靶向抑制CD133+Hep-2细胞Glut-1表达增强喉癌放射敏感性的机制研究

The exact mechanism of the radioresistance of laryngeal carcinoma remains unclear. Recently, cancer stem cells (CSC) were found to play an important role in radioresistance. CD133+ cells are a useful marker for CSCs in human laryngeal carcinoma. We found that increased Glut-1 expression was associated with the radioresistance of laryngeal carcinoma. To our knowledge, no study has examined the relationship between Glut-1 expression in laryngeal CSCs and radioresistance. Previously, we found that antisense Glut-1 decreases the expression of Glut-1 in laryngeal carcinoma, which improves the radiosensitivity of laryngeal carcinoma. In a preliminary study, we also found that CD133+ cells proliferated more. Glut-1 mRNA and protein levels were higher in CD133+ cells than in CD133- cells. Therefore, we speculated that the inhibition of Glut-1 expression in laryngeal CD133+Hep-2 cells might improve the radiosensitivity of laryngeal carcinoma. To test this hypothesis, we assessed the change in CD133+ Hep-2 cells and Glut-1 expression in laryngeal carcinoma cells before and after radiotherapy to determine whether CD133+Hep-2 cells are associated with the radioresistance of laryngeal carcinoma. We found that the radioresistance of laryngeal carcinoma was associated with increased Glut-1 expression in CD133+ Hep-2 cells. Based on these results, we developed a new hypothesis regarding the preservation of laryngeal function in patients with laryngeal carcinoma via targeted inhibition of Glut-1 expression in CD133+ Hep-2 cells to improve the radiosensitivity of laryngeal carcinoma.

喉癌放射抵抗的确切机制尚未阐明，近年来发现肿瘤干细胞在肿瘤放射抵抗中起重要作用，已证实喉癌CDl33+细胞具有肿瘤干细胞特性。本课题组首次发现喉癌细胞放射抵抗与Glut-1表达有关。而CD133+细胞Glut-1表达与喉癌放射抵抗的关系，迄今国内外尚无报道。前期研究我们发现反义Glut-1能降低喉癌细胞Glut-1表达继而提高喉癌放射敏感性，预实验发现CD133+喉癌细胞Glut-1高表达。因此，我们提出抑制CD133+喉癌细胞Glut-1表达可提高喉癌放射敏感性的假说。为证实这一假说，我们将通过检测喉癌细胞放射前后CD133+ 喉癌细胞及Glut-1表达变化，验证CD133+喉癌细胞与放射抵抗有关，明确这种放射抵抗与CD133+喉癌细胞Glut-1表达密切相关；从体内外实验研究靶向抑制CD133+ 喉癌细胞Glut-1表达而提高喉癌放射敏感性的机制，为喉癌患者保留喉功能治疗提供新理论。

目的：研究靶向抑制CD133+Hep-2细胞Glut-1表达在喉癌放射增敏中的作用.方法：（1）检测54例术后接受放射治疗的喉癌患者喉癌组织中CD133、Glut-1, HIF-1α, PI3K, p-Akt表达。（2）致瘤体内实验：在X线10Gy照射、反义GLUT-1、CD133和GLUT-1表达情况（3）研究CD133+喉癌细胞中Glut-1表达对喉癌放射敏感性影响及机理.结果：（1）CD133未检测到，GLUT-1与喉癌的预后差有关；（3）在10Gy照射下GLUT-1、CD133表达较对照组增高(p﹤0.05)，在反义GLUT-1抑制GLUT-1、CD133表达较对照组及10Gy照射组下降；（3）体外研究发现siRNA-GLUT1可以提高CD133+ Hep2对放射线的敏感性，随着放射剂量的增加，对CD133+ Hep2细胞增殖的抑制作用增强。转染前，CD133+ Hep-2细胞对于X射线不敏感，在4Gy照射时产生放射抵抗，转染后CD133+ Hep-2细胞凋亡率增高明显；在转染siRNA-GLUT1前，4Gy X射线增加CD133+ Hep2细胞侵袭能力，转染siRNA-GLUT1之后，X射线辐照后细胞侵袭能力明显降低；siRNA-GLUT-1转染CD133+ Hep-2细胞后能提高其放射敏感性；成功制备喉癌Hep-2放射抵抗细胞Hep-2R; 各剂量射线照射并不能有效抑制CD133+Hep2R细胞增殖；转染siRNA-GLUT1后，CD133+Hep2R细胞增殖受到抑制；在siRNA-GLUT1的作用下，12Gy抑制CD133+Hep2R细胞增殖、细胞凋亡; 在转染siRNA-GLUT1前，8Gy量的X射线增加CD133+ Hep2R细胞侵袭能力，转染siRNA-GLUT1之后，X射线辐照后细胞侵袭能力明显降低；转染siRNA-GLUT1后，GLUT1转录和蛋白表达明显降低，X射线组均明显降低；（4）成功建立喉癌Hep-2、Hep-2R细胞接种裸鼠移植瘤模型，CD133+-Hep2细胞敲降GLUT1组肿瘤大小、体重均显著低于CD133+-Hep2R细胞敲降GLUT1组，表现在凋亡增加，GLUT-1表达下降。.结论：喉癌的放射抵抗或不敏感与喉癌CD133+细胞GLUT-1表达增高有关，靶向抑制CD133+细胞GLUT-1表达能增强其放射敏感性。