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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 enhances the decay of Lin28a mRNA through interaction with an ARE within the Lin28a mRNA 3′-UTR. (A and B) TTP destabilizes Lin28a mRNA. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 for 24 h. (A) The level of TTP protein was determined by western blot assays. (B) Expression of Lin28a mRNA in PA1 cells was determined by qRT-PCR and mRNA half-life was calculated from the non-linear regression of the mRNA levels at the indicated times after the addition of 5.0 µg/ml actinomycin D. A one-phase model of exponential decay was used to derive the indicated mRNA decay curves. Results shown on the graph represent the means ± SD of three independent experiments (**P < 0.01; ***P < 0.001). (C) Inhibition of TTP by siRNA enhances Lin28a mRNA stability. HCT116 cells were transfected with siRNA against TTP-specific (TTP-siRNA) or scRNA for 24 h. Expression of Lin28a mRNA in HCT116 cells was determined by qRT-PCR and mRNA half-life was calculated as described in (B). Results shown on the graph represent the means ± SD of three independent experiments (*P < 0.05). (D) Lin28a mRNA half-life in PA1 cell with low TTP level is longer than that in AGS cells with high TTP level. Expression of Lin28a mRNA in PA1 and AGS cells was determined by qRT-PCR and mRNA half-life was calculated as described in (B). Results shown on the graph represent the means ± SD of three independent experiments (***P < 0.001). (E and F) The first AUUUA pentamer (ARE1) within the Lin28a 3′-UTR is essential for the inhibitory effect of TTP. (E) Schematic representation of the luciferase reporter constructs used in this study. Fragments (Frag) and oligonucleotides (Oligo) derived from the Lin28a mRNA 3′-UTR were cloned downstream of the luciferase reporter gene in the psiCHECK2 luciferase expression vector. White circles, wild-type (W) pentameric motif AUUUA; gray circles, mutated (M) motif AGCA. (F) PA1 cells were co-transfected with pcDNA6/V5-TTP and a psiCHECK2 luciferase reporter constructs containing fragments (left panel) or oligonucleotides (right panel) derived from the Lin28a mRNA 3′-UTR as described in (E) for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells transfected with the Frag-ARE1 luciferase construct alone or Oligo-ARE1W alone were set to 1.0. Results shown represent the means ± SD of three independent experiments (*P < 0.05; **P < 0.01). ns, not significant. (G and H) TTP decreases the luciferase activity of luciferase reporter gene containing the Lin28b 3′-UTR. (G) Schematic representation of the luciferase reporter constructs used in this study. Fragments (Frag) derived from the Lin28b mRNA 3′-UTR were cloned downstream of the luciferase reporter gene in the psiCHECK2 luciferase expression vector. White circles, wild-type pentameric motif AUUUA. (H) PA1 cells were co-transfected with pcDNA6/V5-TTP and a psiCHECK2 luciferase reporter constructs containing fragments derived from the Lin28b mRNA 3′-UTR as described in (G) for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells transfected with each Frag-ARE luciferase construct alone was set to 1.0. Results shown represent the means ± SD of three independent experiments (**P < 0.01; ***P < 0.001). (I) Ribonucleoprotein immunoprecipitation assay. PA1 cells were cotransfected with pcDNA6/V5-TTP and psiCHECK2 luciferase reporter constructs containing Lin28a Oligo-ARE1W. psiCHECK2 luciferase reporter construct containing mutant ARE1, Oligo-ARE1M was used as a negative control. At 24 h after transfection, the ribonucleoprotein complexes containing TTP were immunoprecipitated with protein G-agarose and anti-V5 or a control antibody. The luciferase mRNA in the immunoprecipitates was amplified by RT-PCR. The presence of TTP in the immunoprecipitates was detected by western blot with anti-V5 antibody. (J and K) RNA EMSA was performed by mixing cytoplasmic extracts containing 3.0 µg of total protein from pcDNA6/V5-TTP-transfected PA1 cells (J) or HCT116 cells (K) with 80 fmol biotinylated wild-type Oligo-ARE1W (WT) or mutant Oligo-ARE1M (MUT) probe. Anti-V5 (J), anti-TTP (K) or control antibody was added to the reaction mixtures. Position of the TTP containing bands (TTP) and super-shifted bands (SS) are indicated.
