lncRNA HOTAIR对肝癌细胞HCCLM3放射敏感性的影响  

Effect of lncRNA HOTAIR on the radiosensitivity of HCCLM3 cells

作　　者：翟金俊[1] 杜贤荣[2] 李彩霞[1] Zhai Jinjun;Du Xianrong;Li Caixia(Emergency Department,Shanxi People′s Hospital,Taiyuan 030000,China;Oncology Department,Shanxi People′s Hospital,Taiyuan 030000,China)

机构地区：[1]山西省人民医院急诊科,太原030000 [2]山西省人民医院肿瘤科,太原030000

出　　处：《中华医学杂志》2020年第18期1419-1425,共7页National Medical Journal of China

摘　　要：目的探讨下调长链非编码RNA(lncRNA)HOX转录反义RNA(HOTAIR)靶向微小RNA761(miR-761)对肝癌细胞HCCLM3放射敏感性的影响。方法实时荧光定量PCR测定HOTAIR在肝癌细胞HuH-7、SNU-449、HCCLM3和正常肝细胞L-02中的表达差异。HCCLM3细胞分成空白对照、Sh-NC(转染shRNA阴性对照)、Sh-HOTAIR(转染HOTAIR shRNA)、RAD+Sh-NC(转染shRNA阴性对照并经8Gy剂量照射)、RAD+Sh-HOTAIR(转染HOTAIR shRNA并经8Gy剂量照射),流式细胞术检测细胞凋亡,Western印迹检测Bcl-2相关X蛋白(Bax)、C-Caspase-3蛋白表达。Sh-NC、Sh-HOTAIR细胞经过0、2、4、6、8 Gy照射处理,平板克隆实验测定放射敏感性。生物信息学软件预测miR-761可能为HOTAIR的靶基因,荧光素酶报告系统鉴定靶向关系。将miR-761抑制物、HOTAIR shRNA以及抑制物阴性对照、HOTAIR shRNA分别共转染到HCCLM3细胞中,同样使用上述方法测定细胞凋亡和Bax、C-含半胱氨酸的天冬氨酸蛋白水解酶-3蛋白表达和细胞存活分数。结果肝癌细胞HuH-7、SNU-449、HCCLM3中HOTAIR表达水平均高于正常肝细胞L-02(1.85±0.12、2.27±0.23、2.68±0.15比1.00±0.09,均P<0.05)。与Sh-NC比较,Sh-HOTAIR、RAD+Sh-NC细胞凋亡率和Bax、C-Caspase-3蛋白水平较高[凋亡率:(13.47±1.32)%、(12.84±1.19)%比(2.98±0.27)%;Bax蛋白:0.74±0.08、0.72±0.06比0.42±0.06;C-Caspase-3蛋白:0.56±0.06、0.54±0.08比0.25±0.04;均P<0.05]。与Sh-HOTAIR、RAD+Sh-NC比较,RAD+Sh-HOTAIR细胞凋亡率和Bax、C-Caspase-3蛋白水平较高[凋亡率:(22.57±2.36)%比(13.47±1.32)%、(12.84±1.19)%;Bax蛋白:0.99±0.11比0.74±0.08、0.72±0.06;C-Caspase-3蛋白:1.03±0.12比0.56±0.06、0.54±0.08;均P<0.05]。与Sh-NC比较,Sh-HOTAIR细胞存活分数较低,细胞放射敏感性较高(P<0.05)。HOTAIR靶向负调控miR-761表达。与共转染抑制物阴性对照、HOTAIR shRNA的细胞比较,共转染miR-761抑制物、HOTAIR shRNA的细胞经过放射处理后,细胞凋亡率较低[(10.24±1.32)%比(21.84±2.01)%],细�Objective To investigate the effect of down-regulating long non-coding RNA(lncRNA)and HOX transcript antisense RNA(HOTAIR)targeting miR-761 on the radiosensitivity of HCCLM3.Methods The expression of HOTAIR in liver cancer cells HuH-7,SNU-449,HCCLM3 and normal liver cells L-02 were measured by real-time quantitative PCR.HCCLM3 cells were divided into control,Sh-NC(transfected shRNA negative control),Sh-HOTAIR(transfected HOTAIR shRNA),RAD+Sh-NC(transfected shRNA negative control and irradiated with 8Gy dose),and RAD+Sh-HOTAIR(HOTAIR shRNA was transfected and irradiated with 8Gy dose)group.Apoptosis was detected by flow cytometry,Bcl-2 Associated X Protein(Bx-2),cleaved cysteine-containing Cleaved cysteinyl aspartate specific proteinase 3(C-Caspase-3)protein expression.Sh-NC,Sh-HOTAIR cells were irradiated with 0,2,4,6,8 Gy,and plate-clone experiments were used to determine radiosensitivity.Bioinformatics software predicted that miR-761 might be a target gene of HOTAIR,and the luciferase reporter system identified the targeting relationship.The miR-761 inhibitor,HOTAIR shRNA and inhibitor negative control,and HOTAIR shRNA were co-transfected into HCCLM3 cells,respectively.Cell apoptosis and Bax and C-cysteine-containing aspartate proteins were also measured using the above method,as well as the hydrolase-3 protein expression and cell survival fraction.Results The expression levels of HOTAIR in liver cancer cells HuH-7,SNU-449,and HCCLM3 were higher than those in normal liver cells L-02(1.85±0.12,2.27±0.23,2.68±0.15 vs 1.00±0.09,P<0.05).Compared with Sh-NC,the apoptosis rate of Sh-HOTAIR,RAD+Sh-NC cells and Bax,C-Caspase-3 protein levels are higher[Apoptotic rate:(13.47±1.32)%,(12.84±1.19)%vs(2.98±0.27)%;Bax protein:0.74±0.08,0.72±0.06 vs 0.42±0.06;C-Caspase-3 protein:0.56±0.06,0.54±0.08 vs 0.25±0.04,all P<0.05].Compared with Sh-HOTAIR and RAD+Sh-NC,RAD+Sh-HOTAIR cell apoptosis rate and Bax,C-Caspase-3 protein levels are higher[apoptosis rate:(22.57±2.36)%vs(13.47±1.32)%,(12.84±1.19)%,Bax protein:0.

关 键 词：HOX转录反义RNA 肝细胞癌 放射敏感性 微小RNA761 

分 类 号：R735[医药卫生—肿瘤]