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Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p.

Nguyen HT, Jia W, Beedle AM, Kennedy EJ, Murph MM - PLoS ONE (2015)

Bottom Line: The 3´-untranslated region (3´-UTR) of ATF3 was a predicted, putative target for miR-30c-2-3p, which we confirmed as a bona-fide interaction using a luciferase reporter assay.Furthermore, the presence of anti-miR-30c-2-3p enhanced ATF3 mRNA and protein after lysophosphatidic acid stimulation.Thus, the data suggest that after the expression of ATF3 and miR-30c-2-3p are elicited by lysophosphatidic acid, subsequently miR-30c-2-3p negatively regulates the expression of ATF3 through post-transcriptional silencing, which prevents further ATF3-related outcomes as a consequence of lysophosphatidic acid signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, College of Pharmacy, 240 W. Green Street, Athens, Georgia 30602, United States of America.

ABSTRACT
Although microRNAs (miRNAs) are small, non-protein-coding entities, they have important roles in post-transcriptional regulation of most of the human genome. These small entities generate fine-tuning adjustments in the expression of mRNA, which can mildly or massively affect the abundance of proteins. Previously, we found that the expression of miR-30c-2-3p is induced by lysophosphatidic acid and has an important role in the regulation of cell proliferation in ovarian cancer cells. The goal here is to confirm that ATF3 mRNA is a target of miR-30c-2-3p silencing, thereby further establishing the functional role of miR-30c-2-3p. Using a combination of bioinformatics, qRT-PCR, immunoblotting and luciferase assays, we uncovered a regulatory pathway between miR-30c-2-3p and the expression of the transcription factor, ATF3. Lysophosphatidic acids triggers the expression of both miR-30c-2-3p and ATF3, which peak at 1 h and are absent 8 h post stimulation in SKOV-3 and OVCAR-3 serous ovarian cancer cells. The 3´-untranslated region (3´-UTR) of ATF3 was a predicted, putative target for miR-30c-2-3p, which we confirmed as a bona-fide interaction using a luciferase reporter assay. Specific mutations introduced into the predicted site of interaction between miR-30c-2-3p and the 3´-UTR of ATF3 alleviated the suppression of the luciferase signal. Furthermore, the presence of anti-miR-30c-2-3p enhanced ATF3 mRNA and protein after lysophosphatidic acid stimulation. Thus, the data suggest that after the expression of ATF3 and miR-30c-2-3p are elicited by lysophosphatidic acid, subsequently miR-30c-2-3p negatively regulates the expression of ATF3 through post-transcriptional silencing, which prevents further ATF3-related outcomes as a consequence of lysophosphatidic acid signaling.

No MeSH data available.


Related in: MedlinePlus

Lysophosphatidic acid induces the expression of ATF3.(A) SKOV-3 cells were treated with 5 μM lysophosphatidic acid (LPA) for the times indicated prior to assessment of ATF3 protein. (B) SKOV-3 and OVCAR-3 cells were treated with 5 μM LPA for the times indicated prior to qRT-PCR measurement and normalization of ATF3 transcripts. SKOV-3 cells were treated with increasing concentrations of LPA for 1 h before measuring ATF3 mRNA (C) or protein (D). ***p<0.001, comparing the indicated condition to time 0 or untreated control.
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pone.0139489.g001: Lysophosphatidic acid induces the expression of ATF3.(A) SKOV-3 cells were treated with 5 μM lysophosphatidic acid (LPA) for the times indicated prior to assessment of ATF3 protein. (B) SKOV-3 and OVCAR-3 cells were treated with 5 μM LPA for the times indicated prior to qRT-PCR measurement and normalization of ATF3 transcripts. SKOV-3 cells were treated with increasing concentrations of LPA for 1 h before measuring ATF3 mRNA (C) or protein (D). ***p<0.001, comparing the indicated condition to time 0 or untreated control.

Mentions: To commence our investigation, we added lysophosphatidic acid (5 μM) to SKOV-3 ovarian cancer cells and measured an increase in ATF3 protein levels (Fig 1A) and ATF3 mRNA transcripts (Fig 1B), over 8 h. Since ATF3 expression peaked 1 h after lysophosphatidic acid treatment, we also assessed a range of concentrations (1 to 40 μM) at the 1 h time point and found 5–20 μM to be effective in stimulating ATF3 mRNA transcription (Fig 1C) and ATF3 protein expression (Fig 1D) in SKOV-3 cells. Thus, we selected 5 μM for the remainder of the study because it is the lowest concentration that produces the response. Taken together, this data suggests that in ovarian cancer cells, lysophosphatidic acid stimulation induces the expression of ATF3.


