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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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Co-expression of miRNA-5p and -3p pairs in colon cancer cells. miRNA expression was determined by stem-loop RT-PCR using U6 snRNA as a PCR control. The PCR products were analyzed in 4% agarose gels.
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Fig2: Co-expression of miRNA-5p and -3p pairs in colon cancer cells. miRNA expression was determined by stem-loop RT-PCR using U6 snRNA as a PCR control. The PCR products were analyzed in 4% agarose gels.

Mentions: To focus on co-expression of the 5p/3p miRNAs, data of 5p/3p pairs, with at least one of the pair with log2(fold change) ≥ 1.5 or ≤ −1.5, were extracted from the dataset of the 128 dysregulated miRNAs. A total of 19 pairs (38 miRNAs) that answered to this criterion were obtained (Table 1). The alterations observed in the cancer cells relative to the normal tissues ranged from significant up-regulation by 9.13-fold in miR-21-5p to down-regulation by 3.94-fold in miR-574-3p. Out of the 19 miRNA pairs thus extracted, it is further observed that 14 (73.7%) 5p/3p pairs were co-up-regulated and 3 (15.8%) pairs were co-down-regulated. Only 2 (10.5%) pairs, miR-200b and let-7d, showed reverse directions of dysregulation in the 5p/3p species. Co-expression was validated in three randomly selected miRNA pairs, miR-17, -21, -141, in the co-up-regulated group by stem-loop RT-PCR in all four cervical cancer cell lines and in a normal colon tissue (Figure 2). Up-regulated expression of the three miRNA pairs was evident when the expression levels in the cancer cell lines were compared with the normal tissues. Furthermore, co-expression of the 5p/3p pairs was also shown for miR-21 and -141. However, In the case of miR-17, however, despite the observation that the 3p levels in SK-CO-1 and WiDr were much lower than the 3p species, 5p/3p co-expression was clearly shown for HCT-15 and HT-29. To validate co-regulation, one miRNA pair was randomly chosen from each of the three categories of regulation in Table 1 for direct real-time quantitative RT-PCR assays (Table 2). The data obtained were in excellent agreement with the microarray data, thus supporting the validity of the miRNA profiling data presented. Reverse dysregulation of the let-7d-5p/3p pair was also confirmed. Taken together, the data show frequent co-expression of 5p/3p miRNAs in colon cancer cells, and that the majority (89.5%) of the co-expressed 5p/3p pairs was co-up- or co-down-regulated, strongly suggesting concerted dysregulation of miRNA sister pairs in colon cancer cells.Table 1


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)

Co-expression of miRNA-5p and -3p pairs in colon cancer cells. miRNA expression was determined by stem-loop RT-PCR using U6 snRNA as a PCR control. The PCR products were analyzed in 4% agarose gels.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Co-expression of miRNA-5p and -3p pairs in colon cancer cells. miRNA expression was determined by stem-loop RT-PCR using U6 snRNA as a PCR control. The PCR products were analyzed in 4% agarose gels.
Mentions: To focus on co-expression of the 5p/3p miRNAs, data of 5p/3p pairs, with at least one of the pair with log2(fold change) ≥ 1.5 or ≤ −1.5, were extracted from the dataset of the 128 dysregulated miRNAs. A total of 19 pairs (38 miRNAs) that answered to this criterion were obtained (Table 1). The alterations observed in the cancer cells relative to the normal tissues ranged from significant up-regulation by 9.13-fold in miR-21-5p to down-regulation by 3.94-fold in miR-574-3p. Out of the 19 miRNA pairs thus extracted, it is further observed that 14 (73.7%) 5p/3p pairs were co-up-regulated and 3 (15.8%) pairs were co-down-regulated. Only 2 (10.5%) pairs, miR-200b and let-7d, showed reverse directions of dysregulation in the 5p/3p species. Co-expression was validated in three randomly selected miRNA pairs, miR-17, -21, -141, in the co-up-regulated group by stem-loop RT-PCR in all four cervical cancer cell lines and in a normal colon tissue (Figure 2). Up-regulated expression of the three miRNA pairs was evident when the expression levels in the cancer cell lines were compared with the normal tissues. Furthermore, co-expression of the 5p/3p pairs was also shown for miR-21 and -141. However, In the case of miR-17, however, despite the observation that the 3p levels in SK-CO-1 and WiDr were much lower than the 3p species, 5p/3p co-expression was clearly shown for HCT-15 and HT-29. To validate co-regulation, one miRNA pair was randomly chosen from each of the three categories of regulation in Table 1 for direct real-time quantitative RT-PCR assays (Table 2). The data obtained were in excellent agreement with the microarray data, thus supporting the validity of the miRNA profiling data presented. Reverse dysregulation of the let-7d-5p/3p pair was also confirmed. Taken together, the data show frequent co-expression of 5p/3p miRNAs in colon cancer cells, and that the majority (89.5%) of the co-expressed 5p/3p pairs was co-up- or co-down-regulated, strongly suggesting concerted dysregulation of miRNA sister pairs in colon cancer cells.Table 1

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