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gkr1302-F5: TTP enhances the decay of Lin28a mRNA through interaction with an ARE within the Lin28a mRNA 3′-UTR. (A and B) TTP destabilizes Lin28a mRNA. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 for 24 h. (A) The level of TTP protein was determined by western blot assays. (B) Expression of Lin28a mRNA in PA1 cells was determined by qRT-PCR and mRNA half-life was calculated from the non-linear regression of the mRNA levels at the indicated times after the addition of 5.0 µg/ml actinomycin D. A one-phase model of exponential decay was used to derive the indicated mRNA decay curves. Results shown on the graph represent the means ± SD of three independent experiments (**P < 0.01; ***P < 0.001). (C) Inhibition of TTP by siRNA enhances Lin28a mRNA stability. HCT116 cells were transfected with siRNA against TTP-specific (TTP-siRNA) or scRNA for 24 h. Expression of Lin28a mRNA in HCT116 cells was determined by qRT-PCR and mRNA half-life was calculated as described in (B). Results shown on the graph represent the means ± SD of three independent experiments (*P < 0.05). (D) Lin28a mRNA half-life in PA1 cell with low TTP level is longer than that in AGS cells with high TTP level. Expression of Lin28a mRNA in PA1 and AGS cells was determined by qRT-PCR and mRNA half-life was calculated as described in (B). Results shown on the graph represent the means ± SD of three independent experiments (***P < 0.001). (E and F) The first AUUUA pentamer (ARE1) within the Lin28a 3′-UTR is essential for the inhibitory effect of TTP. (E) Schematic representation of the luciferase reporter constructs used in this study. Fragments (Frag) and oligonucleotides (Oligo) derived from the Lin28a mRNA 3′-UTR were cloned downstream of the luciferase reporter gene in the psiCHECK2 luciferase expression vector. White circles, wild-type (W) pentameric motif AUUUA; gray circles, mutated (M) motif AGCA. (F) PA1 cells were co-transfected with pcDNA6/V5-TTP and a psiCHECK2 luciferase reporter constructs containing fragments (left panel) or oligonucleotides (right panel) derived from the Lin28a mRNA 3′-UTR as described in (E) for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells transfected with the Frag-ARE1 luciferase construct alone or Oligo-ARE1W alone were set to 1.0. Results shown represent the means ± SD of three independent experiments (*P < 0.05; **P < 0.01). ns, not significant. (G and H) TTP decreases the luciferase activity of luciferase reporter gene containing the Lin28b 3′-UTR. (G) Schematic representation of the luciferase reporter constructs used in this study. Fragments (Frag) derived from the Lin28b mRNA 3′-UTR were cloned downstream of the luciferase reporter gene in the psiCHECK2 luciferase expression vector. White circles, wild-type pentameric motif AUUUA. (H) PA1 cells were co-transfected with pcDNA6/V5-TTP and a psiCHECK2 luciferase reporter constructs containing fragments derived from the Lin28b mRNA 3′-UTR as described in (G) for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells transfected with each Frag-ARE luciferase construct alone was set to 1.0. Results shown represent the means ± SD of three independent experiments (**P < 0.01; ***P < 0.001). (I) Ribonucleoprotein immunoprecipitation assay. PA1 cells were cotransfected with pcDNA6/V5-TTP and psiCHECK2 luciferase reporter constructs containing Lin28a Oligo-ARE1W. psiCHECK2 luciferase reporter construct containing mutant ARE1, Oligo-ARE1M was used as a negative control. At 24 h after transfection, the ribonucleoprotein complexes containing TTP were immunoprecipitated with protein G-agarose and anti-V5 or a control antibody. The luciferase mRNA in the immunoprecipitates was amplified by RT-PCR. The presence of TTP in the immunoprecipitates was detected by western blot with anti-V5 antibody. (J and K) RNA EMSA was performed by mixing cytoplasmic extracts containing 3.0 µg of total protein from pcDNA6/V5-TTP-transfected PA1 cells (J) or HCT116 cells (K) with 80 fmol biotinylated wild-type Oligo-ARE1W (WT) or mutant Oligo-ARE1M (MUT) probe. Anti-V5 (J), anti-TTP (K) or control antibody was added to the reaction mixtures. Position of the TTP containing bands (TTP) and super-shifted bands (SS) are indicated.