Lysophosphatidic Acid Mediates Activating Transcription Factor 3 Expression Which Is a Target for Post-Transcriptional Silencing by miR-30c-2-3p.

Nguyen HT, Jia W, Beedle AM, Kennedy EJ, Murph MM - PLoS ONE (2015)

Lysophosphatidic acid induces the expression of ATF3.(A) SKOV-3 cells were treated with 5 μM lysophosphatidic acid (LPA) for the times indicated prior to assessment of ATF3 protein. (B) SKOV-3 and OVCAR-3 cells were treated with 5 μM LPA for the times indicated prior to qRT-PCR measurement and normalization of ATF3 transcripts. SKOV-3 cells were treated with increasing concentrations of LPA for 1 h before measuring ATF3 mRNA (C) or protein (D). ***p<0.001, comparing the indicated condition to time 0 or untreated control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4587950&req=5

pone.0139489.g001: Lysophosphatidic acid induces the expression of ATF3.(A) SKOV-3 cells were treated with 5 μM lysophosphatidic acid (LPA) for the times indicated prior to assessment of ATF3 protein. (B) SKOV-3 and OVCAR-3 cells were treated with 5 μM LPA for the times indicated prior to qRT-PCR measurement and normalization of ATF3 transcripts. SKOV-3 cells were treated with increasing concentrations of LPA for 1 h before measuring ATF3 mRNA (C) or protein (D). ***p<0.001, comparing the indicated condition to time 0 or untreated control.
Mentions: To commence our investigation, we added lysophosphatidic acid (5 μM) to SKOV-3 ovarian cancer cells and measured an increase in ATF3 protein levels (Fig 1A) and ATF3 mRNA transcripts (Fig 1B), over 8 h. Since ATF3 expression peaked 1 h after lysophosphatidic acid treatment, we also assessed a range of concentrations (1 to 40 μM) at the 1 h time point and found 5–20 μM to be effective in stimulating ATF3 mRNA transcription (Fig 1C) and ATF3 protein expression (Fig 1D) in SKOV-3 cells. Thus, we selected 5 μM for the remainder of the study because it is the lowest concentration that produces the response. Taken together, this data suggests that in ovarian cancer cells, lysophosphatidic acid stimulation induces the expression of ATF3.

Bottom Line: The 3´-untranslated region (3´-UTR) of ATF3 was a predicted, putative target for miR-30c-2-3p, which we confirmed as a bona-fide interaction using a luciferase reporter assay.Furthermore, the presence of anti-miR-30c-2-3p enhanced ATF3 mRNA and protein after lysophosphatidic acid stimulation.Thus, the data suggest that after the expression of ATF3 and miR-30c-2-3p are elicited by lysophosphatidic acid, subsequently miR-30c-2-3p negatively regulates the expression of ATF3 through post-transcriptional silencing, which prevents further ATF3-related outcomes as a consequence of lysophosphatidic acid signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, College of Pharmacy, 240 W. Green Street, Athens, Georgia 30602, United States of America.

ABSTRACT
Although microRNAs (miRNAs) are small, non-protein-coding entities, they have important roles in post-transcriptional regulation of most of the human genome. These small entities generate fine-tuning adjustments in the expression of mRNA, which can mildly or massively affect the abundance of proteins. Previously, we found that the expression of miR-30c-2-3p is induced by lysophosphatidic acid and has an important role in the regulation of cell proliferation in ovarian cancer cells. The goal here is to confirm that ATF3 mRNA is a target of miR-30c-2-3p silencing, thereby further establishing the functional role of miR-30c-2-3p. Using a combination of bioinformatics, qRT-PCR, immunoblotting and luciferase assays, we uncovered a regulatory pathway between miR-30c-2-3p and the expression of the transcription factor, ATF3. Lysophosphatidic acids triggers the expression of both miR-30c-2-3p and ATF3, which peak at 1 h and are absent 8 h post stimulation in SKOV-3 and OVCAR-3 serous ovarian cancer cells. The 3´-untranslated region (3´-UTR) of ATF3 was a predicted, putative target for miR-30c-2-3p, which we confirmed as a bona-fide interaction using a luciferase reporter assay. Specific mutations introduced into the predicted site of interaction between miR-30c-2-3p and the 3´-UTR of ATF3 alleviated the suppression of the luciferase signal. Furthermore, the presence of anti-miR-30c-2-3p enhanced ATF3 mRNA and protein after lysophosphatidic acid stimulation. Thus, the data suggest that after the expression of ATF3 and miR-30c-2-3p are elicited by lysophosphatidic acid, subsequently miR-30c-2-3p negatively regulates the expression of ATF3 through post-transcriptional silencing, which prevents further ATF3-related outcomes as a consequence of lysophosphatidic acid signaling.

No MeSH data available.


Related in: MedlinePlus