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ADAM12-L is a direct target of the miR-29 and miR-200 families in breast cancer.

Duhachek-Muggy S, Zolkiewska A - BMC Cancer (2015)

Bottom Line: ADAM12-L, but not ADAM12-S, levels were also significantly diminished by miR-200b/c in SUM1315MO2 cells.In contrast, miR-30b/d did not elicit consistent and significant effects on ADAM12-L expression.Analysis of a publicly available gene expression dataset for 100 breast tumors revealed a statistically significant negative correlation between ADAM12-L and both miR-29b and miR-200c.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA. sduhach1@ksu.edu.

ABSTRACT

Background: ADAM12-L and ADAM12-S represent two major splice variants of human metalloproteinase-disintegrin 12 mRNA, which differ in their 3'-untranslated regions (3'UTRs). ADAM12-L, but not ADAM12-S, has prognostic and chemopredictive values in breast cancer. Expression levels of the two ADAM12 splice variants in clinical samples are highly discordant, suggesting post-transcriptional regulation of the ADAM12 gene. The miR-29, miR-30, and miR-200 families have potential target sites in the ADAM12-L 3'UTR and they may negatively regulate ADAM12-L expression.

Methods: miR-29b/c, miR-30b/d, miR-200b/c, or control miRNA mimics were transfected into SUM159PT, BT549, SUM1315MO2, or Hs578T breast cancer cells. ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR, and ADAM12-L protein was detected by Western blotting. Direct targeting of the ADAM12-L 3'UTR by miRNAs was tested using an ADAM12-L 3'UTR luciferase reporter. The rate of ADAM12-L translation was evaluated by metabolic labeling of cells with (35)S cysteine/methionine. The roles of endogenous miR-29b and miR-200c were tested by transfecting cells with miRNA hairpin inhibitors.

Results: Transfection of miR-29b/c mimics strongly decreased ADAM12-L mRNA levels in SUM159PT and BT549 cells, whereas ADAM12-S levels were not changed. ADAM12-L, but not ADAM12-S, levels were also significantly diminished by miR-200b/c in SUM1315MO2 cells. In Hs578T cells, miR-200b/c mimics impeded translation of ADAM12-L mRNA. Importantly, both miR-29b/c and miR-200b/c strongly decreased steady state levels of ADAM12-L protein in all breast cancer cell lines tested. miR-29b/c and miR-200b/c also significantly decreased the activity of an ADAM12-L 3'UTR reporter, and this effect was abolished when miR-29b/c and miR-200b/c target sequences were mutated. In contrast, miR-30b/d did not elicit consistent and significant effects on ADAM12-L expression. Analysis of a publicly available gene expression dataset for 100 breast tumors revealed a statistically significant negative correlation between ADAM12-L and both miR-29b and miR-200c. Inhibition of endogenous miR-29b and miR-200c in SUM149PT and SUM102PT cells led to increased ADAM12-L expression.

Conclusions: The ADAM12-L 3'UTR is a direct target of miR-29 and miR-200 family members. Since the miR-29 and miR-200 families play important roles in breast cancer progression, these results may help explain the different prognostic and chemopredictive values of ADAM12-L and ADAM12-S in breast cancer.

No MeSH data available.


Related in: MedlinePlus

Relationship between endogenous miR-29b, miR-200c, andADAM12-Lin breast tumors and breast cancer cell lines. (A) Correlation between ADAM12-L and miRNA levels in a set of 100 human breast tumors profiled with the Agilent Whole Human Genome Microarray 4×44K G4112F and the Agilent Human miRNA Microarray 2.0 G4470B, based on ref. [44]. The expression data were retrieved from GEO:GSE19536. Expression values of ADAM12-L were based on the A_23_P202327 probe. The expression values were median-centered for all tumors. Pearson r and P values are shown for each comparison. (B) SUM102PT and SUM149PT cells were transfected with miRNA hairpin inhibitors targeting miR-29b or miR-200c, or with hairpin inhibitor control. ADAM12-L mRNA levels were measured by qRT-PCR and normalized to β-ACTIN. Fold changes in miRNA inhibitor-transfected cells versus control cells were calculated. Graphs represent average values obtained in three independent experiments ± SEM. Statistical significance determined by one-sample t tests. *P < 0.05, **P < 0.01.
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Fig5: Relationship between endogenous miR-29b, miR-200c, andADAM12-Lin breast tumors and breast cancer cell lines. (A) Correlation between ADAM12-L and miRNA levels in a set of 100 human breast tumors profiled with the Agilent Whole Human Genome Microarray 4×44K G4112F and the Agilent Human miRNA Microarray 2.0 G4470B, based on ref. [44]. The expression data were retrieved from GEO:GSE19536. Expression values of ADAM12-L were based on the A_23_P202327 probe. The expression values were median-centered for all tumors. Pearson r and P values are shown for each comparison. (B) SUM102PT and SUM149PT cells were transfected with miRNA hairpin inhibitors targeting miR-29b or miR-200c, or with hairpin inhibitor control. ADAM12-L mRNA levels were measured by qRT-PCR and normalized to β-ACTIN. Fold changes in miRNA inhibitor-transfected cells versus control cells were calculated. Graphs represent average values obtained in three independent experiments ± SEM. Statistical significance determined by one-sample t tests. *P < 0.05, **P < 0.01.

