Published online Dec 28, 2013. doi: 10.3748/wjg.v19.i48.9307
Revised: September 29, 2013
Accepted: December 13, 2013
Published online: December 28, 2013
AIM: 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).
CONCLUSION: Anti-miR-221 can enhance the radiosensitivity of CRC cells by upregulating PTEN.
Core tip: Previous studies have shown that miRNA (miR)-221 expression is elevated in radioresistant colorectal carcinoma (CRC) cells; however, it is unknown whether and how miR-221 controls cellular response to irradiation. We demonstrated that knockdown of miR-221 upregulated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression, and PTEN was identified as a direct target of miR-221 in CRC. Upregulated PTEN expression suppressed AKT activity and increased radiation-induced cell death, enhancing radiosensitivity in CRC cells. This study provides evidence for antioncogenic activity of anti-miR-221 in the irradiation of CRC and may be a useful biomarker or therapeutic target in CRC.