LncRNA LINC00152抵抗替莫唑胺诱导脑胶质瘤细胞凋亡机制探讨

Mechanisms of resistance associated with LncRNA LINC00152 against glioma cells apoptosis induced by Temozolomide

目的LINC00152是异常表达于肝癌、胃癌及结肠癌等恶性肿瘤中的长链非编码RNA(long noncoding RNA,lncRNA),并参与增殖、迁移及凋亡等生物学行为。本研究主要观察LINC00152在替莫唑胺诱导胶质瘤细胞凋亡中的作用及信号机制。方法以脑胶质瘤U251细胞为研究模型,慢病毒转染法外源性上调模型细胞LINC00152表达水平作为转染组,转染空载体作为转染对照组,同时以常规培养的细胞作为空白组;实时荧光定量PCR检测LINC00152表达水平;蛋白质印迹法检测CD133、Nestin、Sox2和Oct-4蛋白表达变化;替莫唑胺诱导模型细胞凋亡,MTT法检测细胞活力变化;结晶紫染色法检测细胞贴壁存活比例;流式细胞术检测细胞凋亡比例。结果空白组、转染对照组和转染组模型细胞中LINC00152RNA相对表达水平分别为0.33±0.02、0.32±0.03和1.58±0.13,与转染对照组相比,转染组LINC00152RNA相对表达水平上调,F=519.308,P<0.001。空白组、转染对照组和转染组模型细胞CD133相对蛋白表达量分别为(5.8±0.4)%、(6.2±0.4)%、(28.8±2.5)%,Nestin相对蛋白表达量分别为(18.7±2.0)%、(20.3±2.0)%和(20.2±1.8)%,Sox2相对蛋白表达量分别为(15.6±1.3)%、(16.7±1.5)%和(50.3±0.2)%,Oct-4相对蛋白表达量分别为(6.1±0.5)%、(5.9±0.6)%和(5.6±0.6)%。与转染对照组相比,转染组细胞CD133(F=237.405)和Sox2(F=879.732)蛋白表达上调,均P<0.001。空白组、转染对照组和转染组模型细胞IC50分别为(47.8±4.2)、(46.3±4.8)、(68.6±7.1)μg/mL,与转染对照组相比,转染组细胞IC50升高,F=15.350,P=0.006。替莫唑胺(68.6μg/mL)处理空白组、转染对照组和转染组模型细胞,各组贴壁存活率为(22.6±3.6)%、(24.8±2.8)%和(48.3±4.2)%,各组总凋亡率分别为(21.9±3.0)%、(20.7±2.6)%和(13.1±1.8)%,与转染对照组相比,转染组细胞贴壁存活率和凋亡率均降低,差异均有统计学意义,均P<0.001。结论LncRNA LINC00152通过维持细胞干性抵抗替莫唑胺诱导的脑胶质瘤U251细胞凋亡。

OBJECTIVE LINC00152 is a long noncoding RNA(LncRNA)that is abnormally expressed in liver,gastric and colon cancers,and is involved in proliferation,migration and apoptosis.In this study,we focused on the role and signaling mechanism of LINC00152 in temozolomide-induced apoptosis in glioma cells.METHODS Glioma U251 cells were used as the study model.The expression level of LINC00152 was up-regulated by lentivirus transfection as the transfection group,the empty vector as the transfection control group and the conventionally cultured cells as the blank group;The expression level of LINC00152 was detected by real-time PCR;the protein expression of CD133,Nestin,Sox2,and Oct-4 was detected by Western blotting assay;apoptosis was induced by temozolomide,and the cell viability was detected by MTT assay;the proportion of cells surviving adherent to the cell wall was detected by crystal violet staining;and the proportion of cells undergoing apoptosis was detected by flow cytometry.RESULTS The relative expression levels of LINC00152 RNA were(0.33±0.02),(0.32±0.03)and(1.58±0.13)in blank group,transfected control group and transfected group,respectively.Compared with the transfected control group,the relative expression level of LINC00152 RNA was up-regulated in the transfected group,F=519.308,P<0.001.In blank group,transfected control group and transfected group,the relative protein expression of CD133 in model cells was(5.8±0.4)%,(6.2±0.4)%and(28.8±2.5)%,the relative protein expression of Nestin was(18.7±2.0)%,(20.3±2.0)%and(20.2±1.8)%,the relative protein expression of Sox2 was(15.6±1.3)%,(16.7±1.5)%and(50.3±0.2)%,and the relative protein expression of Oct-4 was(6.1±0.5)%,(5.9±0.6)%and(5.6±0.6)%.Compared with the transfected control group,CD133(F=237.405)and Sox2(F=879.732)protein expression was significantly up-regulated in the transfected cells,P<0.001.The IC50 of blank group,transfected control group and transfected model cells were(47.8±4.2),(46.3±4.8)and(68.6±7.1)μg/mL,respectively,and compared with transfected control group,the IC50 of transfected cells was significantly higher,F=15.350,P=0.006.Temozolomide(68.6μg/mL)treatment of blank,transfected control and transfected model cells resulted in(22.6±3.6)%,(24.8±2.8)%,(48.3±4.2)%adherence survival rate in each group,and(21.9±3.0)%,(20.7±2.6)%,(13.1±1.8)%total apoptosis rate in each group,respectively.Compared with transfected control group,cell adherence survival rate and apoptosis rate were significantly reduced in transfected group,F=47.51 and F=10.79,P<0.001.CONCLUSION LncRNA LINC00152 resists temozolomide-induced apoptosis in glioma U251 cells by maintaining cell stemness.