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MicroRNA-5p and -3p co-expression and cross-targeting in colon cancer cells.

Choo KB, Soon YL, Nguyen PN, Hiew MS, Huang CJ - J. Biomed. Sci. (2014)

Bottom Line: Nine dysregulated miRNA pairs fell into three miRNA gene families, namely let-7, mir-8/200 and mir-17, which showed frequent cross-targeting in the metastasis process.Focusing on the let-7d-5p/3p pair, the respectively targeted IGF1R and KRAS were shown to be in a reverse relationship with expression of the respective miRNA, which was confirmed in transient transfection assays using let-7d mimic or inhibitor.Targeting of KRAS by let-7d was previous reported; targeting of IGF1R by let-7d-5p was confirmed in luciferase assays in this study.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, Graduate Institute of Biotechnology, School of Agriculture, Chinese Culture University, 55, Hwa-Kang Road, Yang Ming Shan 111, Taipei, Taiwan. hqr2@faculty.pccu.edu.tw.

ABSTRACT

Background: Two mature miRNA species may be generated from the 5' and 3' arms of a pre-miRNA precursor. In most cases, only one species remains while the complementary species is degraded. However, co-existence of miRNA-5p and -3p species is increasingly being reported. In this work, we aimed to systematically investigate co-expression of miRNA-5p/3p in colon cancer cells in a genome-wide analysis, and to examine cross-targeting of the dysregulated miRNAs and 5p/3p species.

Results: Four colon cancer cell lines were examined relative to two normal colon tissues. Of the 1,190 miRNAs analyzed, 92 and 36 were found to be up- or down-regulated, respectively, in cancer cells. Nineteen co-expressed miRNA-5p/3p pairs were further identified suggesting frequent 5p/3p co-accumulation in colon cancer cells. Of these, 14 pairs were co-up-regulated and 3 pairs were co-down-regulated indicating concerted 5p/3p dysregulation. Nine dysregulated miRNA pairs fell into three miRNA gene families, namely let-7, mir-8/200 and mir-17, which showed frequent cross-targeting in the metastasis process. Focusing on the let-7d-5p/3p pair, the respectively targeted IGF1R and KRAS were shown to be in a reverse relationship with expression of the respective miRNA, which was confirmed in transient transfection assays using let-7d mimic or inhibitor. Targeting of KRAS by let-7d was previous reported; targeting of IGF1R by let-7d-5p was confirmed in luciferase assays in this study. The findings of let-7d-5p/3p and multiple other miRNAs targeting IGF1R, KRAS and other metastasis-related factors suggest that 5p/3p miRNAs contribute to cross-targeting of multiple cancer-associated factors and processes possibly to evade functional abolishment when any one of the crucial factors are inactivated.

Conclusions: miRNA-5p/3p species are frequently co-expressed and are coordinately regulated in colon cancer cells. In cancer cells, multiple cross-targeting by the miRNAs, including the co-existing 5p/3p species, frequently occurs in an apparent safe-proof scheme of miRNA regulation of important tumorigenesis processes. Further systematic analysis of co-existing miRNA-5p/3p pairs in clinical tissues is important in elucidating 5p/3p contributions to cancer pathogenesis.

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Targeting of IGF1R by let-7d-5p as shown in luciferase assays. (A) The seed sequences (boxed) of the three putative let-7d-5p-targeted sequences in IGF1R (see also Figure 3 for the designations and locations of IGF1R-1, -2 and -3). The mutated nucleotides are indicated by arrows and lowercase letters. The nucleotide positions of the sequences harboring the targeted site are shown above the sequences in reference to NM_000875. (B) Luciferase assays of transfection of the wild-type (WT) and mutant (Mut) luciferase constructs alone, or in the presence of a let-7d-5p mimic (5p) or a negative control (NC). Relative luciferase activity (RLA) values were calculated relative to the wild-type or mutant construct only, which were arbitrarily set as 1.0. The data were obtained from three independent experiments. **p < 0.001.
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Fig6: Targeting of IGF1R by let-7d-5p as shown in luciferase assays. (A) The seed sequences (boxed) of the three putative let-7d-5p-targeted sequences in IGF1R (see also Figure 3 for the designations and locations of IGF1R-1, -2 and -3). The mutated nucleotides are indicated by arrows and lowercase letters. The nucleotide positions of the sequences harboring the targeted site are shown above the sequences in reference to NM_000875. (B) Luciferase assays of transfection of the wild-type (WT) and mutant (Mut) luciferase constructs alone, or in the presence of a let-7d-5p mimic (5p) or a negative control (NC). Relative luciferase activity (RLA) values were calculated relative to the wild-type or mutant construct only, which were arbitrarily set as 1.0. The data were obtained from three independent experiments. **p < 0.001.

