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Regulation of PP2Cm expression by miRNA-204/211 and miRNA-22 in mouse and human cells.

Pan BF, Gao C, Ren SX, Wang YB, Sun HP, Zhou MY - Acta Pharmacol. Sin. (2015)

Bottom Line: However, the miR-22 binding site was found only in human and not mouse PPM1K 3'UTR.Accordingly, PPM1K 3'UTR activity was suppressed by miR-22 overexpression in human but not mouse cells.These data suggest that different miRNAs contribute to the regulation of PP2Cm expression in a species-specific manner. miR-204 and miR-211 are efficient in both mouse and human cells, while miR-22 regulates PP2Cm expression only in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT

Aim: The mitochondrial targeted 2C-type serine/threonine protein phosphatase (PP2Cm) is encoded by the gene PPM1K and is highly conserved among vertebrates. PP2Cm plays a critical role in branched-chain amino acid catabolism and regulates cell survival. Its expression is dynamically regulated by the nutrient environment and pathological stresses. However, little is known about the molecular mechanism underlying the regulation of PPM1K gene expression. In this study, we aimed to reveal how PPM1K expression is affected by miRNA-mediated post-transcriptional regulation.

Methods: Computational analysis based on conserved miRNA binding motifs was applied to predict the candidate miRNAs that potentially affect PPM1K expression. Dual-luciferase reporter assay was performed to verify the miRNAs' binding sites in the PPM1K gene and their influence on PPM1K 3'UTR activity. We further over-expressed the mimics of these miRNAs in human and mouse cells to examine whether miRNAs affected the mRNA level of PPM1K.

Results: Computational analysis identified numerous miRNAs potentially targeting PPM1K. Luciferase reporter assays demonstrated that the 3'UTR of PPM1K gene contained the recognition sites of miR-204 and miR-211. Overexpression of these miRNAs in human and mouse cells diminished the 3'UTR activity and the endogenous mRNA level of PPM1K. However, the miR-22 binding site was found only in human and not mouse PPM1K 3'UTR. Accordingly, PPM1K 3'UTR activity was suppressed by miR-22 overexpression in human but not mouse cells.

Conclusion: These data suggest that different miRNAs contribute to the regulation of PP2Cm expression in a species-specific manner. miR-204 and miR-211 are efficient in both mouse and human cells, while miR-22 regulates PP2Cm expression only in human cells.

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Candidate miRNAs regulate expression of PPM1K gene in human and mouse cell lines. (A) Summary of putative miRNAs targeting human and mouse PPM1K 3′UTR. (B and C) miR-204 abundance and PP2Cm mRNA level were assessed in HepG2 (B) and NIH 3T3 (C) cells after transfection with miRNA mimics. Data are mean±SEM for three individual samples (bP<0.05).
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fig1: Candidate miRNAs regulate expression of PPM1K gene in human and mouse cell lines. (A) Summary of putative miRNAs targeting human and mouse PPM1K 3′UTR. (B and C) miR-204 abundance and PP2Cm mRNA level were assessed in HepG2 (B) and NIH 3T3 (C) cells after transfection with miRNA mimics. Data are mean±SEM for three individual samples (bP<0.05).

Mentions: We used TargetScan (online database, http://www.targetscan.org/) to find the binding sites of miRNAs potentially targeting the PPM1K gene via its 3′UTR sequence. The human (2214 base pairs in length) and mouse (4216 base pairs in length) 3′UTR sequences of PPM1K were analyzed. Among the miRNA candidates, miR-204, miR-211 and miR-22 are of particular interest. In addition to a less conserved binding site in mouse, miR-204 and miR-211 have conserved binding sites in the 3′UTR region of both the human and mouse PPM1K genes (Figure 1A). The miR-22 conserved binding site was only detected in the 3′UTR region of the human but not the mouse PPM1K gene (Figure 1A).


Regulation of PP2Cm expression by miRNA-204/211 and miRNA-22 in mouse and human cells.

Pan BF, Gao C, Ren SX, Wang YB, Sun HP, Zhou MY - Acta Pharmacol. Sin. (2015)

Candidate miRNAs regulate expression of PPM1K gene in human and mouse cell lines. (A) Summary of putative miRNAs targeting human and mouse PPM1K 3′UTR. (B and C) miR-204 abundance and PP2Cm mRNA level were assessed in HepG2 (B) and NIH 3T3 (C) cells after transfection with miRNA mimics. Data are mean±SEM for three individual samples (bP<0.05).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4816230&req=5

fig1: Candidate miRNAs regulate expression of PPM1K gene in human and mouse cell lines. (A) Summary of putative miRNAs targeting human and mouse PPM1K 3′UTR. (B and C) miR-204 abundance and PP2Cm mRNA level were assessed in HepG2 (B) and NIH 3T3 (C) cells after transfection with miRNA mimics. Data are mean±SEM for three individual samples (bP<0.05).
Mentions: We used TargetScan (online database, http://www.targetscan.org/) to find the binding sites of miRNAs potentially targeting the PPM1K gene via its 3′UTR sequence. The human (2214 base pairs in length) and mouse (4216 base pairs in length) 3′UTR sequences of PPM1K were analyzed. Among the miRNA candidates, miR-204, miR-211 and miR-22 are of particular interest. In addition to a less conserved binding site in mouse, miR-204 and miR-211 have conserved binding sites in the 3′UTR region of both the human and mouse PPM1K genes (Figure 1A). The miR-22 conserved binding site was only detected in the 3′UTR region of the human but not the mouse PPM1K gene (Figure 1A).

Bottom Line: However, the miR-22 binding site was found only in human and not mouse PPM1K 3'UTR.Accordingly, PPM1K 3'UTR activity was suppressed by miR-22 overexpression in human but not mouse cells.These data suggest that different miRNAs contribute to the regulation of PP2Cm expression in a species-specific manner. miR-204 and miR-211 are efficient in both mouse and human cells, while miR-22 regulates PP2Cm expression only in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

ABSTRACT

Aim: The mitochondrial targeted 2C-type serine/threonine protein phosphatase (PP2Cm) is encoded by the gene PPM1K and is highly conserved among vertebrates. PP2Cm plays a critical role in branched-chain amino acid catabolism and regulates cell survival. Its expression is dynamically regulated by the nutrient environment and pathological stresses. However, little is known about the molecular mechanism underlying the regulation of PPM1K gene expression. In this study, we aimed to reveal how PPM1K expression is affected by miRNA-mediated post-transcriptional regulation.

Methods: Computational analysis based on conserved miRNA binding motifs was applied to predict the candidate miRNAs that potentially affect PPM1K expression. Dual-luciferase reporter assay was performed to verify the miRNAs' binding sites in the PPM1K gene and their influence on PPM1K 3'UTR activity. We further over-expressed the mimics of these miRNAs in human and mouse cells to examine whether miRNAs affected the mRNA level of PPM1K.

Results: Computational analysis identified numerous miRNAs potentially targeting PPM1K. Luciferase reporter assays demonstrated that the 3'UTR of PPM1K gene contained the recognition sites of miR-204 and miR-211. Overexpression of these miRNAs in human and mouse cells diminished the 3'UTR activity and the endogenous mRNA level of PPM1K. However, the miR-22 binding site was found only in human and not mouse PPM1K 3'UTR. Accordingly, PPM1K 3'UTR activity was suppressed by miR-22 overexpression in human but not mouse cells.

Conclusion: These data suggest that different miRNAs contribute to the regulation of PP2Cm expression in a species-specific manner. miR-204 and miR-211 are efficient in both mouse and human cells, while miR-22 regulates PP2Cm expression only in human cells.

Show MeSH