siRNA介导的RNAi对肿瘤细胞CD59沉默作用的研究
摘要
目的观察siRNA介导的RNA干扰(RNAi)对宫颈癌细胞株HeLa中CD59基因表达的影响以及裸鼠卵巢癌移植瘤的抑瘤效应,探讨CD59在肿瘤细胞免疫逃逸及相关信号转导通路中的作用。
方法设计并合成3对编码CD59 shRNA的单链寡核苷酸,退火后将双链寡核苷酸与线形pSUPER表达载体连接构建成重组质粒,并以无关序列作为对照,脂质体法转染包装细胞系Phoenix A,收集病毒上清,感染HeLa细胞,RT-PCR和Western blots筛选出抑制效率最高的序列siCD59,荧光染料释放试验检测CD59功能的改变;siCD59稳定转染卵巢癌细胞株A2780,并以siCD59-C作为对照,体外观察CD59基因改变后对补体溶破的抵抗作用的变化;流式细胞仪测定活性caspase-3的表达,Hoechst染色测定细胞核的改变;将两种卵巢癌细胞注射到4-5周龄的雌性裸鼠体内建立裸鼠移植瘤模型,动态观测移植瘤生长情况,计算肿瘤生长率,6-8周处死裸鼠取部分移植瘤组织做免疫组织化学染色检测CD59蛋白的表达。
结果成功构建4条重组载体(包括1条对照),发现其中2条(siCD59-T2 andsiCD59-T3)能够有效抑制CD59基因的表达。RT-PCR结果显示转染siCD59-T2和siCD59-T3(siCD59)组mRNA的表达分别下降到原来的42.60%(P＜0.01)和49.23%(P＜0.01)。Western blot检测CD59蛋白的表达量分别下降到原来的51.61%(P＜0.05)和52.18%(P＜0.05)。在1:5,1:10,1:20不同补体稀释度下,siCD59和siCD59-C染料释放率分别为96.03%和40.40%,86.83%和30.97%,66.83%和19.51%,差别有统计学意义。siCD59组活性caspase-3的表达和siCD59-C相比增加了62.42%,且高致密性核、碎裂核比siCD59-C组多201.19%;同时,siCD59组裸鼠移植瘤的重量(0.27+0.092)明显比siCD59-C组(1.57±0.176)轻,抑瘤率为82.80%;且siCD59组阳性染色细胞(296±70)明显少于siCD59-C(814±1 12)组,差别有统计学意义。
结论siRNA介导的RNAi能有效抑制CD59基因的表达,并能增加补体介导的溶细胞活性及诱导细胞凋亡,抑制裸鼠体内卵巢癌移植瘤的生长,为后续进行肿瘤细胞的研究奠定了基础,可望为肿瘤的免疫治疗开辟新途径。
Objective To investigate the effect of retrovirus-mediated RNA interference(RNAi)on the expression of CD59 gene of human cervix cancer cell line HeLa and human ovary cancer cell line A2780,study the effect of cytolysis and apoptosis and the inhibitory effects of xenografts in nude mice.
Methods Three pairs of single-stranded oligo encoding CD59 shRNA sequence were designed and synthesized.After annealing ds oligo were inserted into lined pSUPER expression vectors to construct recombinant vectors.Then three recombinant vectors and one negative-control vector(siCD59-C)were transfected into the packaging cell Phoenix A using liposome.HeLa cells were infected by the virus supernants which contained the four recombinant vectors.CD59 mRNA and protein level was detected by RT-PCR and Western blots.Its function was analysed by dye release assay.The ovary cancer cell A2780 was transfected with siCD59 and siCD59-C using liposome2000 and the stable strains were selected by using G418-medium,the cell viability and cell damage was tested by MTT and LDH release assay,flow cytometry and Hoechst stain was performed to assess the cell apoptosis when incubated with 8%NHS.Eighteen 4-5 week old nude mice were divided randomly into two groups,the xenografts of ovary cancer cells A2780 siCD59 and siCD59-C were established.All mice were sacrificed after 6-8 w.The weights of the tumors were measured and the tumor growth rates were calculated.The distribution of CD59 protein was detected by immunohistochemistry.
