Abstract
Aim:
To investigate the regulatory effect of microRNA-221 (miR-221) on CDKN1C/p57 expression in colorectal carcinoma (CRC).
Methods:
Thirty four CRC and adjacent non-tumorous tissue samples were collected individually. Total RNA and protein were isolatedand from these samples and four human CRC-derived cell lines (including HT-29, Lovo, SW-480 and Caco2). MiR-221 expression was examined using real-time RT-PCR. CRC cells were treated with or without anti-p57-siRNA prior to the addition of pre-miR-221 or anti-miR-221. The mRNA and protein levels of CDKN1C/p57 were examined using semi-quantitative RT-PCR and Western blot, respectively. CRC cell proliferation and apoptosis were assessed using MTT assay and flow cytometry, respectively. The CDKN1C/p57 3′-UTR fragment was amplified using PCR from the genomic DNA of human colon cells and inserted into a luciferase reporter construct. The reporter construct was then transfected into CRC cells together with pre-miR-221 or anti-miR-221, and the luciferase activity in the transfected cells was examined.
Results:
MiR-221 expression was significantly up-regulated in 90% of CRC samples compared to that in the adjacent non-tumorous tissue, and the expression level was positively correlated to an advanced TNM stage and local invasion. There was no significant difference in CDKN1C/p57 mRNA expression between CRC and corresponding non-tumorous tissues, whereas CDKN1C/p57 protein expression was markedly decreased in the CRC samples. A significant inverse correlation between miR-221 and CDKN1C/p57 expression was found in CRC cells. Moreover, a miR-221-specific inhibitor significantly increased CDKN1C/p57 protein expression in CRC cells. Anti-miR-221 markedly inhibited CRC cell proliferation and induced apoptosis. This inhibitory effect was abolished by pretreatment with anti-p57-siRNA, suggesting that the inhibition was mediated by CDKN1C/p57. A significant increase of the luciferase activity was observed in CRC cells co-transfected with the luciferase reporter construct and anti-miR-221.
Conclusion:
MiR-221 binds to the target site in the 3′-UTR of the CDKN1C/p57 mRNA to inhibit CDKN1C/p57 expression by post-transcriptional gene silencing to promote CRC occurrence and progress, therefore serving as a potential therapeutic target for the prevention and treatment of CRC.
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Acknowledgements
This work was supported by the Natural Science Foundation of Guangdong Province (No 8451051501000390), the Medical Scientific Research Foundation of Guangdong Province (No B2010190) and the Chief Foundation of Nanfang Hospital of Southern Medical University (No 2008C005).
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Sun, K., Wang, W., Zeng, Jj. et al. MicroRNA-221 inhibits CDKN1C/p57 expression in human colorectal carcinoma. Acta Pharmacol Sin 32, 375–384 (2011). https://doi.org/10.1038/aps.2010.206
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DOI: https://doi.org/10.1038/aps.2010.206
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