慢病毒介导的RNA干扰对人非小细胞肺癌化疗多药耐药性的研究
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摘要
目的研究与肿瘤细胞多药耐药性(MDR)相关的两个基因MDR1、MRP对非小细胞肺癌(NSCLC)的多药耐药性的关系。采用RNA干扰技术(RNAi),构建表达Si-MDR1和Si-MRP的重组慢病毒PTM-Si-MDR1、PTM-Si-MRP,转染人肺癌细胞A549及其耐顺铂细胞株A549/DDP,研究化疗药物对RANi后细胞的杀伤效果,探讨治疗NSCLC的新方法。研究共分三部分:第一部分MDR1、MRP基因SiRNA有效位点的筛选;第二部分Si-MDR1、Si-MRP重组慢病毒载体的构建;第三部分Si-MDR1,Si-MRP逆转A549/DDP细胞耐药性的体外及体内研究。
方法(1)MDR1、MRP基因各设计四个干扰位点,合成SiRNA片段,分别连接质粒载体pSUPER并转染人肺癌细胞A549。48小时后,荧光显微镜观察重组质粒对A549细胞的转染效率,荧光定量PCR检测MDR1、MRP在mRNA水平上的表达,筛选出有效干扰位点用于慢病毒包装。(2)采用PTM、pHelper1.0、pHelper0慢病毒载体系统介导Si-MDR1、Si-MRP的表达。转染293T细胞得到大量重组慢病毒,转染Hela细胞进行病毒滴度测定。重组慢病毒转染宿主细胞A549、A549/DDP两天后,流式细胞仪检测转染效率、荧光定量PCR和Western Blot检测MDR1、MRP在mRNA水平和蛋白水平上的干扰效率。(3)三种化疗药物顺铂(DDP)、阿霉素(ADM)、健择(Gemzer)分别作用A549-Si-MDR1及A549/DDP-Si-MDR1细胞24小时后,荧光显微镜观察EGFP阳性细胞形态变化。(4)PTM、PTM-Si-MDR1、PTM-Si-MRP在体外分别感染A549及A549DDP细胞(MOI=10)1周后,加入不同浓度的化疗药物:Gemzer、ADM、DDP,作用48小时后CCK-8试剂盒检测细胞凋亡情况。(5)建立PTM、PTM-Si-MDR1、PTM-Si-MR稳定转染的A549及A549/DDP细胞株,在BALB/c裸鼠体内建立原位肺癌异种移植模型,给予药物后观察动物体重、肿瘤生长及多处转移情况,全面评价MDR表型。
结果(1)成功设计MDR1、MRP基因的SiRNA干扰片段,并构建MDR1、MRP SiRNA筛选质粒,pSUPER-Si-MDR1、pSUPER-Si-MRP筛选有效干扰位点。(2)成功将构建的筛选质粒转染到人肺癌细胞A549及其耐顺铂细胞株A549/DDP中,转染48小时后,荧光显微镜检测绿色荧光蛋白确定转染效率约47.9%。(3)采用荧光定量PCR检测转染48小时后A549细胞中MDR1、MRP在mRNA水平上的表达情况。结果显示,MDR1、MRP基因得到特异高效的沉默,证明pSUPER-Si-MDR1, pSUPER-Si-MRP质粒构建成功,是有效的SiRNA表达质粒。(4)筛选出的有效干扰位点序列用于慢病毒包装。转染293T细胞进行扩增,荧光显微镜及流式细胞仪检测转染效率,结果证明包装好的慢病毒载体能高效转染293T细胞,转染效率85%,并能稳定表达SiRNA。(5)重组慢病毒转染Hela细胞进行滴度测定,为5×108TU/ml。(6)PTM-Si-MDR1、PTM-Si-MRP均以MOI=10转染A549两天后,流式细胞仪检测转染效率,PTM-Si-MDR1转染效率为77.3%,PTM-Si-MRP转染效率80.8%。(7) Real-time PCR、Western Blot检测转染两天的A549细胞MDR1、MRP在mRNA水平和蛋白水平上的表达,计算MDR1抑制效率为72%,MRP抑制效率为69%,抑制效果明显。(8)化疗药物DDP(50ug/ml)、ADM(1ug/ml)、Gemzer(200ug/ml)作用于A549-Si-MDR1细胞(转染一周)24小时后,明场及荧光显微镜下观察细胞形态,发现细胞皱缩,形态发生变化,出现凋亡现象。(9)不同浓度的DDP、ADM、Gemzer分别作用于RNAi后的细胞A549 ( A549-PTM、A549-PTM-Si-MRP、A549-PTM-Si-MDR1)和A549/DDP(A549/DDP-PTM、A549/DDP-PTM-Si-MRP、A549/DDP-PTM-Si-MDR1),48小时后CCK-8法检测细胞活性,结果显示RNAi后,A549/DDP细胞中,三组药物的Si-MRP、Si-MDR干扰组和对照组相比较,其敏感性有明显提高;A549细胞中MRP、MDR干扰组和对照组对药物的敏感性无明显差异。(10)在肺癌原位异种移植裸鼠模型体内,与PTM空载体转染的A549/DDP耐药株相比,稳定转染PTM-Si-MDR1、PTM-Si-MRP至A549/DDP耐药株,可显著降低原发肿瘤大小及对侧肺转移、远处皮下转移率,完全逆转其MDR表型。
结论我们首次把慢病毒介导的RNAi技术用于人肺癌中多药耐药性基因的研究,并证明它能持久抑制人肺癌细胞A549及其耐顺铂细胞株A549/DDP中的MDR1、MRP基因表达,在细胞、基因及蛋白多个水平,通过体外和体内实验多方面证实以MDR1、MRP作为RNAi的靶基因可完全逆转A549/DDP的MDR表型。本研究为肿瘤尤其是NSCLC的临床化疗提供了实验依据和新的思路,结果提示对于继发性耐药的肿瘤病人,应用RNAi等基因治疗方法辅助化疗药物,极有可能在临床上提高缓解率、增强肿瘤细胞对药物的敏感性,延长化疗后患者的生存期。
