抗 miRNA-221 通过上调 PTEN 使人类结直肠癌细胞对辐射敏感。
Anti-miRNA-221 sensitizes human colorectal carcinoma cells to radiation by upregulating PTEN.
机构信息
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.
出版信息
World J Gastroenterol. 2013 Dec 28;19(48):9307-17. doi: 10.3748/wjg.v19.i48.9307.
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.
目的
探讨微小 RNA（miR）-221 对结直肠癌（CRC）细胞放射敏感性的调节作用及其机制。
方法
常规培养人 CRC 细胞系，并使其分别接受 0、2、4、6 和 8 Gy 剂量的 X 射线照射。照射后 24 h 提取细胞总 RNA 和蛋白质，实时逆转录聚合酶链反应（PCR）检测 miR-221 和磷酸酶张力蛋白同源物缺失的第 10 号染色体（PTEN）基因 mRNA 表达的变化。Western 印迹检测细胞中 PTEN 蛋白的变化。用 Lipofectamine 2000 将 pre-miR-221 或 anti-miR-221 加入 Caco2 细胞之前，用抗-PTEN-siRNA 对其进行预处理。照射后采用集落形成实验和流式细胞术分析检测存活细胞分数和增敏增强比。此外，PCR 扩增 PTEN 3'-非翻译区片段并将其插入荧光素酶报告质粒。将构建的荧光素酶报告质粒与 pre-miR-221 或 anti-miR-221 共转染 CRC 细胞，检测转染细胞中的荧光素酶活性。
结果
X 射线辐射剂量对人 Caco2 细胞中 miR-221 和 PTEN 蛋白的表达具有显著影响，呈剂量依赖性。miR-221 的表达水平随照射剂量的增加而逐渐升高，而 PTEN 蛋白的表达水平则逐渐降低。与 pre-miR-221 和阴性对照相比，anti-miR-221 组的 miR-221 表达水平显著降低（P < 0.01）。anti-miR-221 上调了 Caco2 细胞中 PTEN 蛋白的表达并增强了其放射敏感性（P < 0.01）。此外，用抗-PTEN-siRNA 预处理后，这种增强作用被显著抑制，表明放射敏感性的增强确实是由 PTEN 介导的。共转染荧光素酶报告质粒构建体和 anti-miR-221 的 CRC 细胞的荧光素酶活性显著增加（P < 0.01）。
结论
anti-miR-221 通过上调 PTEN 增强 CRC 细胞的放射敏感性。
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