Results Recombinant vectors were successfully constructed and transfected. Two of four recombinant vectors(siCD59-T2 and siCD59-T3)could effectively inhibit the expression of CD59 mRNA and protein.The mRNA expression in siCD59-T2 and siCD59-T3 infected HeLa cells was significantly reduced to 42.60%(P＜0.01)and 49.23 %(P＜0.01),respectively.Western blots demonstrated CD59 protein expression was significantly reduced to 51.61%(P＜0.05)and 52.18%(P＜0.05),respectively.When incubated with fresh normal human serum(8%,v/v)for 1 h at 37℃,the dye release assays between siCD59-T3 group and siCD59-C group suggested that the dye release rate was 6.03%/40.40%,86.83%/30.97%,66.83%/19.51%with 1:5,1:10,1:20 complement dilution.There was difference between them,which suggested that CD59's protection against human complement decreased.The difference among siCD59-T1,siCD59-C,and blank control group had no statistical significance(P＞0.05).The cell viability was decreased and cell damage was increased in siCD59 group compared to siCD59-C transfected cells.Furthermore,this led to the activation of caspase-3 and the hypercondensed nuclei using flow cytometry and Hoechst stain.Meanwhile,the weight of ovary tumor graft in nude mice was significantly decreased in siCD59 group(0.27±0.092) compared to the siCD59-C group(1.57±0.176),the inhibited rate was 82.80%.And the stained cell of CD59 protein of tumor tissue in siCD59 group(296±70)was significantly decreased compared to the siCD59-C group(814±112).There was significant difference between them(P＜0.05).
Conclusion Recombinant retroviral vectors and stable virus-producing packaging cell lines were successfully constructed and identified,CD59 silencing by siRNA increased complement-mediated cytolysis and led to cell apoptosis,inhibited growth of xenografts in nude mice,which may pave the way for studying the role of CD59 in the immune escape of tumors cells as well as in tumor therapy.
方法设计并合成3对编码CD59 shRNA的单链寡核苷酸,退火后将双链寡核苷酸与线形pSUPER表达载体连接构建成重组质粒,并以无关序列作为对照,脂质体法转染包装细胞系Phoenix A,收集病毒上清,感染HeLa细胞,RT-PCR和Western blots筛选出抑制效率最高的序列siCD59,荧光染料释放试验检测CD59功能的改变;siCD59稳定转染卵巢癌细胞株A2780,并以siCD59-C作为对照,体外观察CD59基因改变后对补体溶破的抵抗作用的变化;流式细胞仪测定活性caspase-3的表达,Hoechst染色测定细胞核的改变;将两种卵巢癌细胞注射到4-5周龄的雌性裸鼠体内建立裸鼠移植瘤模型,动态观测移植瘤生长情况,计算肿瘤生长率,6-8周处死裸鼠取部分移植瘤组织做免疫组织化学染色检测CD59蛋白的表达。
结果成功构建4条重组载体(包括1条对照),发现其中2条(siCD59-T2 andsiCD59-T3)能够有效抑制CD59基因的表达。RT-PCR结果显示转染siCD59-T2和siCD59-T3(siCD59)组mRNA的表达分别下降到原来的42.60%(P＜0.01)和49.23%(P＜0.01)。Western blot检测CD59蛋白的表达量分别下降到原来的51.61%(P＜0.05)和52.18%(P＜0.05)。在1:5,1:10,1:20不同补体稀释度下,siCD59和siCD59-C染料释放率分别为96.03%和40.40%,86.83%和30.97%,66.83%和19.51%,差别有统计学意义。siCD59组活性caspase-3的表达和siCD59-C相比增加了62.42%,且高致密性核、碎裂核比siCD59-C组多201.19%;同时,siCD59组裸鼠移植瘤的重量(0.27+0.092)明显比siCD59-C组(1.57±0.176)轻,抑瘤率为82.80%;且siCD59组阳性染色细胞(296±70)明显少于siCD59-C(814±1 12)组,差别有统计学意义。
结论siRNA介导的RNAi能有效抑制CD59基因的表达,并能增加补体介导的溶细胞活性及诱导细胞凋亡,抑制裸鼠体内卵巢癌移植瘤的生长,为后续进行肿瘤细胞的研究奠定了基础,可望为肿瘤的免疫治疗开辟新途径。
Objective To investigate the effect of retrovirus-mediated RNA interference(RNAi)on the expression of CD59 gene of human cervix cancer cell line HeLa and human ovary cancer cell line A2780,study the effect of cytolysis and apoptosis and the inhibitory effects of xenografts in nude mice.
