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MiR-143 and MiR-145 regulate IGF1R to suppress cell proliferation in colorectal cancer.

Su J, Liang H, Yao W, Wang N, Zhang S, Yan X, Feng H, Pang W, Wang Y, Wang X, Fu Z, Liu Y, Zhao C, Zhang J, Zhang CY, Zen K, Chen X, Wang Y - PLoS ONE (2014)

Bottom Line: We identified specific target sites for miR-143 and miR-145 (miR-143/145) in the 3'-untranslated region (3'-UTR) of the IGF1R gene.These miRNAs are members of a cluster of miRNAs that have been reported to exhibit tumor suppressor activity.We demonstrated that the repression of IGF1R by miR-143/145 suppressed the proliferation of Caco2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China.

ABSTRACT
Insulin-like growth factor 1 receptor (IGF1R) is a transmembrane receptor that is activated by insulin-like growth factor 1 (IGF-1) and by a related hormone called IGF-2. It belongs to the large class of tyrosine kinase receptors and plays an important role in colorectal cancer etiology and progression. In this study, we used bioinformatic analyses to search for miRNAs that potentially target IGF1R. We identified specific target sites for miR-143 and miR-145 (miR-143/145) in the 3'-untranslated region (3'-UTR) of the IGF1R gene. These miRNAs are members of a cluster of miRNAs that have been reported to exhibit tumor suppressor activity. Consistent with the bioinformatic analyses, we identified an inverse correlation between miR-143/145 levels and IGF1R protein levels in colorectal cancer tissues. By overexpressing miR-143/145 in Caco2, HT29 and SW480 colorectal cancer cells, we experimentally validated that miR-143/145 directly recognizes the 3'-UTR of the IGF1R transcript and regulates IGF1R expression. Furthermore, the biological consequences of the targeting of IGF1R by miR-143/145 were examined by cell proliferation assays in vitro. We demonstrated that the repression of IGF1R by miR-143/145 suppressed the proliferation of Caco2 cells. Taken together, our findings provide evidence for a role of the miR-143/145 cluster as a tumor suppressor in colorectal cancer through the inhibition of IGF1R translation.

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miR-143/145 directly regulate IGF1R expression at the post-transcriptional level.(A, F and K) Quantitative RT-PCR analysis of miR-143/145 levels in Caco2, HT29 and SW480 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. (B, C; G, H; and L, M) Western blot analysis of IGF1R protein levels in Caco2 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. B, G and L: representative image; C, H and M: quantitative analysis. (D, I and N) Quantitative RT-PCR analysis of IGF1R mRNA levels in Caco2, HT29 and SW480 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. (E, J and O) Direct recognition of the IGF1R 3′-UTR by miR-143/145. Caco2, HT29 and SW480 cells were co-transfected with firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-143/145 binding sites in the IGF1R 3′-UTR and a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. Twenty-four hours after transfection, the cells were assayed using a luciferase assay kit. The results are displayed as the ratio of firefly luciferase activity in the miR-143/145-transfected cells to the activity in the control cells. *P<0.05; **P<0.01.
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pone-0114420-g002: miR-143/145 directly regulate IGF1R expression at the post-transcriptional level.(A, F and K) Quantitative RT-PCR analysis of miR-143/145 levels in Caco2, HT29 and SW480 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. (B, C; G, H; and L, M) Western blot analysis of IGF1R protein levels in Caco2 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. B, G and L: representative image; C, H and M: quantitative analysis. (D, I and N) Quantitative RT-PCR analysis of IGF1R mRNA levels in Caco2, HT29 and SW480 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. (E, J and O) Direct recognition of the IGF1R 3′-UTR by miR-143/145. Caco2, HT29 and SW480 cells were co-transfected with firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-143/145 binding sites in the IGF1R 3′-UTR and a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. Twenty-four hours after transfection, the cells were assayed using a luciferase assay kit. The results are displayed as the ratio of firefly luciferase activity in the miR-143/145-transfected cells to the activity in the control cells. *P<0.05; **P<0.01.

Mentions: The correlation between miR-143/145 and IGF1R was further examined by evaluating IGF1R expression in the human colorectal cancer cell lines Caco2, HT29 and SW480 after overexpression of miR-143/145. In these experiments, overexpression was achieved by transfecting Caco2, HT29 and SW480 cells with pre-miR-143 and/or pre-miR-145, which are synthetic RNA oligonucleotides that mimic the miR-143 and miR-145 precursors. The efficient overexpression of miR-143/145 is shown in Figure 2A, 2F and 2K. As anticipated, overexpression of miR-143 and/or miR-145 significantly suppressed IGF1R protein levels in Caco2, HT29 and SW480 cells (Figure 2B, 2C; 2G, 2H; and 2L, 2M). To determine the regulatory level at which miR-143/145 influenced IGF1R expression, we repeated the above experiments and examined the expression of IGF1R mRNA after transfection. Overexpression of miR-143/145 did not affect IGF1R mRNA stability (Figure 2D, 2I and 2N). These results demonstrated that miR-143/145 specifically regulates IGF1R expression at the post-transcriptional level, which is the most common mechanism for animal miRNAs.


