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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

Different luciferase reporter systems provided conflicting data regarding the effect of curcumin on the EZH2 3′UTR.(A) Curcumin treatment led to increased luciferase activity relative to DMSO treatment as determined by the dual-luciferase reporter assay carried out with p1 (*P < 0.05). (B–D) Curcumin treatment led to a significant decrease in luciferase activity relative to DMSO treatment as determined by the luciferase reporter assay performed with p2 and the control vector pRL-TK, pRL-CMV, or pRL-SV40 (*P < 0.05). (E) Curcumin treatment led to increased luciferase activity relative to DMSO treatment as determined by the luciferase reporter assay carried out with p1-scram (*P < 0.05). (F) Compared to the control treatment with DMSO, treatment with curcumin did not affect luciferase activity when the luciferase reporter assay was performed using p2-scram and the control vector pRL-TK (N/S, not statistically significant; P > 0.05). For each EZH2 3′UTR or scrambled EZH2 3′UTR, luciferase activity (hRluc:hluc or hluc:hRluc) was normalized to 1 relative to the control treatment with DMSO. The data in all of the bar graphs were plotted as the mean ± SEM.
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f3: Different luciferase reporter systems provided conflicting data regarding the effect of curcumin on the EZH2 3′UTR.(A) Curcumin treatment led to increased luciferase activity relative to DMSO treatment as determined by the dual-luciferase reporter assay carried out with p1 (*P < 0.05). (B–D) Curcumin treatment led to a significant decrease in luciferase activity relative to DMSO treatment as determined by the luciferase reporter assay performed with p2 and the control vector pRL-TK, pRL-CMV, or pRL-SV40 (*P < 0.05). (E) Curcumin treatment led to increased luciferase activity relative to DMSO treatment as determined by the luciferase reporter assay carried out with p1-scram (*P < 0.05). (F) Compared to the control treatment with DMSO, treatment with curcumin did not affect luciferase activity when the luciferase reporter assay was performed using p2-scram and the control vector pRL-TK (N/S, not statistically significant; P > 0.05). For each EZH2 3′UTR or scrambled EZH2 3′UTR, luciferase activity (hRluc:hluc or hluc:hRluc) was normalized to 1 relative to the control treatment with DMSO. The data in all of the bar graphs were plotted as the mean ± SEM.

Mentions: To determine whether curcumin regulates EZH2 expression by targeting the 3′UTR of EZH2, luciferase reporter assays were performed with EZH2 3′UTR reporters. Because it was previously reported that the activity of the internal control plasmid can be affected by the presence of co-transfected reporter plasmids used to normalize the transfection efficiency8, we employed two dual-luciferase reporter systems containing exactly the same sequence of the EZH2 3′UTR. To construct the EZH2 3′UTR reporter systems, 263 bp of the EZH2 3′UTR was inserted into the dual-luciferase reporter vector pmiR-RB-REPORTTM between the Sgf I and Not I sites (Fig. 1A), or into the luciferase reporter vector pMIR-REPORTTM Luciferase between the Spe I and Hind III sites (Fig. 1B) to generate pmiR-RB-EZH2 UTR (p1) (Fig. 1C) and pMIR-EZH2 UTR (p2) (Fig. 1D), respectively. A scrambled sequence of the EZH2 3′UTR was inserted into the dual-luciferase reporter vector pmiR-RB-REPORTTM or into the luciferase reporter vector pMIR-REPORTTM Luciferase to generate the negative control constructs pmiR-RB-EZH2 UTRscram (p1-scram) (Fig. 1E) and pMIR-EZH2 UTRscram (p2-scram) (Fig. 1F), respectively. A549 cells were then transfected with the dual-luciferase reporter construct p1, or co-transfected with the luciferase reporter construct p2 and the control vector pRL-TK, pRL-SV40, or pRL-CMV (Fig. 2A–C). Unexpectedly, compared with dimethylsulfoxide (DMSO), curcumin caused a significant increase in EZH2 3′UTR-renilla luciferase activity in A549 cells transfected with the dual-luciferase reporter construct p1 containing the EZH2 3′UTR (Fig. 3A). In contrast, in A549 cells co-transfected with the luciferase reporter construct p2 and the control vector pRL-TK, pRL-CMV, or pRL-SV40, there was a significant reduction in EZH2 3′UTR-firefly luciferase activity in cells treated with curcumin relative to those treated with DMSO (Fig. 3B–D), which is in agreement with the finding that curcumin inhibited EZH2 mRNA and protein expression. Luciferase reporter assay results with NCI-H2170 cells are consistent with that of A549 cells (data not shown).