Methods Three pairs of single-stranded oligo encoding CD59 shRNA sequence were designed and synthesized.After annealing ds oligo were inserted into lined pSUPER expression vectors to construct recombinant vectors.Then three recombinant vectors and one negative-control vector(siCD59-C)were transfected into the packaging cell Phoenix A using liposome.HeLa cells were infected by the virus supernants which contained the four recombinant vectors.CD59 mRNA and protein level was detected by RT-PCR and Western blots.Its function was analysed by dye release assay.The ovary cancer cell A2780 was transfected with siCD59 and siCD59-C using liposome2000 and the stable strains were selected by using G418-medium,the cell viability and cell damage was tested by MTT and LDH release assay,flow cytometry and Hoechst stain was performed to assess the cell apoptosis when incubated with 8%NHS.Eighteen 4-5 week old nude mice were divided randomly into two groups,the xenografts of ovary cancer cells A2780 siCD59 and siCD59-C were established.All mice were sacrificed after 6-8 w.The weights of the tumors were measured and the tumor growth rates were calculated.The distribution of CD59 protein was detected by immunohistochemistry.
Results Recombinant vectors were successfully constructed and transfected. Two of four recombinant vectors(siCD59-T2 and siCD59-T3)could effectively inhibit the expression of CD59 mRNA and protein.The mRNA expression in siCD59-T2 and siCD59-T3 infected HeLa cells was significantly reduced to 42.60%(P＜0.01)and 49.23 %(P＜0.01),respectively.Western blots demonstrated CD59 protein expression was significantly reduced to 51.61%(P＜0.05)and 52.18%(P＜0.05),respectively.When incubated with fresh normal human serum(8%,v/v)for 1 h at 37℃,the dye release assays between siCD59-T3 group and siCD59-C group suggested that the dye release rate was 6.03%/40.40%,86.83%/30.97%,66.83%/19.51%with 1:5,1:10,1:20 complement dilution.There was difference between them,which suggested that CD59's protection against human complement decreased.The difference among siCD59-T1,siCD59-C,and blank control group had no statistical significance(P＞0.05).The cell viability was decreased and cell damage was increased in siCD59 group compared to siCD59-C transfected cells.Furthermore,this led to the activation of caspase-3 and the hypercondensed nuclei using flow cytometry and Hoechst stain.Meanwhile,the weight of ovary tumor graft in nude mice was significantly decreased in siCD59 group(0.27±0.092) compared to the siCD59-C group(1.57±0.176),the inhibited rate was 82.80%.And the stained cell of CD59 protein of tumor tissue in siCD59 group(296±70)was significantly decreased compared to the siCD59-C group(814±112).There was significant difference between them(P＜0.05).
Conclusion Recombinant retroviral vectors and stable virus-producing packaging cell lines were successfully constructed and identified,CD59 silencing by siRNA increased complement-mediated cytolysis and led to cell apoptosis,inhibited growth of xenografts in nude mice,which may pave the way for studying the role of CD59 in the immune escape of tumors cells as well as in tumor therapy.
引文
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