反义miR-221／222抑制Akt通路活性提高胶质瘤细胞放射敏感性的研究

Inhibition of Akt pathway by miR - 221/222 antisense oligonucleotides enhances the radiosensitivity of glioblastoma

目的探讨敲低miR-221／222表达抑制Akt通路活性提高胶质瘤细胞放射敏感性的作用。方法脂质体共转染反义寡聚核苷酸（反义miR-221／222）下调胶质瘤U251，LN-229细胞miR-221与miR-222的表达。用Northern印迹方法测定转染后细胞miR-221、miR-222的表达水平；克隆形成实验验证各组胶质瘤细胞的放射敏感性；Western印迹分析各组胶质瘤细胞的pAkt蛋白表达变化；反义miR-221／222联合X线照射治疗裸鼠皮下移植瘤，观察肿瘤生长水平；Real-time PCR检测治疗后miR-221、miR-222的表达；免疫组化分析肿瘤组织中pAkt蛋白表达水平。结果Northern印迹分析显示反义miR-221／222可以有效地敲低胶质瘤细胞的miR-221和miR-222的表达水平。克隆形成实验证实反义mi-221／222联合X线照射可增加胶质瘤细胞放射敏感性。Western印迹分析显示反义miR-221／222可下调pAkt蛋白表达。经反义miR-221／222转染联合X线照射治疗后，裸鼠皮下移植瘤生长明显受抑，肿瘤组织中miR-221／miR-222表达水平下降，pAkt蛋白表达水平也明显降低。结论反义miR-221／222通过抑制Akt通路活性增加胶质瘤细胞的放射敏感性。

Objective To study the radiation - sensitizing effect of inhibiting of Akt pathway by knocking down miR- 221/222 in the human glioblastoma. Methods miR -221/222 antisense oligonucleotides were transfected into human glioblastoma cells. Northern blot analysis was conducted to detect the expression of miR - 221/222 in control,scrambled oligonucleotide （ODN） transfected cell group and antisense miR- 221/2220DNs transfected cell group. Clonogenic assay was used to measure its radiosensitivity and pAkt expression was examined by Western blot analysis. The therapeutic efficacy of antisense miR-221/222 combined with irradiation on the growth of xenograft tumors in nude mice were also observed. The expression of miR -221/222 was identified in tumor tissue by Real -time polymerase chain reaction （PCR）. The pAkt expression was simultaneously examined by immunohistochemistry assay. Results The expression level of miR -221/222 was significantly decreased in cells transfected with antisense miR -221/222 as compared to the parental cells or cells transfected with scrambled ODN. AS - miR - 221/222 combined with irradiation could synergistically enhance its radiosensitivity. The expression of pAkt was down - regulated in the AS - miR - 221/222 group revealed by Western blot analysis. The growth of xenograft tumors treated with antisense miR-221/222 and X- ray radiation were also inhibited. In antisense miR- 221/222 and X-ray radiation treated tumor tissue,the expression of miR -221/222 and pAkt was down -regulated. Conclusion AS -miR -221/222 may enhance the radiosensitivity of glioblastoma by suppressing the Akt pathway.