title: MicroRNA-221 inhibits CDKN1C/p57 expression in human colorectal carcinoma.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of General Surgery, Nanfang Hospital of Southern Medical University, Guangzhou, China. sunkai9602@sina.com
authors.forename: Kai
authors.initials: K
authors.lastname: Sun
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Jun-jie
authors.initials: JJ
authors.lastname: Zeng
authors.forename: Cheng-tang
authors.initials: CT
authors.lastname: Wu
authors.forename: Shang-tong
authors.initials: ST
authors.lastname: Lei
authors.forename: Guo-xin
authors.initials: GX
authors.lastname: Li
mesh_terms: D020413:3' Untranslated Regions; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000096:biosynthesis / Q000235:genetics*; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D018816:Genes, cdc; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D015854:Up-Regulation
chemical_list: D020413:3' Untranslated Regions; D050761:Cyclin-Dependent Kinase Inhibitor p57; D035683:MicroRNAs; C493964:CDKN1C protein, human; C525709:MIR221, human