Mentions: To determine whether miR-29b/c, miR-30b/d, or miR-200b/c might regulate ADAM12-L expression in breast cancer patients in vivo, we examined the relationship between these miRNAs and ADAM12-L mRNA in a cohort of 100 breast cancer patients for which mRNA/miRNA expression data were publicly available (GEO: GSE19536) [44]. Importantly, the microarray platform used in the referenced study contained an oligoprobe mapping uniquely to the ADAM12-L transcript, without contribution of the ADAM12-S splice variant. There was a significant negative correlation between miR-29b and ADAM12-L (P = 0.0001), between miR-200c and ADAM12-L (P = 0.0002), and a weaker but significant correlation between miR-200b and ADAM12-L (P = 0.0464) (Figure 5A). These results are consistent with a role of miR-29b and miR-200c (and possibly miR-200b) in the regulation of ADAM12-L expression in breast tumors. To further test this hypothesis, we asked whether inhibition of the endogenous miR-29b or miR-200c in SUM102PT and SUM149PT, two basal cell lines with low to moderate expression of miR-29b and miR-200c (see Figure 1D), is sufficient to increase the level of ADAM12-L. We transfected these cells with miRNA hairpin inhibitors to miR-29b and miR-200c (or with control hairpin inhibitor) and assessed the level of ADAM12-L mRNA by qRT-PCR. In SUM102PT cells, miR-29b inhibitor increased the ADAM12-L level by ~80%, and this effect was significant. miR-200b/c inhibitor increased ADAM12-L by ~20%, but this effect did not reach the level of statistical significance (Figure 5B). In SUM149PT cells, miR-29b and miR-200c inhibitors increased ADAM12-L levels by ~50% and ~30%, respectively, and these effects were statistically significant (Figure 5B).Figure 5


ADAM12-L is a direct target of the miR-29 and miR-200 families in breast cancer.

Duhachek-Muggy S, Zolkiewska A - BMC Cancer (2015)

Relationship between endogenous miR-29b, miR-200c, andADAM12-Lin breast tumors and breast cancer cell lines. (A) Correlation between ADAM12-L and miRNA levels in a set of 100 human breast tumors profiled with the Agilent Whole Human Genome Microarray 4×44K G4112F and the Agilent Human miRNA Microarray 2.0 G4470B, based on ref. [44]. The expression data were retrieved from GEO:GSE19536. Expression values of ADAM12-L were based on the A_23_P202327 probe. The expression values were median-centered for all tumors. Pearson r and P values are shown for each comparison. (B) SUM102PT and SUM149PT cells were transfected with miRNA hairpin inhibitors targeting miR-29b or miR-200c, or with hairpin inhibitor control. ADAM12-L mRNA levels were measured by qRT-PCR and normalized to β-ACTIN. Fold changes in miRNA inhibitor-transfected cells versus control cells were calculated. Graphs represent average values obtained in three independent experiments ± SEM. Statistical significance determined by one-sample t tests. *P < 0.05, **P < 0.01.
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Related In: Results  -  Collection

