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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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Overexpression of Lin28a attenuates the effects of TTP on let-7 b levels, CDC34 levels and PA1 cell growth. (A–C) Downregulation of Lin28a by siRNA increases let-7b levels but decreases CDC34 levels in PA1 cells. PA1 cells were transfected with Lin28a-specific (Lin28a-siRNA) or scRNA for 24 h. (A) The level of Lin28a was determined by qRT-PCR (top panel) and western blot assays (bottom panel). The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (B) The level of mature let-7b was measured by qRT-PCR. The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (*P < 0.05). (C) The level of CDC34 was determined by qRT-PCR. The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (D–F) Transfection of Lin28a cDNA without the 3′-UTR abolishes the effects of TTP on expression of let-7b, CDC34 and PA1 cell growth. PA1 cells were transfected with a combination of pcDNA6/V5-TTP and pcDNA3/Flag-Lin28a for 24 h. (D) The levels of TTP and Lin28a were measured by semi-qRT-PCR (top panel) and western blot assays (bottom panel). (E) The level of let-7b was measured by qRT-PCR. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (*P < 0.05; **P < 0.01). (F) Cell viability was assessed by measuring absorbance at 490 nm using a MTS cell proliferation assay. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01; ***P < 0.001).
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gkr1302-F6: Overexpression of Lin28a attenuates the effects of TTP on let-7 b levels, CDC34 levels and PA1 cell growth. (A–C) Downregulation of Lin28a by siRNA increases let-7b levels but decreases CDC34 levels in PA1 cells. PA1 cells were transfected with Lin28a-specific (Lin28a-siRNA) or scRNA for 24 h. (A) The level of Lin28a was determined by qRT-PCR (top panel) and western blot assays (bottom panel). The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (B) The level of mature let-7b was measured by qRT-PCR. The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (*P < 0.05). (C) The level of CDC34 was determined by qRT-PCR. The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (D–F) Transfection of Lin28a cDNA without the 3′-UTR abolishes the effects of TTP on expression of let-7b, CDC34 and PA1 cell growth. PA1 cells were transfected with a combination of pcDNA6/V5-TTP and pcDNA3/Flag-Lin28a for 24 h. (D) The levels of TTP and Lin28a were measured by semi-qRT-PCR (top panel) and western blot assays (bottom panel). (E) The level of let-7b was measured by qRT-PCR. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (*P < 0.05; **P < 0.01). (F) Cell viability was assessed by measuring absorbance at 490 nm using a MTS cell proliferation assay. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01; ***P < 0.001).

Mentions: Based on our results, it was speculated that TTP induces let-7b level through down-regulation of Lin28a. To confirm this hypothesis, we first determined whether knockdown of Lin28a by siRNA enhanced let-7b expression in PA1 cells. Western blot analysis confirmed the inhibition of Lin28a expression in PA1 cells (PA1/Lin28a-siRNA) by siRNA treatment (Figure 6A). As expected, let-7b expression level in PA1/Lin28a-siRNA was increased compared to PA1 cells treated with control siRNA (PA1/scRNA) (Figure 6B). In addition, inhibition of Lin28a decreased the level of CDC34 (Figure 6C). To confirm the involvement of Lin28a for the TTP-induced increase of let-7b level, we co-transfected PA1 cells with pcDNA6/V5-TTP and pcDNA3/Flag-Lin28a, which does not contain the Lin28a 3′-UTR. At 24-h post-transfection, cells were analyzed for the expression of let-7b, CDC34 and proliferation. Overexpression of Lin28a (Figure 6D) abrogated the effect of TTP on the expression of let-7b and CDC34 (Figure 6E). In addition, overexpression of Lin28a restored the growth of PA1/TTP to 87% of PA1/pcDNA (Figure 6F). These results indicate that TTP affects the expression level of let-7b and CDC34 and the cell growth through down-regulation of Lin28a in PA1 cells.Figure 6.


