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MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer.

Ng EK, Tsang WP, Ng SS, Jin HC, Yu J, Li JJ, Röcken C, Ebert MP, Kwok TT, Sung JJ - Br. J. Cancer (2009)

Bottom Line: The functional effect of miR-143 and its targets was investigated in human colon cancer cell lines to confirm miRNA-target association.Restoration of the miR-143 expression in colon cell lines decreased tumour cell growth and soft-agar colony formation, and downregulated the DNMT3A expression in both mRNA and protein levels.These findings elucidated a tumour-suppressive role of miR-143 in the epigenetic aberration of CRC, providing a potential development of miRNA-based targeted approaches for CRC therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Therapeutics, Institute of Digestive Disease, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.

ABSTRACT

Background: MicroRNAs (miRNAs) are 19-25-nucleotides regulatory non-protein-coding RNA molecules that regulate the expressions of a wide variety of genes, including some involved in cancer development. In this study, we investigated the possible role of miR-143 in colorectal cancer (CRC).

Methods: Expression levels of human mature miRNAs were examined using real-time PCR-based expression arrays on paired colorectal carcinomas and adjacent non-cancerous colonic tissues. The downregulation of miR-143 was further evaluated in colon cancer cell lines and in paired CRC and adjacent non-cancerous colonic tissues by qRT-PCR. Potential targets of miR-143 were defined. The functional effect of miR-143 and its targets was investigated in human colon cancer cell lines to confirm miRNA-target association.

Results: Both real-time PCR-based expression arrays and qRT-PCR showed that miR-143 was frequently downregulated in 87.5% (35 of 40) of colorectal carcinoma tissues compared with their adjacent non-cancerous colonic tissues. Using in silico predictions, DNA methyltranferase 3A (DNMT3A) was defined as a potential target of miR-143. Restoration of the miR-143 expression in colon cell lines decreased tumour cell growth and soft-agar colony formation, and downregulated the DNMT3A expression in both mRNA and protein levels. DNMT3A was shown to be a direct target of miR-143 by luciferase reporter assay. Furthermore, the miR-143 expression was observed to be inversely correlated with DNMT3A mRNA and protein expression in CRC tissues.

Conclusion: Our findings suggest that miR-143 regulates DNMT3A in CRC. These findings elucidated a tumour-suppressive role of miR-143 in the epigenetic aberration of CRC, providing a potential development of miRNA-based targeted approaches for CRC therapy.

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Related in: MedlinePlus

DNMT3A is the direct target of miRNA-143. (A) The wild-type (WT) and mutated (MUT) 3′UTR of DNMT3A, with the seed region (bold) and base substitutions (bold and italic), were sub-cloned into luciferase reporter construct and are shown. (B) Ectopic miR-143 expression inhibits wild-type but not mutant DNMT3A 3′UTR reporter activity in 228 and SW480 cells. Cells were co-transfected with miR-143 precursor and with either WT or MUT DNMT3A 3′UTR reporter construct. Luciferase activity assay was performed at 24 h after transfection (Mann–Whitney test, *P<0.05, **P<0.01).
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fig4: DNMT3A is the direct target of miRNA-143. (A) The wild-type (WT) and mutated (MUT) 3′UTR of DNMT3A, with the seed region (bold) and base substitutions (bold and italic), were sub-cloned into luciferase reporter construct and are shown. (B) Ectopic miR-143 expression inhibits wild-type but not mutant DNMT3A 3′UTR reporter activity in 228 and SW480 cells. Cells were co-transfected with miR-143 precursor and with either WT or MUT DNMT3A 3′UTR reporter construct. Luciferase activity assay was performed at 24 h after transfection (Mann–Whitney test, *P<0.05, **P<0.01).

Mentions: To further confirm that DNMT3A is the direct target of miR-143, a segment of the 3′UTR of DNMT3A, with or without point mutations in the seed sequence (Figure 4A), was sub-cloned downstream of the firefly luciferase reporter. The constructs were then co-transfected with miR-143 precursor or with pre-miR control for luciferase activity assays. The relative luciferase activity of the WT construct of DNMT3A 3′UTR in both the colon cancer cells was significantly reduced in the presence of miR-143 (P<0.05 for 228 cells and P<0.01 for SW480 cells; Mann–Whitney test), whereas such a suppressive effect of miR-143 on luciferase activity was not observed in both cells with the MUT construct of DNMT3A 3′UTR (Figure 4B), highlighting a direct and specific interaction of miR-143 on DNMT3A 3′UTR.


MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer.

