lncRNA FOXC2-AS1逆转骨肉瘤细胞对阿霉素耐药性的影响  

Effect of lncRNA FOXC2-AS1 on reversing adriamycin resistance in osteosarcoma cells

作　　者：张义[1] 张擎柱[1] 谷锐[1] 冯震[1] 石利涛[1] 魏俊强[1] 曹向宇[1] 金宇[1] ZHANG Yi;ZHANG Qingzhu;GU Rui;FENG Zhen;SHI Litao;WEI Junqiang;CAO Xiangyu;JIN Yu(Depart-ment of Orthopaedics,Affiliated Hospital of Chengde Medical College,Chengde 067000,China)

机构地区：[1]承德医学院附属医院骨科

出　　处：《临床肿瘤学杂志》2019年第5期385-390,共6页Chinese Clinical Oncology

摘　　要：目的探讨沉默长链非编码RNA FOXC2-AS1(lncRNA FOXC2-AS1)对人骨肉瘤细胞阿霉素(ADM)耐药性的影响及可能作用机制。方法采用ADM长期浓度梯度递增法体外建立ADM耐药细胞株MG-63/ADR,利用实时荧光定量PCR(QPCR)法检测MG-63和MG-63/ADR细胞中FOXC2-AS1的表达水平,采用阴性对照序列和si-FOXC2-AS1序列转染MG-63/ADR细胞作为si-NC组和si-FOXC2-AS1组,另设空白对照组。MTT实验和平板克隆形成实验检测各组细胞对ADM的敏感性,并计算半数抑制浓度(IC50)。Western blotting法检测各组细胞中Notch-1及下游靶因子Hes-1、Hey-1、Hey-2蛋白水平。结果体外成功建立MG-63/ADR耐药细胞株,1、5、25、125、625、3125、15 625 ng/ml ADM对MG-63/ADR细胞的增殖抑制率分别为(5.90±0.48)%、(9.86±0.85)%、(16.75±2.21)%、(29.84±2.98)%、(50.45±4.96)%、(54.82±5.33)%和(57.69±5.76)%,明显低于MG-63细胞。ADM对MG-63和MG-63/ADR的IC50分别为106.55 ng/ml和685.47 ng/ml。MG-63/ADR耐药指数为6.43。si-FOXC2-AS1组FOXC2-AS1的相对表达量为0.44±0.12,低于空白对照组(1.43±0.22)和si-NC组,差异有统计学意义(P<0.05)。1、5、25、125、625、3125和15 625 ng/ml ADM对si-FOXC2-AS1组MG-63/ADR细胞的增殖抑制率分别为(6.36±0.40)%、(13.41±1.05)%、(32.45±2.69)%、(49.73±4.14)%、(64.82±4.67)%、(77.56±5.79)%和(79.50±5.62)%,明显高于空白对照组。转染si-FOXC2-AS1后MG-63/ADR细胞对ADM的敏感性增加了4.25倍。si-FOXC2-AS1组经ADM(100 ng/ml)作用96 h后,MG-63/ADR细胞克隆形成率为(27.64±7.28)%,低于空白对照组的(76.07±17.91)%和si-NC组的(69.83±15.71)%,差异有统计学意义(P<0.05)。si-FOXC2-AS1组MG-63/ADR细胞中Notch-1及其下游分子Hes-1、Hey-1、Hey-2蛋白的表达水平分别为0.67±0.15、0.52±0.10、0.45±0.12和0.38±0.11,均低于空白对照组和si-NC组,差异有统计学意义(P<0.05)。结论沉默lncRNA FOXC2-AS1表达通过抑制Notch-1信号通路的活化,进而增加MG-63/ADR细胞对ADM的敏感性。Objective To investigate the effect of silencing long-chain noncoding RNA FOXC2-AS1(lncRNA FOXC2-AS1) on adriamycin(ADM) resistance in human osteosarcoma cells and its possible mechanism. Methods Adriamycin-resistant cell line MG-63/ADR was established in vitro by ADM long-term concentration gradient increasing method. The expression of FOXC2-AS1 in MG-63 and MG-63/ADR cells was detected by real-time fluorescence quantitative PCR(QPCR). MG-63/ADR cells were transfected with negative control sequence and si-FOXC2-AS1 sequence as si-NC group and si-FOXC2-AS1 group, and blank control group was set up. MTT assay and plate cloning assay were used to detect the sensitivity of cells to ADM, and the half inhibitory concentration(IC50) was calculated. The levels of Notch-1, Hes-1, Hey-1 and Hey-2 were detected by Western blotting. Results MG-63/ADR cell line was successfully obtained in vitro. The inhibition rates of 1,5,25,125,625,3125,15 625 ng/ml ADM on the proliferation of MG-63/ADR cells were(5.90±0.48)%,(9.86±0.85)%,(16.75±2.21)%,(29.84±2.98)%,(50.45±4.96)%,(54.82±5.33)% and(57.69±5.76)% respectively, which were significantly lower than those of MG-63 cells. The IC50 of ADM for MG-63 and MG-63/ADR were 106.55 ng/ml and 685.47 ng/ml, respectively. The resistance index of MG-63/ADR was 6.43. The relative expression of FOXC2-AS1 in the si-FOXC2-AS1 group was 0.44±0.12, which was lower than that in the blank control group(1.43±0.22) and the si-NC group(P<0.05). The inhibition rates of 1,5,25,125,625,3125,15 625 ng/ml ADM on the proliferation of MG-63/ADR cells in si-FOXC2-AS1 group were(6.36±0.40)%,(13.41±1.05)%,(32.45±2.69)%,(49.73±4.14)%,(64.82±4.67)%,(77.56±5.79)% and(79.50±5.62)%, respectively, which were significantly higher than those in the blank control group. The sensitivity of MG-63/ADR cells to ADM increased 4.25 times after transfection of si-FOXC2-AS1. After 96 hours of ADM(100 ng/ml) treatment, the clone formation rate of MG-63/ADR cells in si-FOXC2-AS1 group was(27.64±7.28)%, lower than t

关 键 词：骨肉瘤 长链非编码RNA FOXC2-AS1 Notch-1信号通路 阿霉素耐药 

分 类 号：R738.1[医药卫生—肿瘤]