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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-Lis a poor target for miR-30b/d. (A,B) SUM159PT and SUM1315MO2 cells were transfected with miR-30b mimic, miR-30d 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 (for SUM159PT) or two (for SUM1315MO2) independent experiments ± SEM. Statistical significance was determined by one-sample t tests. *P < 0.05. (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-30b, miR-30d 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 < 0.001. 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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Fig3: ADAM12-Lis a poor target for miR-30b/d. (A,B) SUM159PT and SUM1315MO2 cells were transfected with miR-30b mimic, miR-30d 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 (for SUM159PT) or two (for SUM1315MO2) independent experiments ± SEM. Statistical significance was determined by one-sample t tests. *P < 0.05. (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-30b, miR-30d 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 < 0.001. 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: Similarly, we assessed whether miR-30b/d potentially target ADAM12-L. We transfected miR-30b/d or control mimic into SUM159PT and SUM1315MO2 cells, two claudin-low cell lines with low to moderate endogenous miR-30b/d expression (Figure 1D), and measured the level of ADAM12-L and ADAM12-S mRNA by qRT-PCR. miR-30d exerted a ~30%, statistically significant, down-regulation of ADAM12-L expression in SUM159PT cells and no apparent inhibition of ADAM12-L expression in SUM1315MO2 cells. miR-30b did not diminish ADAM12-L levels in either cell line and neither miRNA mimic affected ADAM12-S expression (Figure 3A). miR-30b/d had a modest effect on ADAM12-L protein in both cell lines (Figure 3B). To test whether miR-30b or miR-30d directly targets the ADAM12-L 3′UTR, we used the luciferase reporter in SUM159PT cells. Transfection of miR-30b mimic elicited a significant decrease in luciferase activity but miR-30d mimic did not (Figure 3C). Destruction of the potential miR-30 target site by mutagenesis eliminated the effect of miR-30b mimic.Figure 3


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-Lis a poor target for miR-30b/d. (A,B) SUM159PT and SUM1315MO2 cells were transfected with miR-30b mimic, miR-30d 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 (for SUM159PT) or two (for SUM1315MO2) independent experiments ± SEM. Statistical significance was determined by one-sample t tests. *P < 0.05. (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-30b, miR-30d 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 < 0.001. 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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Fig3: ADAM12-Lis a poor target for miR-30b/d. (A,B) SUM159PT and SUM1315MO2 cells were transfected with miR-30b mimic, miR-30d 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 (for SUM159PT) or two (for SUM1315MO2) independent experiments ± SEM. Statistical significance was determined by one-sample t tests. *P < 0.05. (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-30b, miR-30d 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 < 0.001. 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: Similarly, we assessed whether miR-30b/d potentially target ADAM12-L. We transfected miR-30b/d or control mimic into SUM159PT and SUM1315MO2 cells, two claudin-low cell lines with low to moderate endogenous miR-30b/d expression (Figure 1D), and measured the level of ADAM12-L and ADAM12-S mRNA by qRT-PCR. miR-30d exerted a ~30%, statistically significant, down-regulation of ADAM12-L expression in SUM159PT cells and no apparent inhibition of ADAM12-L expression in SUM1315MO2 cells. miR-30b did not diminish ADAM12-L levels in either cell line and neither miRNA mimic affected ADAM12-S expression (Figure 3A). miR-30b/d had a modest effect on ADAM12-L protein in both cell lines (Figure 3B). To test whether miR-30b or miR-30d directly targets the ADAM12-L 3′UTR, we used the luciferase reporter in SUM159PT cells. Transfection of miR-30b mimic elicited a significant decrease in luciferase activity but miR-30d mimic did not (Figure 3C). Destruction of the potential miR-30 target site by mutagenesis eliminated the effect of miR-30b mimic.Figure 3

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