MiR-143 and MiR-145 regulate IGF1R to suppress cell proliferation in colorectal cancer.

Su J, Liang H, Yao W, Wang N, Zhang S, Yan X, Feng H, Pang W, Wang Y, Wang X, Fu Z, Liu Y, Zhao C, Zhang J, Zhang CY, Zen K, Chen X, Wang Y - PLoS ONE (2014)

miR-143/145 directly regulate IGF1R expression at the post-transcriptional level.(A, F and K) Quantitative RT-PCR analysis of miR-143/145 levels in Caco2, HT29 and SW480 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. (B, C; G, H; and L, M) Western blot analysis of IGF1R protein levels in Caco2 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. B, G and L: representative image; C, H and M: quantitative analysis. (D, I and N) Quantitative RT-PCR analysis of IGF1R mRNA levels in Caco2, HT29 and SW480 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. (E, J and O) Direct recognition of the IGF1R 3′-UTR by miR-143/145. Caco2, HT29 and SW480 cells were co-transfected with firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-143/145 binding sites in the IGF1R 3′-UTR and a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. Twenty-four hours after transfection, the cells were assayed using a luciferase assay kit. The results are displayed as the ratio of firefly luciferase activity in the miR-143/145-transfected cells to the activity in the control cells. *P<0.05; **P<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4256231&req=5

pone-0114420-g002: miR-143/145 directly regulate IGF1R expression at the post-transcriptional level.(A, F and K) Quantitative RT-PCR analysis of miR-143/145 levels in Caco2, HT29 and SW480 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. (B, C; G, H; and L, M) Western blot analysis of IGF1R protein levels in Caco2 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. B, G and L: representative image; C, H and M: quantitative analysis. (D, I and N) Quantitative RT-PCR analysis of IGF1R mRNA levels in Caco2, HT29 and SW480 cells treated with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. (E, J and O) Direct recognition of the IGF1R 3′-UTR by miR-143/145. Caco2, HT29 and SW480 cells were co-transfected with firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-143/145 binding sites in the IGF1R 3′-UTR and a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. Twenty-four hours after transfection, the cells were assayed using a luciferase assay kit. The results are displayed as the ratio of firefly luciferase activity in the miR-143/145-transfected cells to the activity in the control cells. *P<0.05; **P<0.01.
Mentions: The correlation between miR-143/145 and IGF1R was further examined by evaluating IGF1R expression in the human colorectal cancer cell lines Caco2, HT29 and SW480 after overexpression of miR-143/145. In these experiments, overexpression was achieved by transfecting Caco2, HT29 and SW480 cells with pre-miR-143 and/or pre-miR-145, which are synthetic RNA oligonucleotides that mimic the miR-143 and miR-145 precursors. The efficient overexpression of miR-143/145 is shown in Figure 2A, 2F and 2K. As anticipated, overexpression of miR-143 and/or miR-145 significantly suppressed IGF1R protein levels in Caco2, HT29 and SW480 cells (Figure 2B, 2C; 2G, 2H; and 2L, 2M). To determine the regulatory level at which miR-143/145 influenced IGF1R expression, we repeated the above experiments and examined the expression of IGF1R mRNA after transfection. Overexpression of miR-143/145 did not affect IGF1R mRNA stability (Figure 2D, 2I and 2N). These results demonstrated that miR-143/145 specifically regulates IGF1R expression at the post-transcriptional level, which is the most common mechanism for animal miRNAs.

Bottom Line: We identified specific target sites for miR-143 and miR-145 (miR-143/145) in the 3'-untranslated region (3'-UTR) of the IGF1R gene.These miRNAs are members of a cluster of miRNAs that have been reported to exhibit tumor suppressor activity.We demonstrated that the repression of IGF1R by miR-143/145 suppressed the proliferation of Caco2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China.

ABSTRACT
Insulin-like growth factor 1 receptor (IGF1R) is a transmembrane receptor that is activated by insulin-like growth factor 1 (IGF-1) and by a related hormone called IGF-2. It belongs to the large class of tyrosine kinase receptors and plays an important role in colorectal cancer etiology and progression. In this study, we used bioinformatic analyses to search for miRNAs that potentially target IGF1R. We identified specific target sites for miR-143 and miR-145 (miR-143/145) in the 3'-untranslated region (3'-UTR) of the IGF1R gene. These miRNAs are members of a cluster of miRNAs that have been reported to exhibit tumor suppressor activity. Consistent with the bioinformatic analyses, we identified an inverse correlation between miR-143/145 levels and IGF1R protein levels in colorectal cancer tissues. By overexpressing miR-143/145 in Caco2, HT29 and SW480 colorectal cancer cells, we experimentally validated that miR-143/145 directly recognizes the 3'-UTR of the IGF1R transcript and regulates IGF1R expression. Furthermore, the biological consequences of the targeting of IGF1R by miR-143/145 were examined by cell proliferation assays in vitro. We demonstrated that the repression of IGF1R by miR-143/145 suppressed the proliferation of Caco2 cells. Taken together, our findings provide evidence for a role of the miR-143/145 cluster as a tumor suppressor in colorectal cancer through the inhibition of IGF1R translation.

Show MeSH
Related in: MedlinePlus