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Ectopic over-expression of tristetraprolin in human cancer cells promotes biogenesis of let-7 by down-regulation of Lin28.

Kim CW, Vo MT, Kim HK, Lee HH, Yoon NA, Lee BJ, Min YJ, Joo WD, Cha HJ, Park JW, Cho WJ - Nucleic Acids Res. (2011)

Bottom Line: Tristetraprolin (TTP) is a AU-rich element (ARE) binding protein and exhibits suppressive effects on cell growth through down-regulation of ARE-containing oncogenes.The let-7 microRNA has emerged as a significant factor in tumor suppression.This event is associated with TTP-mediated inhibition of Lin28, which has emerged as a negative modulator of let-7.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Ulsan, Ulsan 680-749, Korea.

ABSTRACT
Tristetraprolin (TTP) is a AU-rich element (ARE) binding protein and exhibits suppressive effects on cell growth through down-regulation of ARE-containing oncogenes. The let-7 microRNA has emerged as a significant factor in tumor suppression. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. In this work, an unexpected link between TTP and let-7 has been found in human cancer cells. TTP promotes an increase in expression of mature let-7, which leads to the inhibition of let-7 target gene CDC34 expression and suppresses cell growth. This event is associated with TTP-mediated inhibition of Lin28, which has emerged as a negative modulator of let-7. Lin28 mRNA contains ARE within its 3'-UTR and TTP enhances the decay of Lin28 mRNA through binding to its 3'-UTR. This suggests that the TTP-mediated down-regulation of Lin28 plays a key role in let-7 miRNA biogenesis in cancer cells.

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TTP negatively regulates the expression of the let-7b target gene CDC34. (A and B) Overexpression of TTP downregulates CDC34 levels. The levels of CDC34 in PA1 cells transfected with pcDNA6/V5-TTP and pcDNA6/V5 was determined by semi-qRT-PCR (A, top panel), western blots (A, bottom panel) and qRT-PCR (B). The data represent the mean ± SD of 3 independent experiments (*P < 0.05). (C and D) Overexpression of let-7b suppresses CDC34 expression. PA1 cells were transfected with 50 nM of let-7b or scrambled miRNA oligonucleotides for 24 h. (C) The levels of let-7b was determined by qRT-PCR. The levels obtained from mock-treated cells (PA1/Mock) were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (D) CDC34 protein levels were measured by western blot assays. (E and F) Knockdown of let-7b abolishes the suppression of CDC34 expression induced by TTP. PA1 cells were cotransfected with pcDNA6/V5-TTP, anti-let-7b oligonucleotides (AS-let-7b) or scrambled oligonucleotides (scRNA) for 24 h. (E) The level of let-7b was determined by qRT-PCR. The levels of let-7b in PA1 cells transfected with pcDNA and scRNA oligonucleotides were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01). (F) The level of CDC34 was determined by western blot assays. (G) Down-regulation of CDC34 by TTP is mediated by let-7b. PA1 cells were transfected with various combination of pcDNA6/V5, pcDNA6/V5-TTP, psiCHECK2-CDC34 3′-UTR WT, psiCHECK2-CDC34 3′-UTR MUT and let-7b for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells co-transfected with the pcDNA6/V5 and psiCHECK2-CDC34 3′-UTR WT was set to 1.0. Results shown represent the means ± SD of three independent experiments (**P < 0.01). ns, not significant.
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gkr1302-F2: TTP negatively regulates the expression of the let-7b target gene CDC34. (A and B) Overexpression of TTP downregulates CDC34 levels. The levels of CDC34 in PA1 cells transfected with pcDNA6/V5-TTP and pcDNA6/V5 was determined by semi-qRT-PCR (A, top panel), western blots (A, bottom panel) and qRT-PCR (B). The data represent the mean ± SD of 3 independent experiments (*P < 0.05). (C and D) Overexpression of let-7b suppresses CDC34 expression. PA1 cells were transfected with 50 nM of let-7b or scrambled miRNA oligonucleotides for 24 h. (C) The levels of let-7b was determined by qRT-PCR. The levels obtained from mock-treated cells (PA1/Mock) were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (D) CDC34 protein levels were measured by western blot assays. (E and F) Knockdown of let-7b abolishes the suppression of CDC34 expression induced by TTP. PA1 cells were cotransfected with pcDNA6/V5-TTP, anti-let-7b oligonucleotides (AS-let-7b) or scrambled oligonucleotides (scRNA) for 24 h. (E) The level of let-7b was determined by qRT-PCR. The levels of let-7b in PA1 cells transfected with pcDNA and scRNA oligonucleotides were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01). (F) The level of CDC34 was determined by western blot assays. (G) Down-regulation of CDC34 by TTP is mediated by let-7b. PA1 cells were transfected with various combination of pcDNA6/V5, pcDNA6/V5-TTP, psiCHECK2-CDC34 3′-UTR WT, psiCHECK2-CDC34 3′-UTR MUT and let-7b for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells co-transfected with the pcDNA6/V5 and psiCHECK2-CDC34 3′-UTR WT was set to 1.0. Results shown represent the means ± SD of three independent experiments (**P < 0.01). ns, not significant.