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Fig5: Relationship between endogenous miR-29b, miR-200c, andADAM12-Lin breast tumors and breast cancer cell lines. (A) Correlation between ADAM12-L and miRNA levels in a set of 100 human breast tumors profiled with the Agilent Whole Human Genome Microarray 4×44K G4112F and the Agilent Human miRNA Microarray 2.0 G4470B, based on ref. [44]. The expression data were retrieved from GEO:GSE19536. Expression values of ADAM12-L were based on the A_23_P202327 probe. The expression values were median-centered for all tumors. Pearson r and P values are shown for each comparison. (B) SUM102PT and SUM149PT cells were transfected with miRNA hairpin inhibitors targeting miR-29b or miR-200c, or with hairpin inhibitor control. ADAM12-L mRNA levels were measured by qRT-PCR and normalized to β-ACTIN. Fold changes in miRNA inhibitor-transfected cells versus control cells were calculated. Graphs represent average values obtained in three independent experiments ± SEM. Statistical significance determined by one-sample t tests. *P < 0.05, **P < 0.01.
Mentions: To determine whether miR-29b/c, miR-30b/d, or miR-200b/c might regulate ADAM12-L expression in breast cancer patients in vivo, we examined the relationship between these miRNAs and ADAM12-L mRNA in a cohort of 100 breast cancer patients for which mRNA/miRNA expression data were publicly available (GEO: GSE19536) [44]. Importantly, the microarray platform used in the referenced study contained an oligoprobe mapping uniquely to the ADAM12-L transcript, without contribution of the ADAM12-S splice variant. There was a significant negative correlation between miR-29b and ADAM12-L (P = 0.0001), between miR-200c and ADAM12-L (P = 0.0002), and a weaker but significant correlation between miR-200b and ADAM12-L (P = 0.0464) (Figure 5A). These results are consistent with a role of miR-29b and miR-200c (and possibly miR-200b) in the regulation of ADAM12-L expression in breast tumors. To further test this hypothesis, we asked whether inhibition of the endogenous miR-29b or miR-200c in SUM102PT and SUM149PT, two basal cell lines with low to moderate expression of miR-29b and miR-200c (see Figure 1D), is sufficient to increase the level of ADAM12-L. We transfected these cells with miRNA hairpin inhibitors to miR-29b and miR-200c (or with control hairpin inhibitor) and assessed the level of ADAM12-L mRNA by qRT-PCR. In SUM102PT cells, miR-29b inhibitor increased the ADAM12-L level by ~80%, and this effect was significant. miR-200b/c inhibitor increased ADAM12-L by ~20%, but this effect did not reach the level of statistical significance (Figure 5B). In SUM149PT cells, miR-29b and miR-200c inhibitors increased ADAM12-L levels by ~50% and ~30%, respectively, and these effects were statistically significant (Figure 5B).Figure 5

Bottom Line: ADAM12-L, but not ADAM12-S, levels were also significantly diminished by miR-200b/c in SUM1315MO2 cells.In contrast, miR-30b/d did not elicit consistent and significant effects on ADAM12-L expression.Analysis of a publicly available gene expression dataset for 100 breast tumors revealed a statistically significant negative correlation between ADAM12-L and both miR-29b and miR-200c.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA. sduhach1@ksu.edu.

ABSTRACT

Background: ADAM12-L and ADAM12-S represent two major splice variants of human metalloproteinase-disintegrin 12 mRNA, which differ in their 3'-untranslated regions (3'UTRs). ADAM12-L, but not ADAM12-S, has prognostic and chemopredictive values in breast cancer. Expression levels of the two ADAM12 splice variants in clinical samples are highly discordant, suggesting post-transcriptional regulation of the ADAM12 gene. The miR-29, miR-30, and miR-200 families have potential target sites in the ADAM12-L 3'UTR and they may negatively regulate ADAM12-L expression.

Methods: miR-29b/c, miR-30b/d, miR-200b/c, or control miRNA mimics were transfected into SUM159PT, BT549, SUM1315MO2, or Hs578T breast cancer cells. ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR, and ADAM12-L protein was detected by Western blotting. Direct targeting of the ADAM12-L 3'UTR by miRNAs was tested using an ADAM12-L 3'UTR luciferase reporter. The rate of ADAM12-L translation was evaluated by metabolic labeling of cells with (35)S cysteine/methionine. The roles of endogenous miR-29b and miR-200c were tested by transfecting cells with miRNA hairpin inhibitors.

Results: Transfection of miR-29b/c mimics strongly decreased ADAM12-L mRNA levels in SUM159PT and BT549 cells, whereas ADAM12-S levels were not changed. ADAM12-L, but not ADAM12-S, levels were also significantly diminished by miR-200b/c in SUM1315MO2 cells. In Hs578T cells, miR-200b/c mimics impeded translation of ADAM12-L mRNA. Importantly, both miR-29b/c and miR-200b/c strongly decreased steady state levels of ADAM12-L protein in all breast cancer cell lines tested. miR-29b/c and miR-200b/c also significantly decreased the activity of an ADAM12-L 3'UTR reporter, and this effect was abolished when miR-29b/c and miR-200b/c target sequences were mutated. In contrast, miR-30b/d did not elicit consistent and significant effects on ADAM12-L expression. Analysis of a publicly available gene expression dataset for 100 breast tumors revealed a statistically significant negative correlation between ADAM12-L and both miR-29b and miR-200c. Inhibition of endogenous miR-29b and miR-200c in SUM149PT and SUM102PT cells led to increased ADAM12-L expression.

Conclusions: The ADAM12-L 3'UTR is a direct target of miR-29 and miR-200 family members. Since the miR-29 and miR-200 families play important roles in breast cancer progression, these results may help explain the different prognostic and chemopredictive values of ADAM12-L and ADAM12-S in breast cancer.

No MeSH data available.


Related in: MedlinePlus