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ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression.

Yoshikawa T, Wu J, Otsuka M, Kishikawa T, Ohno M, Shibata C, Takata A, Han F, Kang YJ, Chen CY, Shyu AB, Han J, Koike K - Nucleic Acids Res. (2015)

Bottom Line: Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors.Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription.ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

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Enhancement of miRNA function by Y27632 is mediated by increased PAIP2 expression. (A) β-globin reporter mRNA levels were determined using a β-globin construct carrying let-7 wt binding sites. Let-7-mediated mRNA decay was enhanced by Y27632 when transcription from this construct was stopped by doxycycline, but not when ActD was applied. β-globin reporter mRNA levels in 293T cells were determined by qRT-PCR 8 h after adding Y27632 with Dox or ActD treatment. Data represent the means ± S.D. of three experiments. *P< 0.05. (B) PAIP2 mRNA levels in 293T cells were determined by qRT-PCR at 6 h after Y27632 treatment. Data represent the means ± S.D. of three experiments. *P< 0.05. Similar results were obtained using Caco2 cells. (C) PAIP2 protein levels in Caco2 cells treated with Y27632 were determined at the indicated time points by western blotting. (D) Confirmation of reduced PAIP2 protein levels in PAIP2-knockdown Caco2 cells. (E, F) Y27632 did not enhance miRNA function in PAIP2-knockdown Caco2 cells. Cells were transfected with a reporter construct and the corresponding miRNA precursor-expressing plasmid [let-7 in (E) and miR-122 in (F)] and the effect of Y27632 was determined. Data represent the means ± S.D. of three experiments. *P< 0.05.
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Figure 3: Enhancement of miRNA function by Y27632 is mediated by increased PAIP2 expression. (A) β-globin reporter mRNA levels were determined using a β-globin construct carrying let-7 wt binding sites. Let-7-mediated mRNA decay was enhanced by Y27632 when transcription from this construct was stopped by doxycycline, but not when ActD was applied. β-globin reporter mRNA levels in 293T cells were determined by qRT-PCR 8 h after adding Y27632 with Dox or ActD treatment. Data represent the means ± S.D. of three experiments. *P< 0.05. (B) PAIP2 mRNA levels in 293T cells were determined by qRT-PCR at 6 h after Y27632 treatment. Data represent the means ± S.D. of three experiments. *P< 0.05. Similar results were obtained using Caco2 cells. (C) PAIP2 protein levels in Caco2 cells treated with Y27632 were determined at the indicated time points by western blotting. (D) Confirmation of reduced PAIP2 protein levels in PAIP2-knockdown Caco2 cells. (E, F) Y27632 did not enhance miRNA function in PAIP2-knockdown Caco2 cells. Cells were transfected with a reporter construct and the corresponding miRNA precursor-expressing plasmid [let-7 in (E) and miR-122 in (F)] and the effect of Y27632 was determined. Data represent the means ± S.D. of three experiments. *P< 0.05.

Mentions: To date, there is no information on the relationship between ROCK inhibition and miRNA function. We noticed that prolonged pretreatment with Y27632 was required to detect increased decay of miRNA-targeted mRNA when ActD was used to block global transcription (Figure 2C). Y27632 had no detectable effect when applied simultaneously with ActD. One possibility is that ROCK inhibition induces de novo protein synthesis and that newly synthesized proteins function to enhance miRNA-targeted mRNA degradation. To evaluate this, we compared the decay of a β-globin reporter mRNA driven by a Tet-off promoter after addition of Dox or ActD with or without Y27632. When transcription of a β-globin reporter mRNA containing wild-type let-7 binding sites was blocked by Dox, the ROCK inhibitor enhanced mRNA decay; however, when transcription was inhibited by ActD, the ROCK inhibitor had no detectable effect (Figure 3A). Thus ROCK inhibitors most likely require de novo protein synthesis to enhance miRNA function.


ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression.