Mentions: To determine whether decreased expression of Lin28a resulted from changes in the stability of Lin28a mRNA, the half life of this mRNA was calculated from the mRNA levels measured by qRT-PCR in PA1 cells transfected with pcDNA6/V5-TTP or control pcDNA6/V5 vector. Overexpression of TTP protein in PA1/TTP cells was confirmed by western blot analysis (Figure 5A). In the control PA1/pcDNA cells, Lin28a mRNA was stable for 2 h after actinomycin D treatment. However, in PA1/TTP cells, the half-life of Lin28a mRNA was 1 h 30 min (Figure 5B). We also determined the half-life of Lin28a mRNA in HCT116 cells treated with siRNA against TTP (HCT116/TTP-siRNA) or control siRNA (HCT116/scRNA). In the control HCT116 and HCT116/scRNA cells, the half-life of Lin28a mRNA was 40 min. However, in HCT116/TTP-siRNA cells, Lin28a mRNA was stable until 3 h after actinomycin D treatment (Figure 5C). To determine whether regulation of Lin28a mRNA decay by TTP is biologically important, we compared the Lin28a mRNA half-life between HCT116 with high TTP levels and PA1 with low TTP level. As shown in Figure 5D, the half-life of Lin28a mRNA in PA1 cells was longer (t1/2 = 4 h 50 min) than that in AGS cells (t1/2 = 35 min). These results indicate that the TTP expression contributes to a decrease in Lin28a levels through destabilization of Lin28a mRNA.Figure 5.


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 enhances the decay of Lin28a mRNA through interaction with an ARE within the Lin28a mRNA 3′-UTR. (A and B) TTP destabilizes Lin28a mRNA. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 for 24 h. (A) The level of TTP protein was determined by western blot assays. (B) Expression of Lin28a mRNA in PA1 cells was determined by qRT-PCR and mRNA half-life was calculated from the non-linear regression of the mRNA levels at the indicated times after the addition of 5.0 µg/ml actinomycin D. A one-phase model of exponential decay was used to derive the indicated mRNA decay curves. Results shown on the graph represent the means ± SD of three independent experiments (**P < 0.01; ***P < 0.001). (C) Inhibition of TTP by siRNA enhances Lin28a mRNA stability. HCT116 cells were transfected with siRNA against TTP-specific (TTP-siRNA) or scRNA for 24 h. Expression of Lin28a mRNA in HCT116 cells was determined by qRT-PCR and mRNA half-life was calculated as described in (B). Results shown on the graph represent the means ± SD of three independent experiments (*P < 0.05). (D) Lin28a mRNA half-life in PA1 cell with low TTP level is longer than that in AGS cells with high TTP level. Expression of Lin28a mRNA in PA1 and AGS cells was determined by qRT-PCR and mRNA half-life was calculated as described in (B). Results shown on the graph represent the means ± SD of three independent experiments (***P < 0.001). (E and F) The first AUUUA pentamer (ARE1) within the Lin28a 3′-UTR is essential for the inhibitory effect of TTP. (E) Schematic representation of the luciferase reporter constructs used in this study. Fragments (Frag) and oligonucleotides (Oligo) derived from the Lin28a mRNA 3′-UTR were cloned downstream of the luciferase reporter gene in the psiCHECK2 luciferase expression vector. White circles, wild-type (W) pentameric motif AUUUA; gray circles, mutated (M) motif AGCA. (F) PA1 cells were co-transfected with pcDNA6/V5-TTP and a psiCHECK2 luciferase reporter constructs containing fragments (left panel) or oligonucleotides (right panel) derived from the Lin28a mRNA 3′-UTR as described in (E) for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells transfected with the Frag-ARE1 luciferase construct alone or Oligo-ARE1W alone were set to 1.0. Results shown represent the means ± SD of three independent experiments (*P < 0.05; **P < 0.01). ns, not significant. (G