敲低长链基因间非编码RNA-p21对乏氧肿瘤细胞放射敏感性的影响

Effect of long intergenic noncoding RNA-p21 knockdown on radiosensitivity of hypoxic tumor cells

目的探讨敲低长链基因间非编码RNA-p21（1incRNA-p21）对乏氧肿瘤细胞放射敏感性的影响及机制。方法实时荧光定量PCR（QRT-PCR）检测人肝癌细胞SMMC7721和脑胶质瘤细胞U251MG乏氧培养不同时间lincRNA—p21的表达。构建lincRNA—p21siRNA慢病毒载体．经慢病毒转染建立稳定敲低lincRNA—p21的SMMC7721和U251MG细胞系。流式细胞术检测乏氧肿瘤细胞周期和凋亡。克隆形成实验检测乏氧肿瘤细胞放射敏感性。Westernblot检测乏氧诱导因子1α（HIF-1α）和GLUT1蛋白含量。结果SMMC7721和U251MG细胞乏氧（1％O2）培养24和48h．1incRNA—p21的表达随培养时间的增加逐渐升高。24和48h与0h组相比，lincRNA—p21表达明显升高（t=5．13、7．49、8．90、10．11，P〈0．05）。稳定转染lincRNA—p21siRNA下调乏氧肿瘤细胞中lincRNA—p21的表达（t=144．81、334．32，P〈0．05）。乏氧（1％02）培养48h，敲低lincRNA-p21细胞SMMC7721和U251MG发生G2／M期阻滞（t=7．05、10．18，P〈0．05）；细胞凋亡明显增加（t=42．27、24．79，P〈0．05）；HIF—1α和GLUT1蛋白含量减少，放射敏感性增强，放射增敏比（SER）约为1．23、1．31。结论乏氧促进肿瘤细胞中lincRNA—p21表达。敲低lincRNA-p21增强乏氧肿瘤细胞放射敏感性，其机制可能与敲低lincRNA—p2l诱导细胞G2／M期阻滞和细胞凋亡，并导致HIF-1α蛋白水平下降有关。

Objective To investigate the effect and mechanisms of lincRNA-p21 knockdown on radiosensitivity of hypoxic tumor cells. Methods The expression of lincRNA-p21 in SMMC7721 and U251MG cells after hypoxia treatment were detected by QRT-PCR. Lentivirus vector carrying lincRNA-p21 siRNA or scrambled oligos were constructed. SMMC7721 and U251MG cells with stable integration of lincRNA-p21 siRNA or scrambled sequence were generated through lentiviral-mediated gene transfer. Cell cycle and apoptosis of hypoxic tumor cells were analyzed by flow cytometry. Radiosensitivity of hypoxic tumor cells were measured by cloning formation assay. The expressions of HIF-1α and GLUT1 protein were detected by Western blot. Results Under hypoxia condition （ 1% 02 ） for 24 and 48 h, lincRNA-p21 strongly increased （ t = 5.13, 7.49, 8.90, 10. 11, P 〈 0.05 ） in SMMC7721 and U251MG cells in a time- dependent manner, but this increase was suppressed in the SMMC7721 and U251MG ceils with stable integration of lincRNA-p21 siRNA （ t = 144. 81, 334. 32, P 〈 0. 05）. When SMMC7721 and U251MG cells were cultured under hypoxia condition （ 1% 02 ） for 48 h, knockdown of lineRNA-p21 induced G2/M phase arrest （t=7.05, 10.18, P〈0.05） and apoptosis （t =42.27, 24. 79, P〈0.05）, reduced the expressions of HIF-1α and GLUT1 protein, and enhanced radiosensitivity with a radiosensitization ratio of 1.23 and 1.31, respectively. Conclusions Hypoxia treatment elevate the expression of lincRNA-p21. Knockdown of lineRNA-p21 enhance radiosensitivity of hypoxic tumor cells accompanied with G2/M phase arrest, apoptosis induction and the decrease of HIF-1α protein expression.