摘要
目的研究短发夹RNA(short hairpin RNA,shRNA)沉默结肠癌相关转录因子2(colon cancer associated transcript 2,CCAT2)对非小细胞肺癌耐顺铂(cisplatin,DDP)细胞株(A549/DDP)的增殖、侵袭和凋亡以及体内肿瘤形成的影响。方法shRNA-CCAT2(sh-CCAT2)或shRNA-阴性对照(shRNA-NC)转染A549/DDP细胞(并设未经处理的A549/DDP细胞为对照组),通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)检测3组A549/DDP细胞CCAT2 mRNA表达;通过MTT实验检测3组A549/DDP细胞经过不同质量浓度(0~8 mg/L)DDP处理后的增殖能力变化,并依此选定2 mg/L DDP进行后续实验。采用克隆形成实验、流式细胞法、Transwell实验检测2 mg/L DDP处理对各组细胞(并设未经处理的A549/DDP细胞为对照组)增殖、凋亡、侵袭的影响,用Western blot检测各组细胞增殖标记蛋白〔Ki67蛋白、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)〕、凋亡标记蛋白(Caspase-3、Caspase-9)、侵袭标记蛋白〔血管内皮生长因子(vascular endothelial growth factor,VEGF)、基质金属蛋白酶-14(matrix metalloproteinase-14,MMP-14)〕蛋白的表达。裸鼠皮下注射A549/DDP细胞、转染shRNA-NC和转染sh-CCAT2的A549/DDP细胞,每3 d腹腔注射DDP 2 mg/kg,连续给药7次,另设对照组(皮下注射A549/DDP细胞,腹腔注射等量生理盐水)。检测每5 d肿瘤体积,共30 d。30 d后处死取肿瘤组织,RT-PCR检测CCAT2 mRNA表达,TUNEL染色检测肿瘤细胞凋亡。结果与对照组和shRNA-NC转染组相比,sh-CCAT2转染A549/DDP细胞后,细胞CCAT2 mRNA表达降低(P<0.01),2~8 mg/L DDP作用后细胞增殖抑制更明显(P<0.01)。与对照组相比,DDP处理的3组细胞克隆形成减少,增殖标记蛋白Ki67和PCNA表达减少(P<0.01);细胞凋亡率增加,凋亡标记蛋白Caspase-3和Caspase-9表达增多(P<0.01);细胞侵袭数目减少,侵袭标记蛋白VEGF和MMP-14表达降低(P<0.01);DDP处理的3组细胞中,sh-CCAT 2转染的细胞上述表现最为明显(P<0.01)。体内实验显示,与对照组相比,3个DDP组肿瘤体积减小,30 d时差异有统计学意义(P<0.01);肿瘤组织CCAT2 mRNA表达减少(P<0.01),细胞凋亡增加(P<0.01)。3个DDP组中,接种sh-CCAT2转染的A549/DDP细胞组上述表现最为明显(P<0.01)。结论sh-CCAT2可以抑制A549/DDP细胞的增殖、诱导细胞凋亡和降低细胞的侵袭能力,从而抑制A549/DDP细胞生长。
Objective To investigate the effects of short hairpin RNA(shRNA)on the proliferation,invasion,apoptosis and tumor formation of non-small cell lung cancer cisplatin-resistant cell line(A549/DDP)via silencing of colon cancer associated transcript 2(CCAT2).Methods TA549/DDP cells were transfected with shRNA-CCAT2(sh-CCAT2)or shRNA-negative control(shRNA-NC),and untransfected A549/DDP cells were used as the control group.CCAT2 mRNA expression in three groups of A549/DDP cells was detected by quantitative real-time PCR(qRT-PCR).The proliferation of three groups of A549/DDP cells treated with different mass concentrations of DDP(0-8 mg/L)was detected by MTT.According to the proliferation experiment results,2 mg/L was selected as DDP concentration for subsequent experiments.The effects of 2 mg/L DDP treatment on the proliferation,apoptosis,and invasion ability of each group of cells(with untreated A549/DDP cells as the control group)were tested by clone formation experiments,flow cytometry analysis and Transwell experiments.The expression levels of cell proliferation marker proteins(Ki67,PCNA),apoptosis marker proteins(Caspase-3,Caspase-9)and invasion marker proteins(VEGF,MMP-14)were detected by Western blot.Nude mice were injected subcutaneously with A549/DDP cells,A549/DDP cells transfected with shRNANC or A549/DDP cells transfected with sh-CCAT2.DDP was intraperitoneally injected at the concentration of 2 mg per kilogram of mice body weight totally for 7 times with an interval of 3 d.A control group was injected subcutaneously with A549/DDP cells,and an equal volume of normal saline instead of DDP was injected intraperitoneally.The tumor volume was detected every 5 d for a total of 30 d.Mice were sacrificed and tumor tissues were taken out 30 d later.CCAT2 mRNA expression level in tumor tissues was detected by RT-PCR,and tumor cell apoptosis was detected by TUNEL staining.Results Compared with the control group and the shRNA-NC transfection group,the expression level of CCAT2 mRNA was decreased in sh-CCAT2 transfected A549/DDP cells(P<0.01).The decrease degree of cell proliferation was more pronounced after treating with 2 to 8 mg/L of DDP(P<0.01).Compared with the control group,in the three groups that treated with DDP,the formation of clones and the expression of proliferation marker proteins Ki67 and PCNA were reduce(P<0.01),while the rate of apoptosis and the expression of apoptosis marker proteins Caspase-3 and Caspase-9 were increased(P<0.01).Also,the number of invasion cell and the expression of invasion marker proteins VEGF and MMP-14 were reduced in the three groups that treated with DDP(P<0.01).Among the three groups of DDP-treated cells,the changes in sh-CCAT2 transfected cells was the most obvious(P<0.01).Compared with the control group,the tumor volume of the three DDP treatment groups was smaller and the differences were statistically significant at 30 d(P<0.01).The expression of CCAT2 mRNA was decreased in tumor tissues(P<0.01),while apoptosis increased(P<0.01).Among the three DDP treatment groups,the A549/DDP cell group transfected with sh-CCAT2 showed the most notable changes(P<0.01).Conclusion sh-CCAT2 can inhibit the proliferation of A549/DDP cells,induce apoptosis and reduce the cell invasion ability,thereby inhibiting the growth of A549/DDP cells.
作者
贺亮
杜泽东
王阳
蒙荣钦
赵文武
HE Liang;DU Ze-dong;WANG Yang;MENG Rong-qin;ZHAO Wen-wu(Department of Oncology,Chengdu 363 Hospital,Chengdu 610041,China;Department of Oncology,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2020年第3期312-319,共8页
Journal of Sichuan University(Medical Sciences)
基金
四川省医学会青年创新课题(No.150035)资助。
