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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 levels of Lin28a in human cancer cells. (A–D) Overexpression of TTP inhibits Lin28a levels in PA1 cells. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 for 24 h. (A) The levels of TTP, Drosha, Dicer, Ago2 and Lin28a proteins were determined by western blot assays. (B) The expression levels of TTP and Lin28a were determined by semi-qRT-PCR. (C) The level of Lin28a was determined 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 < 0.01). (D) Overexpression of TTP decreases Lin28b levels in PA1 cells. The level of TTP and Lin28b were determined by RT-PCR (top panel) and western blot (bottom panel). (E–I) Downregulation of TTP by siRNA increases Lin28a levels and decreases let-7b in HCT116 cells. HCT116 cells were transfected with TTP-specific (TTP-siRNA) or scRNA. After 24 h, the levels of TTP and Lin28a were determined by qRT-PCR (E and F) and western blot assays (G). The level of let-7b was determined by qRT-PCR (H) and cell viability was assessed by measuring absorbance at 490 nm using a MTS cell proliferation assay (I). The levels obtained from mock-transfected HCT116 cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01; ***P < 0.001). ns, not significant. (J) The level of TTP protein is inversely correlated with those of Lin28a protein within several human cell lines. Levels of TTP and Lin28a proteins were determined by western blot analysis in PA1 (ovarian teratocarcinoma), MCF7 (breast adenocarcinoma), AGS (gastric adenocarcinoma), K562 (erythroleukemia), HepG2 (hepatocellular carcinoma) and NT2 (neuronally committed teratocarcinoma) cells. β-Actin was detected as the loading control. (K) TTP expression level is inversely correlated with that of Lin28a in human ovarian tissues. Representative TTP and Lin28a immunohistochemical staining in normal and ovarian adenocarcinoma tissues. Normal ovarian tissues showed strong immunoreactivity for TTP, where as the ovarian adenocarcinoma showed strong positive staining for Lin28a.
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gkr1302-F4: TTP negatively regulates the levels of Lin28a in human cancer cells. (A–D) Overexpression of TTP inhibits Lin28a levels in PA1 cells. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 for 24 h. (A) The levels of TTP, Drosha, Dicer, Ago2 and Lin28a proteins were determined by western blot assays. (B) The expression levels of TTP and Lin28a were determined by semi-qRT-PCR. (C) The level of Lin28a was determined 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 < 0.01). (D) Overexpression of TTP decreases Lin28b levels in PA1 cells. The level of TTP and Lin28b were determined by RT-PCR (top panel) and western blot (bottom panel). (E–I) Downregulation of TTP by siRNA increases Lin28a levels and decreases let-7b in HCT116 cells. HCT116 cells were transfected with TTP-specific (TTP-siRNA) or scRNA. After 24 h, the levels of TTP and Lin28a were determined by qRT-PCR (E and F) and western blot assays (G). The level of let-7b was determined by qRT-PCR (H) and cell viability was assessed by measuring absorbance at 490 nm using a MTS cell proliferation assay (I). The levels obtained from mock-transfected HCT116 cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01; ***P < 0.001). ns, not significant. (J) The level of TTP protein is inversely correlated with those of Lin28a protein within several human cell lines. Levels of TTP and Lin28a proteins were determined by western blot analysis in PA1 (ovarian teratocarcinoma), MCF7 (breast adenocarcinoma), AGS (gastric adenocarcinoma), K562 (erythroleukemia), HepG2 (hepatocellular carcinoma) and NT2 (neuronally committed teratocarcinoma) cells. β-Actin was detected as the loading control. (K) TTP expression level is inversely correlated with that of Lin28a in human ovarian tissues. Representative TTP and Lin28a immunohistochemical staining in normal and ovarian adenocarcinoma tissues. Normal ovarian tissues showed strong immunoreactivity for TTP, where as the ovarian adenocarcinoma showed strong positive staining for Lin28a.

Mentions: TTP overexpression did not affect the levels of pri- and pre-let-7b but increased mature let-7b level (Figure 1C). Thus, we hypothesized that TTP controls let-7b biogenesis by modulating the expression level of proteins involved in the post-transcriptional regulation of let-7b biogenesis. The core components involved in the post-transcriptional regulation of miRNA biogenesis are Drosha, Dicer, Argonaute (AGO) and Lin28 (6); as such, we analyzed the levels of these known let-7b regulatory factors in PA1/TTP and PA1/pcDNA cells. Western blot analysis revealed that while TTP overexpression did not affect the levels of Drosha, Dicer and AGO, it decreased the expression of the negative regulatory factor Lin28a (Figure 4A). TTP-induced reduction of Lin28a level was confirmed by semi-qRT-PCR (Figure 4B) and qRT-PCR (Figure 4C). TTP also decreased Lin28b level in PA1 cells (Figure 4D).Figure 4.


