摘要
目的利用RNA干扰技术建立稳定敲低OPTN蛋白的人SKOV3细胞株和利用CRISPR/Cas 9基因编辑技术建立稳定敲除OPTN蛋白的人SKOV3细胞株,并研究降低OPTN基因表达对细胞抗癌药物敏感性的影响。方法针对OPTN基因设计shRNA和sgRNA的序列,并与慢病毒载体质粒pGPU 6_GFP_Neo和敲除质粒PX 459载体连接产生重组载体。并将重组的质粒测序验证,将测序成功的重组质粒载体与包装质粒(psPAX 2、pMD 2.G)共转染293 T细胞进行病毒包装,收集病毒上清液并感染SKOV3细胞,获得稳定敲低和稳定敲除OPTN基因的SKOV3细胞株。RT-PCR检测敲低组OPTN基因的表达的变化,PCR检测敲除组的基因组DNA的变化,Western blot法分析检测OPTN蛋白的表达情况。MTT法检测DDP对卵巢癌细胞增殖的影响,qRT-PCR检测caspase3、Bax、Bcl-2的mRNA的表达情况,Western blot检测caspase3、Bax、和Bcl-2蛋白的表达情况。结果成功构建慢病毒载体和基因敲除载体,在SKOV3细胞中的OPTN蛋白表达降低(敲减细胞株)或完全敲除(敲除细胞株)。OPTN基因被敲减和敲除的卵巢癌细胞(SKOV3)对顺铂(DDP)的敏感性降低;与对照组相比,DDP对细胞抑制率降低(P<0.05,P<0.01),基因敲除的细胞效果更好。通过qRT-PCR和Western blot检测稳定敲减和敲除OPTN蛋白的SKOV3细胞其凋亡相关基因caspase3、Bax的mRNA和蛋白的表达量降低,而Bcl-2的表达量上升(P<0.05,P<0.01)。结论在卵巢癌中降低或去除OPTN基因表达,能够降低卵巢癌细胞对抗癌药物DDP的敏感性,OPTN可能通过影响凋亡相关途径实现的。
Objective To investigate the effect of reducing OPTN gene expression on the sensitivity of cells to anti-cancer drugs by establishing the human SKOV3 cell line stably knocking down OPTN protein with RNA interference technique and human SKOV3 cell line stably knockout OPTN protein with CRISPR/Cas9 gene editing technique.Methods The sequences of shRNA and sgRNA were designed for OPTN gene and ligated with lentiviral vector plasmid pGPU6_GFP_Neo and knockout plasmid PX459 vector to produce recombinant vector.The recombinant plasmid vector and packaging plasmids(psPAX2 and pMD2.G)were co-transfected into 293T cells for virus pack-aging.The supernatant of virus was collected to infect SKOV3 cells.SKOV3 cell line with stable knockdown and stable knockout of OPTN gene was obtained.RT-PCR was used to detect the changes of OPTN gene expression in knockdown group.PCR was used to detect the changes of genomic DNA in the knockout group.The expression of OPTN protein was detected by Western blot.The effect of DDP on the proliferation of ovarian cancer cells was de-tected by MTT.The expression of mRNA of caspase3,Bax and Bcl-2 was detected by qRT-PCR.The expression of caspase3,Bax and Bcl-2 proteins was detected by Western blot.Results Lentiviral vector and gene knockout vec-tor were constructed successfully.In SKOV3 cells,the expression of OPTN protein in SKOV3 cells decreased(knockout cell line)or knocked out completely(knockout cell line).Ovarian cancer cells(SKOV3)with OPTN gene knockdown and knockout were less sensitive to DDP.Compared with the control group,the inhibition rate of DDP on cells was lower(P<0.05,P<0.01),and the effect of gene knockout cells was better.The expression of apoptosis-related genes Caspase3 and Bax mRNA and protein decreased,while the expression of Bcl-2 increased in SKOV3 cells with stable knockdown and knockout of OPTN protein by qRT-PCR and Western blot(P<0.05,P<0.01).Conclusion Reducing or removing the expression of OPTN gene in ovarian cancer can reduce the sensitiv-ity of ovarian cancer cells to the anticancer drug DDP,which may be achieved by affecting apoptosis-related path-ways.
作者
王鹏
陈曦
徐恰
刘会
郭若文
刘芸
许尹
秦宜德
Wang Peng;Chen Xi;Xu Qia(Dept of Biochemistry and Molecular Biology,School of Basic Medical Science,Anhui Medical University,Hefei 230032;2Dept of Immunology School of Basic Medical Science,Anhui Medical University,Hefei 230032)
出处
《安徽医科大学学报》
CAS
北大核心
2020年第9期1315-1320,共6页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81472448、81601107)。
