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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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Effects of altered endogenous let-7d-5p and -3p levels on IGF1R and KRAS expression. In the experiments, a let-7d-5p inhibitor (A-C) or a let-7d-3p mimic (D-F) was transfected to HCT-15 cells for 48 h before further assays. The let-7d-5p or -3p miRNA (A & D) and the IGF1R and KRAS mRNA levels (B & E) were determined by real-time PCR; the IGF1R and KRAS protein levels (C & F) were determined by western blot analysis. The data shown were derived from three independent experiments. In (C) & (F), a representative western blot of IGF1R or KRAS is also shown. N.C., a validated negative control; R.L., relative levels compared with the mock control. *p < 0.05, **p < 0.01 and N.S. indicates statistically not significant relative to the mock control.
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Fig5: Effects of altered endogenous let-7d-5p and -3p levels on IGF1R and KRAS expression. In the experiments, a let-7d-5p inhibitor (A-C) or a let-7d-3p mimic (D-F) was transfected to HCT-15 cells for 48 h before further assays. The let-7d-5p or -3p miRNA (A & D) and the IGF1R and KRAS mRNA levels (B & E) were determined by real-time PCR; the IGF1R and KRAS protein levels (C & F) were determined by western blot analysis. The data shown were derived from three independent experiments. In (C) & (F), a representative western blot of IGF1R or KRAS is also shown. N.C., a validated negative control; R.L., relative levels compared with the mock control. *p < 0.05, **p < 0.01 and N.S. indicates statistically not significant relative to the mock control.

Mentions: To further validate let-7d-5p/3p targeting at IFG1R and KRAS, effects of altering endogenous miRNA levels on the target mRNAs and proteins were investigated. When the endogenous let-7d-5p level was knockdown by transfection of a specific let-7d-5p inhibitor sequence in HCT-15 cells (Figure 5A), IGF1R mRNA was significantly up-regulated by 2.58 ± 0.59-fold whereas transfection of a negative control with a scrambled sequence had insignificant effects on the IGF1R mRNA levels (Figure 5B). On the other hand, the IGF1R protein level was significantly up-regulated by 1.64-fold relative to the mock control (Figure 5C). Hence, knocking down let-7d-5p had clearly led to up-regulation of IGF1R, consistent with let-7d-5p regulation of IGF1R. When a let-7d-3p-specific mimic sequence was transfected into HCT-15 cells, a 478-fold increase of the let-7d-3p level was achieved (Figure 5D). The increased level of the miRNA was accompanied by significant down-regulation of the KRAS mRNA level to 0.30-fold (Figure 5E), and also significant down-regulation of the KRAS protein to 0.52-fold that of the mock control (Figure 5F), supporting let-7d-3p regulation of KRAS apparently via increased degradation of the KRAS transcript.Figure 5


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)

Effects of altered endogenous let-7d-5p and -3p levels on IGF1R and KRAS expression. In the experiments, a let-7d-5p inhibitor (A-C) or a let-7d-3p mimic (D-F) was transfected to HCT-15 cells for 48 h before further assays. The let-7d-5p or -3p miRNA (A & D) and the IGF1R and KRAS mRNA levels (B & E) were determined by real-time PCR; the IGF1R and KRAS protein levels (C & F) were determined by western blot analysis. The data shown were derived from three independent experiments. In (C) & (F), a representative western blot of IGF1R or KRAS is also shown. N.C., a validated negative control; R.L., relative levels compared with the mock control. *p < 0.05, **p < 0.01 and N.S. indicates statistically not significant relative to the mock control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Effects of altered endogenous let-7d-5p and -3p levels on IGF1R and KRAS expression. In the experiments, a let-7d-5p inhibitor (A-C) or a let-7d-3p mimic (D-F) was transfected to HCT-15 cells for 48 h before further assays. The let-7d-5p or -3p miRNA (A & D) and the IGF1R and KRAS mRNA levels (B & E) were determined by real-time PCR; the IGF1R and KRAS protein levels (C & F) were determined by western blot analysis. The data shown were derived from three independent experiments. In (C) & (F), a representative western blot of IGF1R or KRAS is also shown. N.C., a validated negative control; R.L., relative levels compared with the mock control. *p < 0.05, **p < 0.01 and N.S. indicates statistically not significant relative to the mock control.
Mentions: To further validate let-7d-5p/3p targeting at IFG1R and KRAS, effects of altering endogenous miRNA levels on the target mRNAs and proteins were investigated. When the endogenous let-7d-5p level was knockdown by transfection of a specific let-7d-5p inhibitor sequence in HCT-15 cells (Figure 5A), IGF1R mRNA was significantly up-regulated by 2.58 ± 0.59-fold whereas transfection of a negative control with a scrambled sequence had insignificant effects on the IGF1R mRNA levels (Figure 5B). On the other hand, the IGF1R protein level was significantly up-regulated by 1.64-fold relative to the mock control (Figure 5C). Hence, knocking down let-7d-5p had clearly led to up-regulation of IGF1R, consistent with let-7d-5p regulation of IGF1R. When a let-7d-3p-specific mimic sequence was transfected into HCT-15 cells, a 478-fold increase of the let-7d-3p level was achieved (Figure 5D). The increased level of the miRNA was accompanied by significant down-regulation of the KRAS mRNA level to 0.30-fold (Figure 5E), and also significant down-regulation of the KRAS protein to 0.52-fold that of the mock control (Figure 5F), supporting let-7d-3p regulation of KRAS apparently via increased degradation of the KRAS transcript.Figure 5

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