MicroRNA-221 inhibits CDKN1C/p57 expression in human colorectal carcinoma

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 premiR-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 posttranscriptional gene silencing to promote CRC occurrence and progress, therefore serving as a potential therapeutic target for the prevention and treatment of CRC.

MiR-221/222 Promote the Growth of Malignant Glioma Cells by Regulating Its Target Genes

“…The result document that miR-221-and miR-222-transduced cells progressed more quickly through the cell-cycle phases (19). The same result was also proved in human prostate carcinoma cell lines, human thyroid papillary carcinomas, human colorectal carcinoma, and so on (20)(21)(22). Fig.…”

“…Frenquelli M et al also identified that miR-221 and miR-222 promote cancer cell proliferation in chronic lymphocytic leukemia (19). The same result were also proved in human prostate carcinoma cell lines, human thyroid papillary carcinomas, human colorectal carcinoma, and so on (20)(21)(22). The cell cycle distribution and cell apoptosis were analyzed by flow cytometry.…”

p57Kip2 and Cancer: Time for a Critical Appraisal

“…Recently, 2 miRs (miR-221 and miR-222) were reported to downregulate the expression of both p27 Kip1 and p57 Kip2 transcripts (23)(24)(25)(26)(27)(28). Intriguingly, miR-221 and miR-222 also control the transcripts of several other important genes, including Kit, Gax/Meox2, Pten, Timp3, Bmf, Mdm2, Stat, and Puma.…”

Construction of lentivirus-based inhibitor of hsa-microRNA-338-3p with specific secondary structure

“…The ΔΔC t was calculated by subtracting the ΔC t of the reference sample from the ΔC t of each sample. The fold change was calculated using the equation 2 -ΔΔCt [14] . The Taqman microRNA Assays for U6 RNA was used to normalize the relative abundance of miR-338-3p.…”

“…Discussion miRNAs play a key role in the regulation of basic biological activities, such as the growth, development, and differentiation of organisms, and because of this, unbalanced miRNA expression may contribute to many human diseases, particularly the [14] . miR-338-3p was a recently discovered miRNA that was found to be involved in cellular differentiation.…”