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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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Relationship between miR-143 and DNMT3A. Knockdown of DNMT3A inhibited cell proliferation in 228 and SW480 cells. Cells were transfected with DNMT3A siRNA for 24 h. Transfection with scramble siRNA was used as negative control. (A) The DNMT3A protein level 5 days after transfection was examined by western blot. The experiment has been repeated thrice. (B) Cell proliferation 5 days after transfection was assessed using MTT assay. Relative cell proliferation was compared with the corresponding scramble siRNA transfection by the Mann–Whitney test, *P<0.01. (C) The scatter plot of expression correlation between miR-143 and DNMT3A mRNA in seven colon cancer cell lines. Inverse correlation was obtained using Spearman's correlation, r=−0.78, P<0.05. (D) The scatter plot of expression correlation between miR-143 and DNMT3A mRNA in 10 paired adjacent normal and CRC tissues. Inverse correlation was also obtained by Spearman's correlation, r=−0.59, P<0.01. (E) The DNMT3A protein level was semi-quantified using western blot analysis of CRC tumours (T) and of adjacent normal cells (N). (F) The scatter plot of the fold changes of the miR-143 and DNMT3A protein (Log10 scale at both X axis and Y axis) in seven paired CRC samples (Spearman's correlation, r=0.71, P<0.05).
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fig5: Relationship between miR-143 and DNMT3A. Knockdown of DNMT3A inhibited cell proliferation in 228 and SW480 cells. Cells were transfected with DNMT3A siRNA for 24 h. Transfection with scramble siRNA was used as negative control. (A) The DNMT3A protein level 5 days after transfection was examined by western blot. The experiment has been repeated thrice. (B) Cell proliferation 5 days after transfection was assessed using MTT assay. Relative cell proliferation was compared with the corresponding scramble siRNA transfection by the Mann–Whitney test, *P<0.01. (C) The scatter plot of expression correlation between miR-143 and DNMT3A mRNA in seven colon cancer cell lines. Inverse correlation was obtained using Spearman's correlation, r=−0.78, P<0.05. (D) The scatter plot of expression correlation between miR-143 and DNMT3A mRNA in 10 paired adjacent normal and CRC tissues. Inverse correlation was also obtained by Spearman's correlation, r=−0.59, P<0.01. (E) The DNMT3A protein level was semi-quantified using western blot analysis of CRC tumours (T) and of adjacent normal cells (N). (F) The scatter plot of the fold changes of the miR-143 and DNMT3A protein (Log10 scale at both X axis and Y axis) in seven paired CRC samples (Spearman's correlation, r=0.71, P<0.05).

Mentions: To further establish a link between miR-143 and its downstream target, DNMT3A, tumour cell proliferation after a siRNA-mediated knockdown of DNMT3A was examined. Consistent with the growth inhibitory effect of miRNA-143, cell proliferation of 228 and SW480 cells was significantly reduced by 42 and 44%, respectively, once the DNMT3A protein expression was effectively suppressed by siRNA (all P-values <0.05; Mann–Whitney test; Figure 5A and B).


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)

Relationship between miR-143 and DNMT3A. Knockdown of DNMT3A inhibited cell proliferation in 228 and SW480 cells. Cells were transfected with DNMT3A siRNA for 24 h. Transfection with scramble siRNA was used as negative control. (A) The DNMT3A protein level 5 days after transfection was examined by western blot. The experiment has been repeated thrice. (B) Cell proliferation 5 days after transfection was assessed using MTT assay. Relative cell proliferation was compared with the corresponding scramble siRNA transfection by the Mann–Whitney test, *P<0.01. (C) The scatter plot of expression correlation between miR-143 and DNMT3A mRNA in seven colon cancer cell lines. Inverse correlation was obtained using Spearman's correlation, r=−0.78, P<0.05. (D) The scatter plot of expression correlation between miR-143 and DNMT3A mRNA in 10 paired adjacent normal and CRC tissues. Inverse correlation was also obtained by Spearman's correlation, r=−0.59, P<0.01. (E) The DNMT3A protein level was semi-quantified using western blot analysis of CRC tumours (T) and of adjacent normal cells (N). (F) The scatter plot of the fold changes of the miR-143 and DNMT3A protein (Log10 scale at both X axis and Y axis) in seven paired CRC samples (Spearman's correlation, r=0.71, P<0.05).
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Related In: Results  -  Collection

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

fig5: Relationship between miR-143 and DNMT3A. Knockdown of DNMT3A inhibited cell proliferation in 228 and SW480 cells. Cells were transfected with DNMT3A siRNA for 24 h. Transfection with scramble siRNA was used as negative control. (A) The DNMT3A protein level 5 days after transfection was examined by western blot. The experiment has been repeated thrice. (B) Cell proliferation 5 days after transfection was assessed using MTT assay. Relative cell proliferation was compared with the corresponding scramble siRNA transfection by the Mann–Whitney test, *P<0.01. (C) The scatter plot of expression correlation between miR-143 and DNMT3A mRNA in seven colon cancer cell lines. Inverse correlation was obtained using Spearman's correlation, r=−0.78, P<0.05. (D) The scatter plot of expression correlation between miR-143 and DNMT3A mRNA in 10 paired adjacent normal and CRC tissues. Inverse correlation was also obtained by Spearman's correlation, r=−0.59, P<0.01. (E) The DNMT3A protein level was semi-quantified using western blot analysis of CRC tumours (T) and of adjacent normal cells (N). (F) The scatter plot of the fold changes of the miR-143 and DNMT3A protein (Log10 scale at both X axis and Y axis) in seven paired CRC samples (Spearman's correlation, r=0.71, P<0.05).
Mentions: To further establish a link between miR-143 and its downstream target, DNMT3A, tumour cell proliferation after a siRNA-mediated knockdown of DNMT3A was examined. Consistent with the growth inhibitory effect of miRNA-143, cell proliferation of 228 and SW480 cells was significantly reduced by 42 and 44%, respectively, once the DNMT3A protein expression was effectively suppressed by siRNA (all P-values <0.05; Mann–Whitney test; Figure 5A and B).

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