Objective Multi-drug resistance gene MDR1 and multi-drug resistance related protein gene MRP are two factors generating multi-drug resistance, which were selected in this research to study the effects of multi-drug resistance in non small cell lung cancer (NSCLC). Using RNA interference, we constructed two recombinants mediated by lentivirus vector PTM-Si-MDR1 and PTM-Si-MRP to express Si-MDR1 and Si-MRP. After transfected these recombinants to human lung cancer cell A549 and DDP-resistant cell A549/DDP, we studied their resistance to Gemzer, ADM, and DDP. We expected to find a novel way to cure NSCLC by our research. This research involved three parts: (1) Screening of efficient interfering sites of Si-MDR1 and Si-MRP. (2) Construction of Si-MDR1 and Si-MRP recombinant lentivirus. (3) Drug resistance of A549-Si-MDR1 and A549-Si-MRP cells.
Methods (1) Four interfering sites of MDR1 and MRP gene were designed respectively, then chemosynthesised SiRNA fragments and transfected to human lung cancer cell A549 linked with vector pSUPER. The transfection efficiency was detected by fluorescence microscope; MDR1 and MRP gene expression in A549 cell were detected by real-time PCR 48 hours after transfection. The efficient sites for packing recombinant lentivirus were screened. (2) Using the lentivirus system of PTM, pHelper1.0 and pHelper2.0 to mediate Si-MDR1 and Si-MRP expression. 293T cells were transfected for amplification and Hela cells were transfected for lentivirus titer determination. After host cells A549 and A549/DDP transfected for 2 days, transfection efficiency was determined by FACS; the suppression of MDR1 and MRP gene in mRNA level and protein level were determined by real-time PCR and Western Blot respectively. (3) A549-Si-MDR1 and A549/DDP-Si- MDR1 cell treated with three drugs (DDP, ADM and Gemzer) for 24 hours, then the morphous of EGFP positive cells were detected by fluorescence microscope. (4)One week after PTM, PTM-Si-MDR1, and PTM-Si-MRP transfected into A549 and A549/DDP cells (MOI=10), the cells treated with DDP, ADM and Gemzer with different dose for 48 hours, then cytoactive was determined using CCK-8 kit. (5) Follwing the establishment of stable transfected A549 and A549/DDP cell lines by PTM, PTM-Si-MDR1, and PTM-Si-MRP lentivirus, the primary lung cancer xenograft model was established in BALB/c nude mice. After administrating chemotheraphy drugs, the MDR phenotype was assessed in vivo by primary tumor weight calculation and multi-site metastasis assessment.