and H) TTP decreases the luciferase activity of luciferase reporter gene containing the Lin28b 3′-UTR. (G) Schematic representation of the luciferase reporter constructs used in this study. Fragments (Frag) derived from the Lin28b mRNA 3′-UTR were cloned downstream of the luciferase reporter gene in the psiCHECK2 luciferase expression vector. White circles, wild-type pentameric motif AUUUA. (H) PA1 cells were co-transfected with pcDNA6/V5-TTP and a psiCHECK2 luciferase reporter constructs containing fragments derived from the Lin28b mRNA 3′-UTR as described in (G) for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells transfected with each Frag-ARE luciferase construct alone was set to 1.0. Results shown represent the means ± SD of three independent experiments (**P < 0.01; ***P < 0.001). (I) Ribonucleoprotein immunoprecipitation assay. PA1 cells were cotransfected with pcDNA6/V5-TTP and psiCHECK2 luciferase reporter constructs containing Lin28a Oligo-ARE1W. psiCHECK2 luciferase reporter construct containing mutant ARE1, Oligo-ARE1M was used as a negative control. At 24 h after transfection, the ribonucleoprotein complexes containing TTP were immunoprecipitated with protein G-agarose and anti-V5 or a control antibody. The luciferase mRNA in the immunoprecipitates was amplified by RT-PCR. The presence of TTP in the immunoprecipitates was detected by western blot with anti-V5 antibody. (J and K) RNA EMSA was performed by mixing cytoplasmic extracts containing 3.0 µg of total protein from pcDNA6/V5-TTP-transfected PA1 cells (J) or HCT116 cells (K) with 80 fmol biotinylated wild-type Oligo-ARE1W (WT) or mutant Oligo-ARE1M (MUT) probe. Anti-V5 (J), anti-TTP (K) or control antibody was added to the reaction mixtures. Position of the TTP containing bands (TTP) and super-shifted bands (SS) are indicated.
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gkr1302-F5: TTP enhances the decay of Lin28a mRNA through interaction with an ARE within the Lin28a mRNA 3′-UTR. (A and B) TTP destabilizes Lin28a mRNA. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 for 24 h. (A) The level of TTP protein was determined by western blot assays. (B) Expression of Lin28a mRNA in PA1 cells was determined by qRT-PCR and mRNA half-life was calculated from the non-linear regression of the mRNA levels at the indicated times after the addition of 5.0 µg/ml actinomycin D. A one-phase model of exponential decay was used to derive the indicated mRNA decay curves. Results shown on the graph represent the means ± SD of three independent experiments (**P < 0.01; ***P < 0.001). (C) Inhibition of TTP by siRNA enhances Lin28a mRNA stability. HCT116 cells were transfected with siRNA against TTP-specific (TTP-siRNA) or scRNA for 24 h. Expression of Lin28a mRNA in HCT116 cells was determined by qRT-PCR and mRNA half-life was calculated as described in (B). Results shown on the graph represent the means ± SD of three independent experiments (*P < 0.05). (D) Lin28a mRNA half-life in PA1 cell with low TTP level is longer than that in AGS cells with high TTP level. Expression of Lin28a mRNA in PA1 and AGS cells was determined by qRT-PCR and mRNA half-life was calculated as described in (B). Results shown on the graph represent the means ± SD of three independent experiments (***P < 0.001). (E and F) The first AUUUA pentamer (ARE1) within the Lin28a 3′-UTR is essential for the inhibitory effect of TTP. (E) Schematic representation of the luciferase reporter constructs used in this study. Fragments (Frag) and oligonucleotides (Oligo) derived from the Lin28a mRNA 3′-UTR were cloned downstream of the luciferase reporter gene in the psiCHECK2 luciferase expression vector. White circles, wild-type (W) pentameric motif AUUUA; gray circles, mutated (M) motif AGCA. (F) PA1 cells