Mentions: Targeting of KRAS by let-7d has been confirmed in a number of previous studies although the 5p or 3p species was not specified [33,34]. Our analysis clearly supports that it is the let-7d-3p that is targeting KRAS (Figure 3). On the other hand, further experimental evidences were needed to support let-7d-5p targeting IGF1R, and to determine which, or if all, of the three predicted target sites in the 3’-UTR of the IGF1R mRNA (Figure 3) is targeted. To achieve this goal, luciferase assays were performed using the pGL-3-Control vector in which about 200-bp oligonucleotides harboring each of the three let-7d-5p targeting sites in the IGF1R sequence (see Methods, and Additional file 3) were inserted at the 3’-end of the luciferase gene of the vector as previously described [24]. Mutants in the seed sequences of the miRNA target sites were also created (Figure 6A). The wild-type and the mutant constructs were transfected into HCT-15 cells alone, or in the presence of a let-7d-5p mimic for miRNA over-expression, or a negative control (NC) oligonucleotide (Figure 6B). The results showed that on over-expressing let-7d-5p, the presence of the second predicted let-7d-5p site, designated as IGF1R-2, inserted in the luciferase vector clearly led to significant down-regulated luciferase activities to 46% of the wild-type level; on the other hand, when the IGF1R-2 site was mutated, no appreciably effects on the luciferase activities were observed (Figure 6B, middle panel). IGF1R-1 and -3 sites were apparently not targeted by let-7d-5p, although the IGF1R-3 site showed a 10% decrease in luciferase activities. Our collective data confirmed that let-7d-5p targeted IGF1R at one of the three predicted sites to result in down-regulated IGF1R expression in colon cancer cells.Figure 6


MicroRNA-5p and -3p co-expression and cross-targeting in colon cancer cells.

Choo KB, Soon YL, Nguyen PN, Hiew MS, Huang CJ - J. Biomed. Sci. (2014)

Targeting of IGF1R by let-7d-5p as shown in luciferase assays. (A) The seed sequences (boxed) of the three putative let-7d-5p-targeted sequences in IGF1R (see also Figure 3 for the designations and locations of IGF1R-1, -2 and -3). The mutated nucleotides are indicated by arrows and lowercase letters. The nucleotide positions of the sequences harboring the targeted site are shown above the sequences in reference to NM_000875. (B) Luciferase assays of transfection of the wild-type (WT) and mutant (Mut) luciferase constructs alone, or in the presence of a let-7d-5p mimic (5p) or a negative control (NC). Relative luciferase activity (RLA) values were calculated relative to the wild-type or mutant construct only, which were arbitrarily set as 1.0. The data were obtained from three independent experiments. **p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig6: Targeting of IGF1R by let-7d-5p as shown in luciferase assays. (A) The seed sequences (boxed) of the three putative let-7d-5p-targeted sequences in IGF1R (see also Figure 3 for the designations and locations of IGF1R-1, -2 and -3). The mutated nucleotides are indicated by arrows and lowercase letters. The nucleotide positions of the sequences harboring the targeted site are shown above the sequences in reference to NM_000875. (B) Luciferase assays of transfection of the wild-type (WT) and mutant (Mut) luciferase constructs alone, or in the presence of a let-7d-5p mimic (5p) or a negative control (NC). Relative luciferase activity (RLA) values were calculated relative to the wild-type or mutant construct only, which were arbitrarily set as 1.0. The data were obtained from three independent experiments. **p < 0.001.
Mentions: Targeting of KRAS by let-7d has been confirmed in a number of previous studies although the 5p or 3p species was not specified [33,34]. Our analysis clearly supports that it is the let-7d-3p that is targeting KRAS (Figure 3). On the other hand, further experimental evidences were needed to support let-7d-5p targeting IGF1R, and to determine which, or if all, of the three predicted target sites in the 3’-UTR of the IGF1R mRNA (Figure 3) is targeted. To achieve this goal, luciferase assays were performed using the pGL-3-Control vector in which about 200-bp oligonucleotides harboring each of the three let-7d-5p targeting sites in the IGF1R sequence (see Methods, and Additional file 3) were inserted at the 3’-end of the luciferase gene of the vector as previously described [24]. Mutants in the seed sequences of the miRNA target sites were also created (Figure 6A). The wild-type and the mutant constructs were transfected into HCT-15 cells alone, or in the presence of a let-7d-5p mimic for miRNA over-expression, or a negative control (NC) oligonucleotide (Figure 6B). The results showed that on over-expressing let-7d-5p, the presence of the second predicted let-7d-5p site, designated as IGF1R-2, inserted in the luciferase vector clearly led to significant down-regulated luciferase activities to 46% of the wild-type level; on the other hand, when the IGF1R-2 site was mutated, no appreciably effects on the luciferase activities were observed (Figure 6B, middle panel). IGF1R-1 and -3 sites were apparently not targeted by let-7d-5p, although the IGF1R-3 site showed a 10% decrease in luciferase activities. Our collective data confirmed that let-7d-5p targeted IGF1R at one of the three predicted sites to result in down-regulated IGF1R expression in colon cancer cells.Figure 6