Ng EK, Tsang WP, Ng SS, Jin HC, Yu J, Li JJ, Röcken C, Ebert MP, Kwok TT, Sung JJ - Br. J. Cancer (2009)

DNMT3A is the direct target of miRNA-143. (A) The wild-type (WT) and mutated (MUT) 3′UTR of DNMT3A, with the seed region (bold) and base substitutions (bold and italic), were sub-cloned into luciferase reporter construct and are shown. (B) Ectopic miR-143 expression inhibits wild-type but not mutant DNMT3A 3′UTR reporter activity in 228 and SW480 cells. Cells were co-transfected with miR-143 precursor and with either WT or MUT DNMT3A 3′UTR reporter construct. Luciferase activity assay was performed at 24 h after transfection (Mann–Whitney test, *P<0.05, **P<0.01).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2736825&req=5

fig4: DNMT3A is the direct target of miRNA-143. (A) The wild-type (WT) and mutated (MUT) 3′UTR of DNMT3A, with the seed region (bold) and base substitutions (bold and italic), were sub-cloned into luciferase reporter construct and are shown. (B) Ectopic miR-143 expression inhibits wild-type but not mutant DNMT3A 3′UTR reporter activity in 228 and SW480 cells. Cells were co-transfected with miR-143 precursor and with either WT or MUT DNMT3A 3′UTR reporter construct. Luciferase activity assay was performed at 24 h after transfection (Mann–Whitney test, *P<0.05, **P<0.01).
Mentions: To further confirm that DNMT3A is the direct target of miR-143, a segment of the 3′UTR of DNMT3A, with or without point mutations in the seed sequence (Figure 4A), was sub-cloned downstream of the firefly luciferase reporter. The constructs were then co-transfected with miR-143 precursor or with pre-miR control for luciferase activity assays. The relative luciferase activity of the WT construct of DNMT3A 3′UTR in both the colon cancer cells was significantly reduced in the presence of miR-143 (P<0.05 for 228 cells and P<0.01 for SW480 cells; Mann–Whitney test), whereas such a suppressive effect of miR-143 on luciferase activity was not observed in both cells with the MUT construct of DNMT3A 3′UTR (Figure 4B), highlighting a direct and specific interaction of miR-143 on DNMT3A 3′UTR.

Bottom Line: The functional effect of miR-143 and its targets was investigated in human colon cancer cell lines to confirm miRNA-target association.Restoration of the miR-143 expression in colon cell lines decreased tumour cell growth and soft-agar colony formation, and downregulated the DNMT3A expression in both mRNA and protein levels.These findings elucidated a tumour-suppressive role of miR-143 in the epigenetic aberration of CRC, providing a potential development of miRNA-based targeted approaches for CRC therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine and Therapeutics, Institute of Digestive Disease, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China.

ABSTRACT

Background: MicroRNAs (miRNAs) are 19-25-nucleotides regulatory non-protein-coding RNA molecules that regulate the expressions of a wide variety of genes, including some involved in cancer development. In this study, we investigated the possible role of miR-143 in colorectal cancer (CRC).

Methods: Expression levels of human mature miRNAs were examined using real-time PCR-based expression arrays on paired colorectal carcinomas and adjacent non-cancerous colonic tissues. The downregulation of miR-143 was further evaluated in colon cancer cell lines and in paired CRC and adjacent non-cancerous colonic tissues by qRT-PCR. Potential targets of miR-143 were defined. The functional effect of miR-143 and its targets was investigated in human colon cancer cell lines to confirm miRNA-target association.

Results: Both real-time PCR-based expression arrays and qRT-PCR showed that miR-143 was frequently downregulated in 87.5% (35 of 40) of colorectal carcinoma tissues compared with their adjacent non-cancerous colonic tissues. Using in silico predictions, DNA methyltranferase 3A (DNMT3A) was defined as a potential target of miR-143. Restoration of the miR-143 expression in colon cell lines decreased tumour cell growth and soft-agar colony formation, and downregulated the DNMT3A expression in both mRNA and protein levels. DNMT3A was shown to be a direct target of miR-143 by luciferase reporter assay. Furthermore, the miR-143 expression was observed to be inversely correlated with DNMT3A mRNA and protein expression in CRC tissues.

Conclusion: Our findings suggest that miR-143 regulates DNMT3A in CRC. These findings elucidated a tumour-suppressive role of miR-143 in the epigenetic aberration of CRC, providing a potential development of miRNA-based targeted approaches for CRC therapy.

Show MeSH
Related in: MedlinePlus