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)

Different luciferase reporter systems provided conflicting data regarding the effect of curcumin on the EZH2 3′UTR.(A) Curcumin treatment led to increased luciferase activity relative to DMSO treatment as determined by the dual-luciferase reporter assay carried out with p1 (*P < 0.05). (B–D) Curcumin treatment led to a significant decrease in luciferase activity relative to DMSO treatment as determined by the luciferase reporter assay performed with p2 and the control vector pRL-TK, pRL-CMV, or pRL-SV40 (*P < 0.05). (E) Curcumin treatment led to increased luciferase activity relative to DMSO treatment as determined by the luciferase reporter assay carried out with p1-scram (*P < 0.05). (F) Compared to the control treatment with DMSO, treatment with curcumin did not affect luciferase activity when the luciferase reporter assay was performed using p2-scram and the control vector pRL-TK (N/S, not statistically significant; P > 0.05). For each EZH2 3′UTR or scrambled EZH2 3′UTR, luciferase activity (hRluc:hluc or hluc:hRluc) was normalized to 1 relative to the control treatment with DMSO. 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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f3: Different luciferase reporter systems provided conflicting data regarding the effect of curcumin on the EZH2 3′UTR.(A) Curcumin treatment led to increased luciferase activity relative to DMSO treatment as determined by the dual-luciferase reporter assay carried out with p1 (*P < 0.05). (B–D) Curcumin treatment led to a significant decrease in luciferase activity relative to DMSO treatment as determined by the luciferase reporter assay performed with p2 and the control vector pRL-TK, pRL-CMV, or pRL-SV40 (*P < 0.05). (E) Curcumin treatment led to increased luciferase activity relative to DMSO treatment as determined by the luciferase reporter assay carried out with p1-scram (*P < 0.05). (F) Compared to the control treatment with DMSO, treatment with curcumin did not affect luciferase activity when the luciferase reporter assay was performed using p2-scram and the control vector pRL-TK (N/S, not statistically significant; P > 0.05). For each EZH2 3′UTR or scrambled EZH2 3′UTR, luciferase activity (hRluc:hluc or hluc:hRluc) was normalized to 1 relative to the control treatment with DMSO. The data in all of the bar graphs were plotted as the mean ± SEM.
Mentions: To determine whether curcumin regulates EZH2 expression by targeting the 3′UTR of EZH2, luciferase reporter assays were performed with EZH2 3′UTR reporters. Because it was previously reported that the activity of the internal control plasmid can be affected by the presence of co-transfected reporter plasmids used to normalize the transfection efficiency8, we employed two dual-luciferase reporter systems containing exactly the same sequence of the EZH2 3′UTR. To construct the EZH2 3′UTR reporter systems, 263 bp of the EZH2 3′UTR was inserted into the dual-luciferase reporter vector pmiR-RB-REPORTTM between the Sgf I and Not I sites (Fig. 1A), or into the luciferase reporter vector pMIR-REPORTTM Luciferase between the Spe I and Hind III sites (Fig. 1B) to generate pmiR-RB-EZH2 UTR (p1) (Fig. 1C) and pMIR-EZH2 UTR (p2) (Fig. 1D), respectively. A scrambled sequence of the EZH2 3′UTR was inserted into the dual-luciferase reporter vector pmiR-RB-REPORTTM or into the luciferase reporter vector pMIR-REPORTTM Luciferase to generate the negative control constructs pmiR-RB-EZH2 UTRscram (p1-scram) (Fig. 1E) and pMIR-EZH2 UTRscram (p2-scram) (Fig. 1F), respectively. A549 cells were then transfected with the dual-luciferase reporter construct p1, or co-transfected with the luciferase reporter construct p2 and the control vector pRL-TK, pRL-SV40, or pRL-CMV (Fig. 2A–C). Unexpectedly, compared with dimethylsulfoxide (DMSO), curcumin caused a significant increase in EZH2 3′UTR-renilla luciferase activity in A549 cells transfected with the dual-luciferase reporter construct p1 containing the EZH2 3′UTR (Fig. 3A). In contrast, in A549 cells co-transfected with the luciferase reporter construct p2 and the control vector pRL-TK, pRL-CMV, or pRL-SV40, there was a significant reduction in EZH2 3′UTR-firefly luciferase activity in cells treated with curcumin relative to those treated with DMSO (Fig. 3B–D), which is in agreement with the finding that curcumin inhibited EZH2 mRNA and protein expression. Luciferase reporter assay results with NCI-H2170 cells are consistent with that of A549 cells (data not shown).

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