Mentions: Our next goal was to determine whether TTP controls the expression levels of let-7 target genes. It has been reported that let-7 functions as a tumor suppressor by down-regulating K-Ras, cyclin D, CDC34 and c-Myc, as well as several genes involved in cell cycle and cell division control (31–34). To determine the effects of TTP overexpression on the expression of let-7 target genes, we analyzed the expression level of K-Ras, cyclin D, CDC34 and c-Myc by RT-PCR and western blots in PA1 cells transfected with pcDNA6/V5-TTP or the control pcDNA6/V5 vector. We also analyzed the expression level of TTP-target genes including VEGF, COX2 and c-Fos genes (26,27,29). Interestingly, while overexpression of TTP resulted in significant decrease in the expression level of CDC34 (Figure 2A and B), it did not affect the expression level of any of the other genes (Supplementary Figure S2). Down-regulation of CDC34 by TTP overexpression was confirmed by semi-quantitative RT-PCR (semi-qRT-PCR) (Figure 2A, top panel), western blots (Figure 2A, bottom panel) and qRT-PCR (Figure 2B). Coinciding with a previous report (34), transfection of let-7b decreased the level of CDC34 (Figure 2C and D). On the contrary, down-regulation of let-7b by treatment with anti-let-7b oligonucleotides (AS-let-7b) (Figure 2E), which act as competitive inhibitors of let-7b, attenuated the TTP-induced decrease of CDC34 (Figure 2F). To determine whether the inhibitory effect of TTP on CDC34 is mediated by increased let-7b, we prepared a luciferase reporter gene linked to the wild-type CDC34 3′-UTR (CDC34 3′-UTR WT) or mutant CDC34 3′-UTR (CDC34 3′-UTR MUT) with point mutations in let-7b target sites in the CDC34 3′-UTR. Let-7b was also used as a control. While TTP overexpression or let-7b treatment significantly decreased the luciferase activity of luciferase reporter gene containing wild-type CDC34 3′-UTR, it did not affect that containing mutant CDC34 3′-UTR (Figure 2G). Collectively, these results indicate that TTP inhibits CDC34 expression and let-7b mediates the effect.Figure 2.


Ectopic over-expression of tristetraprolin in human cancer cells promotes biogenesis of let-7 by down-regulation of Lin28.

Kim CW, Vo MT, Kim HK, Lee HH, Yoon NA, Lee BJ, Min YJ, Joo WD, Cha HJ, Park JW, Cho WJ - Nucleic Acids Res. (2011)

