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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 let-7d-5p and -3p at the IGF1R and KRAS mRNAs, respectively. The IGF1R and KRAS mRNAs are depicted showing the coding sequence (CDS), the 5’- and 3’-UTR (horizontal lines) and the polyA sequence (An). The three let-7d-5p target sites in the 3’-UTR of IGF1R transcript and the single let-7d-3p site in the KRAS mRNA are shown by solid vertical bars. The seed sequences of the miRNA alignment with the target transcripts are boxed. mfe, minimal free energy.
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Fig3: Targeting of let-7d-5p and -3p at the IGF1R and KRAS mRNAs, respectively. The IGF1R and KRAS mRNAs are depicted showing the coding sequence (CDS), the 5’- and 3’-UTR (horizontal lines) and the polyA sequence (An). The three let-7d-5p target sites in the 3’-UTR of IGF1R transcript and the single let-7d-3p site in the KRAS mRNA are shown by solid vertical bars. The seed sequences of the miRNA alignment with the target transcripts are boxed. mfe, minimal free energy.

Mentions: IGF1R and KRAS targeting by let-7d-5p and 3p was further experimentally validated since the two miRNA species were differentially expressed in colon cancer cells in reverse directions: let-7d-5p was up-regulated whereas let-7d-3p was down-regulated (see Tables 1 and 2). Hafner et al. [32] previously demonstrated let-7d regulation of IGF1R but which of the 5p/3p species was involved was not specified. Bioinformatics interrogation has revealed putative let-7d-5p targeting of the 7,088-bp 3’-untranslated region (3’-UTR) of IGF1R mRNA at three different locations (Figure 3). Likewise, let-7d-3p is predicted to target at a unique site at the 3’-terminus of the 4,549-bp 3’-UTR of the KRAS mRNA (Figure 3). The putative miRNA sites of the two mRNAs are highly conserved in mammals (Figure 3). To examine post-transcriptional regulation, the IGF1R and KRAS protein levels were first examined in the colon cancer cell line and in normal colon tissues (Figure 4). Results showed that the IGF1R-α and -β subunits, which are cleavage product of the same IGF1R precursor, were 0.33- and 0.53-fold, respectively, of the levels in normal cells, and both were significantly down-regulated in cancer cells (Table 5). On the other hand, the KRAS protein was significantly up-regulated in cancer cells by up to 3.57-fold despite down-regulated mRNA levels (Figure 4 and Table 5). A spurious band which has never been described in the literature appeared in the KRAS western blot of the normal colon cancer tissue. The band was likely a KRAS isoform, but the exact nature was not further determined.Figure 3


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 let-7d-5p and -3p at the IGF1R and KRAS mRNAs, respectively. The IGF1R and KRAS mRNAs are depicted showing the coding sequence (CDS), the 5’- and 3’-UTR (horizontal lines) and the polyA sequence (An). The three let-7d-5p target sites in the 3’-UTR of IGF1R transcript and the single let-7d-3p site in the KRAS mRNA are shown by solid vertical bars. The seed sequences of the miRNA alignment with the target transcripts are boxed. mfe, minimal free energy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4195866&req=5

Fig3: Targeting of let-7d-5p and -3p at the IGF1R and KRAS mRNAs, respectively. The IGF1R and KRAS mRNAs are depicted showing the coding sequence (CDS), the 5’- and 3’-UTR (horizontal lines) and the polyA sequence (An). The three let-7d-5p target sites in the 3’-UTR of IGF1R transcript and the single let-7d-3p site in the KRAS mRNA are shown by solid vertical bars. The seed sequences of the miRNA alignment with the target transcripts are boxed. mfe, minimal free energy.
Mentions: IGF1R and KRAS targeting by let-7d-5p and 3p was further experimentally validated since the two miRNA species were differentially expressed in colon cancer cells in reverse directions: let-7d-5p was up-regulated whereas let-7d-3p was down-regulated (see Tables 1 and 2). Hafner et al. [32] previously demonstrated let-7d regulation of IGF1R but which of the 5p/3p species was involved was not specified. Bioinformatics interrogation has revealed putative let-7d-5p targeting of the 7,088-bp 3’-untranslated region (3’-UTR) of IGF1R mRNA at three different locations (Figure 3). Likewise, let-7d-3p is predicted to target at a unique site at the 3’-terminus of the 4,549-bp 3’-UTR of the KRAS mRNA (Figure 3). The putative miRNA sites of the two mRNAs are highly conserved in mammals (Figure 3). To examine post-transcriptional regulation, the IGF1R and KRAS protein levels were first examined in the colon cancer cell line and in normal colon tissues (Figure 4). Results showed that the IGF1R-α and -β subunits, which are cleavage product of the same IGF1R precursor, were 0.33- and 0.53-fold, respectively, of the levels in normal cells, and both were significantly down-regulated in cancer cells (Table 5). On the other hand, the KRAS protein was significantly up-regulated in cancer cells by up to 3.57-fold despite down-regulated mRNA levels (Figure 4 and Table 5). A spurious band which has never been described in the literature appeared in the KRAS western blot of the normal colon cancer tissue. The band was likely a KRAS isoform, but the exact nature was not further determined.Figure 3

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