital, 158 Shangtang Road, Hangzhou, Zhejiang 310014, China.

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.

No MeSH data available.


Related in: MedlinePlus