Yoshikawa T, Wu J, Otsuka M, Kishikawa T, Ohno M, Shibata C, Takata A, Han F, Kang YJ, Chen CY, Shyu AB, Han J, Koike K - Nucleic Acids Res. (2015)

Enhancement of miRNA function by Y27632 is mediated by increased PAIP2 expression. (A) β-globin reporter mRNA levels were determined using a β-globin construct carrying let-7 wt binding sites. Let-7-mediated mRNA decay was enhanced by Y27632 when transcription from this construct was stopped by doxycycline, but not when ActD was applied. β-globin reporter mRNA levels in 293T cells were determined by qRT-PCR 8 h after adding Y27632 with Dox or ActD treatment. Data represent the means ± S.D. of three experiments. *P< 0.05. (B) PAIP2 mRNA levels in 293T cells were determined by qRT-PCR at 6 h after Y27632 treatment. Data represent the means ± S.D. of three experiments. *P< 0.05. Similar results were obtained using Caco2 cells. (C) PAIP2 protein levels in Caco2 cells treated with Y27632 were determined at the indicated time points by western blotting. (D) Confirmation of reduced PAIP2 protein levels in PAIP2-knockdown Caco2 cells. (E, F) Y27632 did not enhance miRNA function in PAIP2-knockdown Caco2 cells. Cells were transfected with a reporter construct and the corresponding miRNA precursor-expressing plasmid [let-7 in (E) and miR-122 in (F)] and the effect of Y27632 was determined. Data represent the means ± S.D. of three experiments. *P< 0.05.
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Figure 3: Enhancement of miRNA function by Y27632 is mediated by increased PAIP2 expression. (A) β-globin reporter mRNA levels were determined using a β-globin construct carrying let-7 wt binding sites. Let-7-mediated mRNA decay was enhanced by Y27632 when transcription from this construct was stopped by doxycycline, but not when ActD was applied. β-globin reporter mRNA levels in 293T cells were determined by qRT-PCR 8 h after adding Y27632 with Dox or ActD treatment. Data represent the means ± S.D. of three experiments. *P< 0.05. (B) PAIP2 mRNA levels in 293T cells were determined by qRT-PCR at 6 h after Y27632 treatment. Data represent the means ± S.D. of three experiments. *P< 0.05. Similar results were obtained using Caco2 cells. (C) PAIP2 protein levels in Caco2 cells treated with Y27632 were determined at the indicated time points by western blotting. (D) Confirmation of reduced PAIP2 protein levels in PAIP2-knockdown Caco2 cells. (E, F) Y27632 did not enhance miRNA function in PAIP2-knockdown Caco2 cells. Cells were transfected with a reporter construct and the corresponding miRNA precursor-expressing plasmid [let-7 in (E) and miR-122 in (F)] and the effect of Y27632 was determined. Data represent the means ± S.D. of three experiments. *P< 0.05.
Mentions: To date, there is no information on the relationship between ROCK inhibition and miRNA function. We noticed that prolonged pretreatment with Y27632 was required to detect increased decay of miRNA-targeted mRNA when ActD was used to block global transcription (Figure 2C). Y27632 had no detectable effect when applied simultaneously with ActD. One possibility is that ROCK inhibition induces de novo protein synthesis and that newly synthesized proteins function to enhance miRNA-targeted mRNA degradation. To evaluate this, we compared the decay of a β-globin reporter mRNA driven by a Tet-off promoter after addition of Dox or ActD with or without Y27632. When transcription of a β-globin reporter mRNA containing wild-type let-7 binding sites was blocked by Dox, the ROCK inhibitor enhanced mRNA decay; however, when transcription was inhibited by ActD, the ROCK inhibitor had no detectable effect (Figure 3A). Thus ROCK inhibitors most likely require de novo protein synthesis to enhance miRNA function.

Bottom Line: Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors.Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription.ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655, Japan.

Show MeSH
Related in: MedlinePlus