TTP negatively regulates the expression of the let-7b target gene CDC34. (A and B) Overexpression of TTP downregulates CDC34 levels. The levels of CDC34 in PA1 cells transfected with pcDNA6/V5-TTP and pcDNA6/V5 was determined by semi-qRT-PCR (A, top panel), western blots (A, bottom panel) and qRT-PCR (B). The data represent the mean ± SD of 3 independent experiments (*P < 0.05). (C and D) Overexpression of let-7b suppresses CDC34 expression. PA1 cells were transfected with 50 nM of let-7b or scrambled miRNA oligonucleotides for 24 h. (C) The levels of let-7b was determined by qRT-PCR. The levels obtained from mock-treated cells (PA1/Mock) were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (D) CDC34 protein levels were measured by western blot assays. (E and F) Knockdown of let-7b abolishes the suppression of CDC34 expression induced by TTP. PA1 cells were cotransfected with pcDNA6/V5-TTP, anti-let-7b oligonucleotides (AS-let-7b) or scrambled oligonucleotides (scRNA) for 24 h. (E) The level of let-7b was determined by qRT-PCR. The levels of let-7b in PA1 cells transfected with pcDNA and scRNA oligonucleotides were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01). (F) The level of CDC34 was determined by western blot assays. (G) Down-regulation of CDC34 by TTP is mediated by let-7b. PA1 cells were transfected with various combination of pcDNA6/V5, pcDNA6/V5-TTP, psiCHECK2-CDC34 3′-UTR WT, psiCHECK2-CDC34 3′-UTR MUT and let-7b for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells co-transfected with the pcDNA6/V5 and psiCHECK2-CDC34 3′-UTR WT was set to 1.0. Results shown represent the means ± SD of three independent experiments (**P < 0.01). ns, not significant.
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gkr1302-F2: TTP negatively regulates the expression of the let-7b target gene CDC34. (A and B) Overexpression of TTP downregulates CDC34 levels. The levels of CDC34 in PA1 cells transfected with pcDNA6/V5-TTP and pcDNA6/V5 was determined by semi-qRT-PCR (A, top panel), western blots (A, bottom panel) and qRT-PCR (B). The data represent the mean ± SD of 3 independent experiments (*P < 0.05). (C and D) Overexpression of let-7b suppresses CDC34 expression. PA1 cells were transfected with 50 nM of let-7b or scrambled miRNA oligonucleotides for 24 h. (C) The levels of let-7b was determined by qRT-PCR. The levels obtained from mock-treated cells (PA1/Mock) were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (D) CDC34 protein levels were measured by western blot assays. (E and F) Knockdown of let-7b abolishes the suppression of CDC34 expression induced by TTP. PA1 cells were cotransfected with pcDNA6/V5-TTP, anti-let-7b oligonucleotides (AS-let-7b) or scrambled oligonucleotides (scRNA) for 24 h. (E) The level of let-7b was determined by qRT-PCR. The levels of let-7b in PA1 cells transfected with pcDNA and scRNA oligonucleotides were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01). (F) The level of CDC34 was determined by western blot assays. (G) Down-regulation of CDC34 by TTP is mediated by let-7b. PA1 cells were transfected with various combination of pcDNA6/V5, pcDNA6/V5-TTP, psiCHECK2-CDC34 3′-UTR WT, psiCHECK2-CDC34 3′-UTR MUT and let-7b for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells co-transfected with the pcDNA6/V5 and psiCHECK2-CDC34 3′-UTR WT was set to 1.0. Results shown represent the means ± SD of three independent experiments (**P < 0.01). ns, not significant.
Mentions: Our next goal was to determine whether TTP controls the expression levels of let-7 target genes. It has been reported that let-7 functions as a tumor suppressor by down-regulating K-Ras, cyclin D, CDC34 and c-Myc, as well as several genes involved in cell cycle and cell division control (31–34). To determine the effects of TTP overexpression on the expression of let-7 target genes, we analyzed the expression level of K-Ras, cyclin D, CDC34 and c-Myc by RT-PCR and western blots in PA1 cells transfected with pcDNA6/V5-TTP or the control pcDNA6/V5 vector. We also analyzed the expression level of TTP-target genes including VEGF, COX2 and c-Fos genes (26,27,29). Interestingly, while overexpression of TTP resulted in significant decrease in the expression level of CDC34 (Figure 2A and B), it did not affect the expression level of any of the other genes (Supplementary Figure S2). Down-regulation of CDC34 by TTP overexpression was confirmed by semi-quantitative RT-PCR (semi-qRT-PCR) (Figure 2A, top panel), western blots (Figure 2A, bottom panel) and qRT-PCR (Figure 2B). Coinciding with a previous report (34), transfection of let-7b decreased the level of CDC34 (Figure 2C and D). On the contrary, down-regulation of let-7b by treatment with anti-let-7b oligonucleotides (AS-let-7b) (Figure 2E), which act as competitive inhibitors of let-7b, attenuated the TTP-induced decrease of CDC34 (Figure 2F). To determine whether the inhibitory effect of TTP on CDC34 is mediated by increased let-7b, we prepared a luciferase reporter gene linked to the wild-type CDC34 3′-UTR (CDC34 3′-UTR WT) or mutant CDC34 3′-UTR (CDC34 3′-UTR MUT) with point mutations in let-7b target sites in the CDC34 3′-UTR. Let-7b was also used as a control. While TTP overexpression or let-7b treatment significantly decreased the luciferase activity of luciferase reporter gene containing wild-type CDC34 3′-UTR, it did not affect that containing mutant CDC34 3′-UTR (Figure 2G). Collectively, these results indicate that TTP inhibits CDC34 expression and let-7b mediates the effect.Figure 2.

Bottom Line: Tristetraprolin (TTP) is a AU-rich element (ARE) binding protein and exhibits suppressive effects on cell growth through down-regulation of ARE-containing oncogenes.The let-7 microRNA has emerged as a significant factor in tumor suppression.This event is associated with TTP-mediated inhibition of Lin28, which has emerged as a negative modulator of let-7.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Ulsan, Ulsan 680-749, Korea.

ABSTRACT
Tristetraprolin (TTP) is a AU-rich element (ARE) binding protein and exhibits suppressive effects on cell growth through down-regulation of ARE-containing oncogenes. The let-7 microRNA has emerged as a significant factor in tumor suppression. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. In this work, an unexpected link between TTP and let-7 has been found in human cancer cells. TTP promotes an increase in expression of mature let-7, which leads to the inhibition of let-7 target gene CDC34 expression and suppresses cell growth. This event is associated with TTP-mediated inhibition of Lin28, which has emerged as a negative modulator of let-7. Lin28 mRNA contains ARE within its 3'-UTR and TTP enhances the decay of Lin28 mRNA through binding to its 3'-UTR. This suggests that the TTP-mediated down-regulation of Lin28 plays a key role in let-7 miRNA biogenesis in cancer cells.

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Related in: MedlinePlus