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 levels of Lin28a in human cancer cells. (A–D) Overexpression of TTP inhibits Lin28a levels in PA1 cells. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 for 24 h. (A) The levels of TTP, Drosha, Dicer, Ago2 and Lin28a proteins were determined by western blot assays. (B) The expression levels of TTP and Lin28a were determined by semi-qRT-PCR. (C) The level of Lin28a was determined 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 < 0.01). (D) Overexpression of TTP decreases Lin28b levels in PA1 cells. The level of TTP and Lin28b were determined by RT-PCR (top panel) and western blot (bottom panel). (E–I) Downregulation of TTP by siRNA increases Lin28a levels and decreases let-7b in HCT116 cells. HCT116 cells were transfected with TTP-specific (TTP-siRNA) or scRNA. After 24 h, the levels of TTP and Lin28a were determined by qRT-PCR (E and F) and western blot assays (G). The level of let-7b was determined by qRT-PCR (H) and cell viability was assessed by measuring absorbance at 490 nm using a MTS cell proliferation assay (I). The levels obtained from mock-transfected HCT116 cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01; ***P < 0.001). ns, not significant. (J) The level of TTP protein is inversely correlated with those of Lin28a protein within several human cell lines. Levels of TTP and Lin28a proteins were determined by western blot analysis in PA1 (ovarian teratocarcinoma), MCF7 (breast adenocarcinoma), AGS (gastric adenocarcinoma), K562 (erythroleukemia), HepG2 (hepatocellular carcinoma) and NT2 (neuronally committed teratocarcinoma) cells. β-Actin was detected as the loading control. (K) TTP expression level is inversely correlated with that of Lin28a in human ovarian tissues. Representative TTP and Lin28a immunohistochemical staining in normal and ovarian adenocarcinoma tissues. Normal ovarian tissues showed strong immunoreactivity for TTP, where as the ovarian adenocarcinoma showed strong positive staining for Lin28a.
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gkr1302-F4: TTP negatively regulates the levels of Lin28a in human cancer cells. (A–D) Overexpression of TTP inhibits Lin28a levels in PA1 cells. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 for 24 h. (A) The levels of TTP, Drosha, Dicer, Ago2 and Lin28a proteins were determined by western blot assays. (B) The expression levels of TTP and Lin28a were determined by semi-qRT-PCR. (C) The level of Lin28a was determined 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 < 0.01). (D) Overexpression of TTP decreases Lin28b levels in PA1 cells. The level of TTP and Lin28b were determined by RT-PCR (top panel) and western blot (bottom panel). (E–I) Downregulation of TTP by siRNA increases Lin28a levels and decreases let-7b in HCT116 cells. HCT116 cells were transfected with TTP-specific (TTP-siRNA) or scRNA. After 24 h, the levels of TTP and Lin28a were determined by qRT-PCR (E and F) and western blot assays (G). The level of let-7b was determined by qRT-PCR (H) and cell viability was assessed by measuring absorbance at 490 nm using a MTS cell proliferation assay (I). The levels obtained from mock-transfected HCT116 cells were set to 1.0. Data are presented as the mean ± SD (n = 3) (**P < 0.01; ***P < 0.001). ns, not significant. (J) The level of TTP protein is inversely correlated with those of Lin28a protein within several human cell lines. Levels of TTP and Lin28a proteins were determined by western blot analysis in PA1 (ovarian teratocarcinoma), MCF7 (breast adenocarcinoma), AGS (gastric adenocarcinoma), K562 (erythroleukemia), HepG2 (hepatocellular carcinoma) and NT2 (neuronally committed teratocarcinoma) cells. β-Actin was detected as the loading control. (K) TTP expression level is inversely correlated with that of Lin28a in human ovarian tissues. Representative TTP and Lin28a immunohistochemical staining in normal and ovarian adenocarcinoma tissues. Normal ovarian tissues showed strong immunoreactivity for TTP, where as the ovarian adenocarcinoma showed strong positive staining for Lin28a.
Mentions: TTP overexpression did not affect the levels of pri- and pre-let-7b but increased mature let-7b level (Figure 1C). Thus, we hypothesized that TTP controls let-7b biogenesis by modulating the expression level of proteins involved in the post-transcriptional regulation of let-7b biogenesis. The core components involved in the post-transcriptional regulation of miRNA biogenesis are Drosha, Dicer, Argonaute (AGO) and Lin28 (6); as such, we analyzed the levels of these known let-7b regulatory factors in PA1/TTP and PA1/pcDNA cells. Western blot analysis revealed that while TTP overexpression did not affect the levels of Drosha, Dicer and AGO, it decreased the expression of the negative regulatory factor Lin28a (Figure 4A). TTP-induced reduction of Lin28a level was confirmed by semi-qRT-PCR (Figure 4B) and qRT-PCR (Figure 4C). TTP also decreased Lin28b level in PA1 cells (Figure 4D).Figure 4.

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