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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

ADAM12-L, but notADAM12-S, is a target for miR-29b/c. (A,B) SUM159PT and BT549 cells were transfected with miR-29b mimic, miR-29c mimic, or mimic control. (A)ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR and normalized to β-ACTIN. Fold changes in miRNA-transfected cells versus control cells were calculated. Graphs represent average values obtained in three independent experiments ± SEM. Statistical significance was determined by one-sample t tests. **P < 0.01, ***P < 0.001, ****P < 0.0001. (B) Cell lysates were enriched for glycoproteins and analyzed by Western blotting using an anti-ADAM12-L antibody. The nascent, full-length form and the mature, processed form are indicated. A Ponceau S-stained band in the glycoprotein-enriched fraction and tubulin in total cell lysates were used as loading controls. (C)Upper SUM159PT cells were transfected with miR-29b, miR-29c mimics, or mimic control and then with the indicated ADAM12-L 3′UTR reporter or an empty vector and a Renilla luciferase control vector. The firefly luciferase activity was measured after 48 h and was normalized to Renilla luciferase activity and to the empty vector. Graph shows the average values for at least two independent experiments ± SEM. Significance was determined by one-sample t tests. ****P < 0.0001. Lower Three nucleotides in the putative miRNA target site (shown in bold) were mutated to destroy the site. The mutated residues are shown in red above the wild-type sequence. The position in the ADAM12-L 3′UTR relative to the stop codon is indicated.
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Fig2: ADAM12-L, but notADAM12-S, is a target for miR-29b/c. (A,B) SUM159PT and BT549 cells were transfected with miR-29b mimic, miR-29c mimic, or mimic control. (A)ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR and normalized to β-ACTIN. Fold changes in miRNA-transfected cells versus control cells were calculated. Graphs represent average values obtained in three independent experiments ± SEM. Statistical significance was determined by one-sample t tests. **P < 0.01, ***P < 0.001, ****P < 0.0001. (B) Cell lysates were enriched for glycoproteins and analyzed by Western blotting using an anti-ADAM12-L antibody. The nascent, full-length form and the mature, processed form are indicated. A Ponceau S-stained band in the glycoprotein-enriched fraction and tubulin in total cell lysates were used as loading controls. (C)Upper SUM159PT cells were transfected with miR-29b, miR-29c mimics, or mimic control and then with the indicated ADAM12-L 3′UTR reporter or an empty vector and a Renilla luciferase control vector. The firefly luciferase activity was measured after 48 h and was normalized to Renilla luciferase activity and to the empty vector. Graph shows the average values for at least two independent experiments ± SEM. Significance was determined by one-sample t tests. ****P < 0.0001. Lower Three nucleotides in the putative miRNA target site (shown in bold) were mutated to destroy the site. The mutated residues are shown in red above the wild-type sequence. The position in the ADAM12-L 3′UTR relative to the stop codon is indicated.

Mentions: To determine whether low levels of miR-29b/c are required for high expression of ADAM12-L in claudin-low cell lines, we utilized SUM159PT and BT549 cells, two representative claudin-low cell lines with low endogenous levels of miR-29b/c (see Figure 1D). Cells were transfected with miR-29b/c or control miRNA mimics, and the levels of ADAM12-L and ADAM12-S mRNAs were measured three days later by qRT-PCR. We found that miR-29b/c mimics decreased the level of ADAM12-L by ~70%, and that this effect was statistically significant (Figure 2A). ADAM12-S expression was not significantly altered by transfection with miR-29b/c mimics. In parallel experiments, we examined the effects of miR-29b/c on ADAM12-L protein expression by immunoblotting. We observed that both miR-29b and miR-29c strongly diminished the level of ADAM12-L protein in both cell lines (Figure 2B). Testing the effect of miRNAs on the expression level of the ADAM12-S isoform was not possible because specific antibodies against ADAM12-S are not currently available. Decreased ADAM12-L protein and mRNA levels after transfection of miR-29b/c suggested that these miRNAs might be directly targeting the ADAM12-L 3′UTR. To examine this possibility, we performed a miRNA target reporter luciferase assay using the pMirTarget reporter vector comprising a ~3-kb region of the ADAM12-L 3′UTR down-stream of the firefly luciferase gene. An approximately 50-60% reduction in the luciferase activity was observed in miR-29b/c mimic-transfected SUM159PT cells compared to control mimic-transfected cells (Figure 2C). Disruption of the predicted miR-29 target site by site-directed mutagenesis largely diminished the effects of miR-29b/c.Figure 2


