敲低circ_0000620表达对肺腺癌A549细胞凋亡和顺铂敏感性的影响

Effects of silencing circ_0000620 expression on apoptosis and cisplatin sensitivity of lung adenocarcinoma A549 cells

目的:探讨敲低circ_0000620表达对肺腺癌A549和顺铂耐药A549(A549/DDP)细胞凋亡和顺铂敏感性的影响。方法:设计针对circ_0000620的siRNA靶序列,分别构建敲低circ_0000620表达的A549和A549/DDP细胞株(实验组),并构建感染sh-NC(阴性对照)的细胞株(对照组),qRT-PCR检测circ_0000620表达量,验证敲低效果。敲低circ_0000620后,利用CCK-8方法检测顺铂对各组细胞活力的影响,并计算顺铂药物半数抑制浓度(IC_(50));利用Caspase-3/7活性和Tunel方法检测敲低circ_0000620表达后A549和A549/DDP细胞的凋亡率。利用敲低circ_0000620表达后A549和对照A549细胞接种BALB/c裸鼠,7 d成瘤后,腹腔注射顺铂(2 mg/kg)进行药物干预,观察肿瘤的生长曲线,28 d后处死裸鼠,称量瘤重。结果:qRT-PCR筛选得到有效抑制circ_0000620表达的siRNA靶序列(siRNA-2 5’-GAAGAATGATATCCTTGGAAC-3’),并用于后续实验。顺铂敏感性实验结果显示实验组IC_(50)降低,顺铂敏感性增加(P<0.05)。细胞凋亡实验结果显示,敲低细胞circ_0000620表达,实验组细胞Caspase-3/7活性升高,凋亡率升高,顺铂敏感性增加(P<0.05)。顺铂干预裸鼠荷瘤实验结果显示,与对照组相比,实验组瘤体体积和瘤重明显降低(P<0.05)。结论:筛选得到有效抑制circ_0000620表达的siRNA靶序列;敲低circ_0000620的表达可提高细胞对顺铂的敏感性。

Aim:To investigate the effects of circ_0000620 shRNA silencing on cell apoptosis and cisplatin sensitivity of lung adenocarcinoma A549.Methods:A549 and A549/DDP cell lines with knockdown of the expression of circ_0000620 were constructed as experiment groups.At the same time,cell lines infected by sh-NC were used as negative control.qRT-PCR was applied to verify the knockdown efficiency.CCK-8 technique was utilized to assess cell viability and determine the median inhibitory concentration(IC 50)to cisplatin.The apoptosis rate of circ_0000620 knockdown A549 and A549/DDP cells was detected by Caspase-3/7 activity and Tunel method.In the nude mice tumor-bearing model experiment,A549 cells with circ_0000620 knockdown or control were injected into nude BALB/c mice.After 7 days when tumors formed,cisplatin(2 mg/kg)was intraperitoneally injected.The growth curve of the transplanted tumor was observed.The nude mice were sacrificed at 28 days and tumor weight was measured.Results:qRT-PCR discovered that siRNA-2(5’-GAAGAAT GATATCCTTGGAAC-3’)could efficiently inhibit the expression of circ_0000620,which was selected for subsequent experiments.The results of CCK-8 test showed that IC 50 was decreased in experiment group,while cisplatin sensitivity increased(P<0.05).Compared with control groups,both Caspase-3/7 activity and apoptosis rate were increased in response to cisplatin treatment in the experiment groups and the sensitivity of cisplatin was promoted(P<0.05).The tumor volume and weight in the experimental group were dramatically reduced when compared with the control group after cisplatin intervention(P<0.05).Conclusions:An efficient circ_0000620 siRNA sequence has been identified.Silencing the expression of circ_0000620 can promote the sensitivity of A549 cells to cisplatin.