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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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EdU proliferation assay analysis of the effect of IGF1R-targeted miR-143/145 on the growth of colorectal cancer cells.The red fluorescent cells are in the S phase of mitosis, and the blue fluorescent cells represent all of the cells. (A and B) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. A: representative image; B: ratio of EdU-positive Caco2 cells. (C and D) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control siRNA, IGF1R siRNA, control vector or the IGF1R overexpression vector. C: representative image; D: ratio of EdU-positive Caco2 cells. (E and F) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control plus control vector, a scrambled control plus IFG1R overexpression vector, pre-miR-143/145 plus control vector, or pre-miR-143/145 plus IFG1R overexpression vector. E: representative image; F: ratio of EdU-positive Caco2 cells. *P<0.05; **P<0.01.
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pone-0114420-g004: EdU proliferation assay analysis of the effect of IGF1R-targeted miR-143/145 on the growth of colorectal cancer cells.The red fluorescent cells are in the S phase of mitosis, and the blue fluorescent cells represent all of the cells. (A and B) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. A: representative image; B: ratio of EdU-positive Caco2 cells. (C and D) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control siRNA, IGF1R siRNA, control vector or the IGF1R overexpression vector. C: representative image; D: ratio of EdU-positive Caco2 cells. (E and F) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control plus control vector, a scrambled control plus IFG1R overexpression vector, pre-miR-143/145 plus control vector, or pre-miR-143/145 plus IFG1R overexpression vector. E: representative image; F: ratio of EdU-positive Caco2 cells. *P<0.05; **P<0.01.

Mentions: To further test the biological effect of IGF1R-targeted miR-143/145 on the growth of colorectal cancer cells, the effect of miR-143/145 and IGF1R on cell proliferation was examined with an EdU assay, an immunochemical detection method that measures nucleotide analogue incorporation into newly replicated DNA. Consistent with the results from the CCK-8 assay, the percentage of EdU-positive cells was significantly lower in cells transfected with pre-miR-143/145 (Figure 4A and 4B). Similarly, significantly more EdU-positive cells were observed in the cells with IGF1R overexpression, whereas IGF1R-siRNA transfected cells showed the opposite effect on cell proliferation (Figure 4C and 4D). These results again demonstrated that decreased IGF1R levels yielded the same phenotype observed by miR-143/145 overexpression. To further examine the functional relationship between miR-143/145 and IGF1R, Caco2 cells were simultaneously transfected with pre- miR-143/145 and the IGF1R overexpression plasmid. As expected, overexpression of IGF1R dramatically rescued the suppressive effect of miR-143/145 on cell proliferation (Figure 4E and 4F). Taken together, the results demonstrate that miR-143/145 suppress cell proliferation by silencing IGF1R.


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)

EdU proliferation assay analysis of the effect of IGF1R-targeted miR-143/145 on the growth of colorectal cancer cells.The red fluorescent cells are in the S phase of mitosis, and the blue fluorescent cells represent all of the cells. (A and B) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. A: representative image; B: ratio of EdU-positive Caco2 cells. (C and D) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control siRNA, IGF1R siRNA, control vector or the IGF1R overexpression vector. C: representative image; D: ratio of EdU-positive Caco2 cells. (E and F) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control plus control vector, a scrambled control plus IFG1R overexpression vector, pre-miR-143/145 plus control vector, or pre-miR-143/145 plus IFG1R overexpression vector. E: representative image; F: ratio of EdU-positive Caco2 cells. *P<0.05; **P<0.01.
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pone-0114420-g004: EdU proliferation assay analysis of the effect of IGF1R-targeted miR-143/145 on the growth of colorectal cancer cells.The red fluorescent cells are in the S phase of mitosis, and the blue fluorescent cells represent all of the cells. (A and B) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control, pre-miR-143, pre-miR-145 or both pre-miR-143 and pre-miR-145. A: representative image; B: ratio of EdU-positive Caco2 cells. (C and D) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control siRNA, IGF1R siRNA, control vector or the IGF1R overexpression vector. C: representative image; D: ratio of EdU-positive Caco2 cells. (E and F) The EdU proliferation assay was performed 48 h after the transfection of Caco2 cells with a scrambled control plus control vector, a scrambled control plus IFG1R overexpression vector, pre-miR-143/145 plus control vector, or pre-miR-143/145 plus IFG1R overexpression vector. E: representative image; F: ratio of EdU-positive Caco2 cells. *P<0.05; **P<0.01.
Mentions: To further test the biological effect of IGF1R-targeted miR-143/145 on the growth of colorectal cancer cells, the effect of miR-143/145 and IGF1R on cell proliferation was examined with an EdU assay, an immunochemical detection method that measures nucleotide analogue incorporation into newly replicated DNA. Consistent with the results from the CCK-8 assay, the percentage of EdU-positive cells was significantly lower in cells transfected with pre-miR-143/145 (Figure 4A and 4B). Similarly, significantly more EdU-positive cells were observed in the cells with IGF1R overexpression, whereas IGF1R-siRNA transfected cells showed the opposite effect on cell proliferation (Figure 4C and 4D). These results again demonstrated that decreased IGF1R levels yielded the same phenotype observed by miR-143/145 overexpression. To further examine the functional relationship between miR-143/145 and IGF1R, Caco2 cells were simultaneously transfected with pre- miR-143/145 and the IGF1R overexpression plasmid. As expected, overexpression of IGF1R dramatically rescued the suppressive effect of miR-143/145 on cell proliferation (Figure 4E and 4F). Taken together, the results demonstrate that miR-143/145 suppress cell proliferation by silencing IGF1R.

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