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)

ADAM12-L, but notADAM12-S, is a target for miR-29b/c. (A,B) SUM159PT and BT549 cells were transfected with miR-29b mimic, miR-29c mimic, or mimic control. (A)ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR and normalized to β-ACTIN. Fold changes in miRNA-transfected cells versus control cells were calculated. Graphs represent average values obtained in three independent experiments ± SEM. Statistical significance was determined by one-sample t tests. **P < 0.01, ***P < 0.001, ****P < 0.0001. (B) Cell lysates were enriched for glycoproteins and analyzed by Western blotting using an anti-ADAM12-L antibody. The nascent, full-length form and the mature, processed form are indicated. A Ponceau S-stained band in the glycoprotein-enriched fraction and tubulin in total cell lysates were used as loading controls. (C)Upper SUM159PT cells were transfected with miR-29b, miR-29c mimics, or mimic control and then with the indicated ADAM12-L 3′UTR reporter or an empty vector and a Renilla luciferase control vector. The firefly luciferase activity was measured after 48 h and was normalized to Renilla luciferase activity and to the empty vector. Graph shows the average values for at least two independent experiments ± SEM. Significance was determined by one-sample t tests. ****P < 0.0001. Lower Three nucleotides in the putative miRNA target site (shown in bold) were mutated to destroy the site. The mutated residues are shown in red above the wild-type sequence. The position in the ADAM12-L 3′UTR relative to the stop codon is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4352249&req=5

Fig2: ADAM12-L, but notADAM12-S, is a target for miR-29b/c. (A,B) SUM159PT and BT549 cells were transfected with miR-29b mimic, miR-29c mimic, or mimic control. (A)ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR and normalized to β-ACTIN. Fold changes in miRNA-transfected cells versus control cells were calculated. Graphs represent average values obtained in three independent experiments ± SEM. Statistical significance was determined by one-sample t tests. **P < 0.01, ***P < 0.001, ****P < 0.0001. (B) Cell lysates were enriched for glycoproteins and analyzed by Western blotting using an anti-ADAM12-L antibody. The nascent, full-length form and the mature, processed form are indicated. A Ponceau S-stained band in the glycoprotein-enriched fraction and tubulin in total cell lysates were used as loading controls. (C)Upper SUM159PT cells were transfected with miR-29b, miR-29c mimics, or mimic control and then with the indicated ADAM12-L 3′UTR reporter or an empty vector and a Renilla luciferase control vector. The firefly luciferase activity was measured after 48 h and was normalized to Renilla luciferase activity and to the empty vector. Graph shows the average values for at least two independent experiments ± SEM. Significance was determined by one-sample t tests. ****P < 0.0001. Lower Three nucleotides in the putative miRNA target site (shown in bold) were mutated to destroy the site. The mutated residues are shown in red above the wild-type sequence. The position in the ADAM12-L 3′UTR relative to the stop codon is indicated.
Mentions: To determine whether low levels of miR-29b/c are required for high expression of ADAM12-L in claudin-low cell lines, we utilized SUM159PT and BT549 cells, two representative claudin-low cell lines with low endogenous levels of miR-29b/c (see Figure 1D). Cells were transfected with miR-29b/c or control miRNA mimics, and the levels of ADAM12-L and ADAM12-S mRNAs were measured three days later by qRT-PCR. We found that miR-29b/c mimics decreased the level of ADAM12-L by ~70%, and that this effect was statistically significant (Figure 2A). ADAM12-S expression was not significantly altered by transfection with miR-29b/c mimics. In parallel experiments, we examined the effects of miR-29b/c on ADAM12-L protein expression by immunoblotting. We observed that both miR-29b and miR-29c strongly diminished the level of ADAM12-L protein in both cell lines (Figure 2B). Testing the effect of miRNAs on the expression level of the ADAM12-S isoform was not possible because specific antibodies against ADAM12-S are not currently available. Decreased ADAM12-L protein and mRNA levels after transfection of miR-29b/c suggested that these miRNAs might be directly targeting the ADAM12-L 3′UTR. To examine this possibility, we performed a miRNA target reporter luciferase assay using the pMirTarget reporter vector comprising a ~3-kb region of the ADAM12-L 3′UTR down-stream of the firefly luciferase gene. An approximately 50-60% reduction in the luciferase activity was observed in miR-29b/c mimic-transfected SUM159PT cells compared to control mimic-transfected cells (Figure 2C). Disruption of the predicted miR-29 target site by site-directed mutagenesis largely diminished the effects of miR-29b/c.Figure 2

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