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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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Transfection of CDC34 cDNA without the 3′-UTR (or overexpression of CDC34) attenuates the inhibitory effect of TTP on the growth of PA1 cells. (A) Overexpression of TTP suppresses the growth of PA1 cells. PA1 cells transfected with pcDNA6/V5-TTP or pcDNA6/V5 were seeded at 1.0 × 104 cells per well in 96-well plates. Cell viability was assessed at 24 h after seeding by measuring absorbance at 490 nm using a MTS cell proliferation assay. The values obtained from PA1/Mock cells were set to 1.0. The data represent the mean ± SD of three independent experiments (***P < 0.001). ns, not significant. (B–D) Transfection of CDC34 cDNA without 3′-UTR abrogates the suppressive effect of TTP on PA1 growth. PA1 cells were transfected with a combination of pcDNA6/V5-TTP and pCMVT2B/CDC34 for 24 h. (B) The levels of TTP and CDC34 proteins were measured by western blot assays. (C) CDC34 levels were 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 < 0.01; ***P < 0.001). (D) 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).
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gkr1302-F3: Transfection of CDC34 cDNA without the 3′-UTR (or overexpression of CDC34) attenuates the inhibitory effect of TTP on the growth of PA1 cells. (A) Overexpression of TTP suppresses the growth of PA1 cells. PA1 cells transfected with pcDNA6/V5-TTP or pcDNA6/V5 were seeded at 1.0 × 104 cells per well in 96-well plates. Cell viability was assessed at 24 h after seeding by measuring absorbance at 490 nm using a MTS cell proliferation assay. The values obtained from PA1/Mock cells were set to 1.0. The data represent the mean ± SD of three independent experiments (***P < 0.001). ns, not significant. (B–D) Transfection of CDC34 cDNA without 3′-UTR abrogates the suppressive effect of TTP on PA1 growth. PA1 cells were transfected with a combination of pcDNA6/V5-TTP and pCMVT2B/CDC34 for 24 h. (B) The levels of TTP and CDC34 proteins were measured by western blot assays. (C) CDC34 levels were 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 < 0.01; ***P < 0.001). (D) 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).

Mentions: We next examined whether TTP overexpression affects the proliferation of PA1 cells in vitro. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 and we evaluated the effects of TTP overexpression on cellular metabolic activity using MTS cell proliferation assay. The results showed that TTP overexpression decreased the MTS value (Figure 3A). It has been reported that TTP overexpression can induce apoptosis (35). However, overexpression of TTP did not induce apoptosis in PA1 cells (Supplementary Figure S3), indicating that decrease in the MTS value is resulted from TTP-induced suppression of the cell growth. TTP expression can suppress cell growth through destabilization of VEGF, COX2, and c-Fos, c-Myc, and cyclin D mRNAs (26,27,29). However, as shown in Supplementary Figure S2, there was no difference in expression of these genes between PA1/pcDNA and PA1/TTP cells. Also, TTP overexpression did not lead to a change in the expression level of several genes involved in the control of cell cycle such as CCNA1, CCNB1, CCNB2, CCND1, CCND2, CCND3 and CDK2 (Supplementary Figure S2). CDC34 is an E2 ubiquitin-conjugating enzyme that supports cell growth by facilitating the proteasome-mediated degradation of multiple cell cycle regulators (36). To determine whether the TTP-induced inhibition of cell growth was mediated by a reduction in CDC34 levels, we transfected PA1 cells with CDC34 cDNA, whose expression is not affected by TTP and let-7b because the transcript does not contain a CDC34 3′-UTR. Overexpression of CDC34 (Figure 3B and C) increased the growth of PA1/TTP to 92% of PA1/pcDNA (Figure 3D). These results show that TTP expression suppresses the growth of PA1 cells through down-regulation of CDC34.Figure 3.


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)

Transfection of CDC34 cDNA without the 3′-UTR (or overexpression of CDC34) attenuates the inhibitory effect of TTP on the growth of PA1 cells. (A) Overexpression of TTP suppresses the growth of PA1 cells. PA1 cells transfected with pcDNA6/V5-TTP or pcDNA6/V5 were seeded at 1.0 × 104 cells per well in 96-well plates. Cell viability was assessed at 24 h after seeding by measuring absorbance at 490 nm using a MTS cell proliferation assay. The values obtained from PA1/Mock cells were set to 1.0. The data represent the mean ± SD of three independent experiments (***P < 0.001). ns, not significant. (B–D) Transfection of CDC34 cDNA without 3′-UTR abrogates the suppressive effect of TTP on PA1 growth. PA1 cells were transfected with a combination of pcDNA6/V5-TTP and pCMVT2B/CDC34 for 24 h. (B) The levels of TTP and CDC34 proteins were measured by western blot assays. (C) CDC34 levels were 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 < 0.01; ***P < 0.001). (D) 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).
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gkr1302-F3: Transfection of CDC34 cDNA without the 3′-UTR (or overexpression of CDC34) attenuates the inhibitory effect of TTP on the growth of PA1 cells. (A) Overexpression of TTP suppresses the growth of PA1 cells. PA1 cells transfected with pcDNA6/V5-TTP or pcDNA6/V5 were seeded at 1.0 × 104 cells per well in 96-well plates. Cell viability was assessed at 24 h after seeding by measuring absorbance at 490 nm using a MTS cell proliferation assay. The values obtained from PA1/Mock cells were set to 1.0. The data represent the mean ± SD of three independent experiments (***P < 0.001). ns, not significant. (B–D) Transfection of CDC34 cDNA without 3′-UTR abrogates the suppressive effect of TTP on PA1 growth. PA1 cells were transfected with a combination of pcDNA6/V5-TTP and pCMVT2B/CDC34 for 24 h. (B) The levels of TTP and CDC34 proteins were measured by western blot assays. (C) CDC34 levels were 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 < 0.01; ***P < 0.001). (D) 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).
Mentions: We next examined whether TTP overexpression affects the proliferation of PA1 cells in vitro. PA1 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5 and we evaluated the effects of TTP overexpression on cellular metabolic activity using MTS cell proliferation assay. The results showed that TTP overexpression decreased the MTS value (Figure 3A). It has been reported that TTP overexpression can induce apoptosis (35). However, overexpression of TTP did not induce apoptosis in PA1 cells (Supplementary Figure S3), indicating that decrease in the MTS value is resulted from TTP-induced suppression of the cell growth. TTP expression can suppress cell growth through destabilization of VEGF, COX2, and c-Fos, c-Myc, and cyclin D mRNAs (26,27,29). However, as shown in Supplementary Figure S2, there was no difference in expression of these genes between PA1/pcDNA and PA1/TTP cells. Also, TTP overexpression did not lead to a change in the expression level of several genes involved in the control of cell cycle such as CCNA1, CCNB1, CCNB2, CCND1, CCND2, CCND3 and CDK2 (Supplementary Figure S2). CDC34 is an E2 ubiquitin-conjugating enzyme that supports cell growth by facilitating the proteasome-mediated degradation of multiple cell cycle regulators (36). To determine whether the TTP-induced inhibition of cell growth was mediated by a reduction in CDC34 levels, we transfected PA1 cells with CDC34 cDNA, whose expression is not affected by TTP and let-7b because the transcript does not contain a CDC34 3′-UTR. Overexpression of CDC34 (Figure 3B and C) increased the growth of PA1/TTP to 92% of PA1/pcDNA (Figure 3D). These results show that TTP expression suppresses the growth of PA1 cells through down-regulation of CDC34.Figure 3.

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