title: Anti-miRNA-221 sensitizes human colorectal carcinoma cells to radiation by upregulating PTEN.
abstract: OBJECTIVE
  To investigate the regulative effect of miRNA (miR)-221 on colorectal carcinoma (CRC) cell radiosensitivity and the underlying mechanisms.
  
  
  METHODS
  A human CRC-derived cell line was cultured conventionally and exposed to different doses of X-rays (0, 2, 4, 6 and 8 Gy). The total RNA and protein of the cells were extracted 24 h after irradiation, and the alteration of miR-221 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene mRNA expression was detected by real-time reverse transcriptase polymerase chain reaction (PCR). The protein alteration of PTEN in the cells was detected by Western blotting. Caco2 cells were pretreated with or without anti-PTEN-siRNA prior to the addition of pre-miR-221 or anti-miR-221 using Lipofectamine 2000. Colony formation assay and flow cytometry analysis were used to measure the surviving cell fraction and the sensitizing enhancement ratio after irradiation. Additionally, PTEN 3'-untranslated region fragment was PCR amplified and inserted into a luciferase reporter plasmid. The luciferase reporter plasmid 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 detected.
  
  
  RESULTS
  The X-ray radiation dose had a significant effect on the expression of miR-221 and PTEN protein in human Caco2 cells in a dose-dependent manner. The miR-221 expression level improved gradually with the increase in irradiation dose, while the PTEN protein expression level reduced gradually. miR-221 expression was significantly reduced in the anti-miR-221 group compared with the pre-miR-221 and negative control groups (P < 0.01). Anti-miR-221 upregulated expression of PTEN protein and enhanced the radiosensitivity of Caco2 cells (P < 0.01). Moreover, the inhibitory effect was dramatically abolished by pretreatment with anti-PTEN-siRNA, suggesting that the enhancement of radiosensitivity was indeed mediated by PTEN. A significant increase of luciferase activity was detected in CRC cells that were cotransfected with the luciferase reporter plasmid construct and anti-miR-221 (P < 0.01).
  
  
  CONCLUSIONS
  Anti-miR-221 can enhance the radiosensitivity of CRC cells by upregulating PTEN.
authors.affiliation: Qi Xue, Kai Sun, Hai-Jun Deng, Shang-Tong Lei, Jing-Qing Dong, Guo-Xin Li, Department of General Surgery, Nanfang Hospital of Southern Medical University, Guangzhou 510515, Guangdong Province, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Xue
authors.affiliation: Qi Xue, Kai Sun, Hai-Jun Deng, Shang-Tong Lei, Jing-Qing Dong, Guo-Xin Li, Department of General Surgery, Nanfang Hospital of Southern Medical University, Guangzhou 510515, Guangdong Province, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Sun
authors.affiliation: Qi Xue, Kai Sun, Hai-Jun Deng, Shang-Tong Lei, Jing-Qing Dong, Guo-Xin Li, Department of General Surgery, Nanfang Hospital of Southern Medical University, Guangzhou 510515, Guangdong Province, China.
authors.forename: Hai-Jun
authors.initials: HJ
authors.lastname: Deng
authors.affiliation: Qi Xue, Kai Sun, Hai-Jun Deng, Shang-Tong Lei, Jing-Qing Dong, Guo-Xin Li, Department of General Surgery, Nanfang Hospital of Southern Medical University, Guangzhou 510515, Guangdong Province, China.
authors.forename: Shang-Tong
authors.initials: ST
authors.lastname: Lei
authors.affiliation: Qi Xue, Kai Sun, Hai-Jun Deng, Shang-Tong Lei, Jing-Qing Dong, Guo-Xin Li, Department of General Surgery, Nanfang Hospital of Southern Medical University, Guangzhou 510515, Guangdong Province, China.
authors.forename: Jing-Qing
authors.initials: JQ
authors.lastname: Dong
authors.affiliation: Qi Xue, Kai Sun, Hai-Jun Deng, Shang-Tong Lei, Jing-Qing Dong, Guo-Xin Li, Department of General Surgery, Nanfang Hospital of Southern Medical University, Guangzhou 510515, Guangdong Province, China.
authors.forename: Guo-Xin
authors.initials: GX
authors.lastname: Li
mesh_terms: D018938:Caco-2 Cells; D049109:Cell Proliferation / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D015179:Colorectal Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D004307:Dose-Response Relationship, Radiation; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D019073:HT29 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism*; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D011836:Radiation Tolerance; D015398:Signal Transduction / Q000528:radiation effects; D014162:Transfection; D015854:Up-Regulation
chemical_list: D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; D051059:PTEN Phosphohydrolase; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; C525709:MIR221, human; C494929:PTEN protein, human
keywords: Colorectal carcinoma; Phosphatase and tensin homolog deleted on chromosome 10; Radiosensitivity; miR-221