were co-transfected with pcDNA6/V5-TTP and a psiCHECK2 luciferase reporter constructs containing fragments (left panel) or oligonucleotides (right panel) derived from the Lin28a mRNA 3′-UTR as described in (E) for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells transfected with the Frag-ARE1 luciferase construct alone or Oligo-ARE1W alone were set to 1.0. Results shown represent the means ± SD of three independent experiments (*P < 0.05; **P < 0.01). ns, not significant. (G and H) TTP decreases the luciferase activity of luciferase reporter gene containing the Lin28b 3′-UTR. (G) Schematic representation of the luciferase reporter constructs used in this study. Fragments (Frag) derived from the Lin28b mRNA 3′-UTR were cloned downstream of the luciferase reporter gene in the psiCHECK2 luciferase expression vector. White circles, wild-type pentameric motif AUUUA. (H) PA1 cells were co-transfected with pcDNA6/V5-TTP and a psiCHECK2 luciferase reporter constructs containing fragments derived from the Lin28b mRNA 3′-UTR as described in (G) for 24 h. After normalizing luciferase activity, the luciferase activity obtained from PA1 cells transfected with each Frag-ARE luciferase construct alone was set to 1.0. Results shown represent the means ± SD of three independent experiments (**P < 0.01; ***P < 0.001). (I) Ribonucleoprotein immunoprecipitation assay. PA1 cells were cotransfected with pcDNA6/V5-TTP and psiCHECK2 luciferase reporter constructs containing Lin28a Oligo-ARE1W. psiCHECK2 luciferase reporter construct containing mutant ARE1, Oligo-ARE1M was used as a negative control. At 24 h after transfection, the ribonucleoprotein complexes containing TTP were immunoprecipitated with protein G-agarose and anti-V5 or a control antibody. The luciferase mRNA in the immunoprecipitates was amplified by RT-PCR. The presence of TTP in the immunoprecipitates was detected by western blot with anti-V5 antibody. (J and K) RNA EMSA was performed by mixing cytoplasmic extracts containing 3.0 µg of total protein from pcDNA6/V5-TTP-transfected PA1 cells (J) or HCT116 cells (K) with 80 fmol biotinylated wild-type Oligo-ARE1W (WT) or mutant Oligo-ARE1M (MUT) probe. Anti-V5 (J), anti-TTP (K) or control antibody was added to the reaction mixtures. Position of the TTP containing bands (TTP) and super-shifted bands (SS) are indicated.
Mentions: To determine whether decreased expression of Lin28a resulted from changes in the stability of Lin28a mRNA, the half life of this mRNA was calculated from the mRNA levels measured by qRT-PCR in PA1 cells transfected with pcDNA6/V5-TTP or control pcDNA6/V5 vector. Overexpression of TTP protein in PA1/TTP cells was confirmed by western blot analysis (Figure 5A). In the control PA1/pcDNA cells, Lin28a mRNA was stable for 2 h after actinomycin D treatment. However, in PA1/TTP cells, the half-life of Lin28a mRNA was 1 h 30 min (Figure 5B). We also determined the half-life of Lin28a mRNA in HCT116 cells treated with siRNA against TTP (HCT116/TTP-siRNA) or control siRNA (HCT116/scRNA). In the control HCT116 and HCT116/scRNA cells, the half-life of Lin28a mRNA was 40 min. However, in HCT116/TTP-siRNA cells, Lin28a mRNA was stable until 3 h after actinomycin D treatment (Figure 5C). To determine whether regulation of Lin28a mRNA decay by TTP is biologically important, we compared the Lin28a mRNA half-life between HCT116 with high TTP levels and PA1 with low TTP level. As shown in Figure 5D, the half-life of Lin28a mRNA in PA1 cells was longer (t1/2 = 4 h 50 min) than that in AGS cells (t1/2 = 35 min). These results indicate that the TTP expression contributes to a decrease in Lin28a levels through destabilization of Lin28a mRNA.Figure 5.

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.

Show MeSH
Related in: MedlinePlus