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)

Overexpression of Lin28a attenuates the effects of TTP on let-7 b levels, CDC34 levels and PA1 cell growth. (A–C) Downregulation of Lin28a by siRNA increases let-7b levels but decreases CDC34 levels in PA1 cells. PA1 cells were transfected with Lin28a-specific (Lin28a-siRNA) or scRNA for 24 h. (A) The level of Lin28a was determined by qRT-PCR (top panel) and western blot assays (bottom panel). The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (B) The level of mature let-7b was measured by qRT-PCR. The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (*P < 0.05). (C) The level of CDC34 was determined by qRT-PCR. The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (D–F) Transfection of Lin28a cDNA without the 3′-UTR abolishes the effects of TTP on expression of let-7b, CDC34 and PA1 cell growth. PA1 cells were transfected with a combination of pcDNA6/V5-TTP and pcDNA3/Flag-Lin28a for 24 h. (D) The levels of TTP and Lin28a were measured by semi-qRT-PCR (top panel) and western blot assays (bottom panel). (E) The level of let-7b was measured by qRT-PCR. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (*P < 0.05; **P < 0.01). (F) Cell viability was assessed by measuring absorbance at 490 nm using a MTS cell proliferation assay. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01; ***P < 0.001).
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gkr1302-F6: Overexpression of Lin28a attenuates the effects of TTP on let-7 b levels, CDC34 levels and PA1 cell growth. (A–C) Downregulation of Lin28a by siRNA increases let-7b levels but decreases CDC34 levels in PA1 cells. PA1 cells were transfected with Lin28a-specific (Lin28a-siRNA) or scRNA for 24 h. (A) The level of Lin28a was determined by qRT-PCR (top panel) and western blot assays (bottom panel). The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (B) The level of mature let-7b was measured by qRT-PCR. The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (*P < 0.05). (C) The level of CDC34 was determined by qRT-PCR. The levels obtained from PA1/scRNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (***P < 0.001). (D–F) Transfection of Lin28a cDNA without the 3′-UTR abolishes the effects of TTP on expression of let-7b, CDC34 and PA1 cell growth. PA1 cells were transfected with a combination of pcDNA6/V5-TTP and pcDNA3/Flag-Lin28a for 24 h. (D) The levels of TTP and Lin28a were measured by semi-qRT-PCR (top panel) and western blot assays (bottom panel). (E) The level of let-7b was measured by qRT-PCR. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (*P < 0.05; **P < 0.01). (F) Cell viability was assessed by measuring absorbance at 490 nm using a MTS cell proliferation assay. The levels obtained from PA1/pcDNA cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01; ***P < 0.001).
Mentions: Based on our results, it was speculated that TTP induces let-7b level through down-regulation of Lin28a. To confirm this hypothesis, we first determined whether knockdown of Lin28a by siRNA enhanced let-7b expression in PA1 cells. Western blot analysis confirmed the inhibition of Lin28a expression in PA1 cells (PA1/Lin28a-siRNA) by siRNA treatment (Figure 6A). As expected, let-7b expression level in PA1/Lin28a-siRNA was increased compared to PA1 cells treated with control siRNA (PA1/scRNA) (Figure 6B). In addition, inhibition of Lin28a decreased the level of CDC34 (Figure 6C). To confirm the involvement of Lin28a for the TTP-induced increase of let-7b level, we co-transfected PA1 cells with pcDNA6/V5-TTP and pcDNA3/Flag-Lin28a, which does not contain the Lin28a 3′-UTR. At 24-h post-transfection, cells were analyzed for the expression of let-7b, CDC34 and proliferation. Overexpression of Lin28a (Figure 6D) abrogated the effect of TTP on the expression of let-7b and CDC34 (Figure 6E). In addition, overexpression of Lin28a restored the growth of PA1/TTP to 87% of PA1/pcDNA (Figure 6F). These results indicate that TTP affects the expression level of let-7b and CDC34 and the cell growth through down-regulation of Lin28a in PA1 cells.Figure 6.

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