Bottom Line: Nine dysregulated miRNA pairs fell into three miRNA gene families, namely let-7, mir-8/200 and mir-17, which showed frequent cross-targeting in the metastasis process.Focusing on the let-7d-5p/3p pair, the respectively targeted IGF1R and KRAS were shown to be in a reverse relationship with expression of the respective miRNA, which was confirmed in transient transfection assays using let-7d mimic or inhibitor.Targeting of KRAS by let-7d was previous reported; targeting of IGF1R by let-7d-5p was confirmed in luciferase assays in this study.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, Graduate Institute of Biotechnology, School of Agriculture, Chinese Culture University, 55, Hwa-Kang Road, Yang Ming Shan 111, Taipei, Taiwan. hqr2@faculty.pccu.edu.tw.

ABSTRACT

Background: Two mature miRNA species may be generated from the 5' and 3' arms of a pre-miRNA precursor. In most cases, only one species remains while the complementary species is degraded. However, co-existence of miRNA-5p and -3p species is increasingly being reported. In this work, we aimed to systematically investigate co-expression of miRNA-5p/3p in colon cancer cells in a genome-wide analysis, and to examine cross-targeting of the dysregulated miRNAs and 5p/3p species.

Results: Four colon cancer cell lines were examined relative to two normal colon tissues. Of the 1,190 miRNAs analyzed, 92 and 36 were found to be up- or down-regulated, respectively, in cancer cells. Nineteen co-expressed miRNA-5p/3p pairs were further identified suggesting frequent 5p/3p co-accumulation in colon cancer cells. Of these, 14 pairs were co-up-regulated and 3 pairs were co-down-regulated indicating concerted 5p/3p dysregulation. Nine dysregulated miRNA pairs fell into three miRNA gene families, namely let-7, mir-8/200 and mir-17, which showed frequent cross-targeting in the metastasis process. Focusing on the let-7d-5p/3p pair, the respectively targeted IGF1R and KRAS were shown to be in a reverse relationship with expression of the respective miRNA, which was confirmed in transient transfection assays using let-7d mimic or inhibitor. Targeting of KRAS by let-7d was previous reported; targeting of IGF1R by let-7d-5p was confirmed in luciferase assays in this study. The findings of let-7d-5p/3p and multiple other miRNAs targeting IGF1R, KRAS and other metastasis-related factors suggest that 5p/3p miRNAs contribute to cross-targeting of multiple cancer-associated factors and processes possibly to evade functional abolishment when any one of the crucial factors are inactivated.

Conclusions: miRNA-5p/3p species are frequently co-expressed and are coordinately regulated in colon cancer cells. In cancer cells, multiple cross-targeting by the miRNAs, including the co-existing 5p/3p species, frequently occurs in an apparent safe-proof scheme of miRNA regulation of important tumorigenesis processes. Further systematic analysis of co-existing miRNA-5p/3p pairs in clinical tissues is important in elucidating 5p/3p contributions to cancer pathogenesis.

Show MeSH
Related in: MedlinePlus