Results (1) Designed SiRNA fragments of MDR1 and MRP genes, constructed screening plasmids pSUPER-Si-MDR1 and pSUPER-Si-MRP to select efficient interfering sites. (2) Transfected the recombinant plasmids to human lung cancer cell A549 and DDP-resistant cell A549/DDP. Transfection efficiency was about 47.9%, determined by fluorescence microscope. (3) Determined MDR1 and MRP gene expression in A549 cell in mRNA level by real-time PCR, 48 hours after transfection. MDR1 and MRP were silenced specificly and efficiently. Thus the plasmid pSUPER-Si-MDR1 and pSUPER-Si-MRP were successful to express SiRNA. (4) Packed two lentivirus vectors using the efficient interfering sites, transfected into 293T cell for amplification. Transfection efficiency was high to 85%, determined by fluorescence microscope and FACS. Results proved that the plasmids could transfect 293T with high efficiency and express SiRNA stably. (5) Recombinant lentivirus titers were determined, being about 5×108TU/ml. (6) A549 and A549/DDP were transfected by PTM-Si-MDR1, PTM-Si-MRP and PTM (blank vector), the transfection efficiencies of PTM-Si-MDR1 and PTM-Si-MRP were 77.3% and 80.8% respectively, determined by FACS. (7) The MDR1 and MRP gene expression in A549 were determined by real-time PCR after transfection 2 days. Suppression efficiencies of MDR1 and MRP were 72% and 69%, which were both obvious. (8) Being treated with three chemotherapy drugs: DDP (50ug/ml), ADM(1ug/ml)and Gemzer (200ug/ml) for 24 hours, A549-SiMDR1 cells (one week after transfection) shrinked and lost morphous, which were typical characters of apoptosis. (9) A549 cells (A549-PTM, A549-Si-MRP, A549-Si-MDR1) and A549/DDPcells (A549/DDP-PTM, A549/DDP-Si-MRP, A549/DDP- Si-MDR1) were treated with DDP, ADM and Gemzer with different doses, and determined cytoactive of each cell group after 48 hours. Results showed that, the sensitivity of A549/DDP to 3 drugs were all boosted. (10) As compared with PTM-transfected A549/DDP group, the primary tumer weight and metastasis ratio of contralater lung and distant organs were all decreased significantly in both the stable-transfected PTM-Si-MDR1 and PTM-Si-MRP A549/DDP group. These date indicated that the MDR phenotype was completely reversed by RNAi technology targeting MDR1 and MRP mRNAs.
Conclutions The lentivirus vector-mediated RNA interference can suppress MDR1 and MRP gene expression remarkably in A549 and DDP-resistant A549/DDP cells to last, and can improve the effects on killing cancer cells. Our research offered experimental prove to raise NSCLC chemotherapy effect clinically, and to prolong the survival time of patients after chemotherapy.
方法(1)MDR1、MRP基因各设计四个干扰位点,合成SiRNA片段,分别连接质粒载体pSUPER并转染人肺癌细胞A549。48小时后,荧光显微镜观察重组质粒对A549细胞的转染效率,荧光定量PCR检测MDR1、MRP在mRNA水平上的表达,筛选出有效干扰位点用于慢病毒包装。(2)采用PTM、pHelper1.0、pHelper0慢病毒载体系统介导Si-MDR1、Si-MRP的表达。转染293T细胞得到大量重组慢病毒,转染Hela细胞进行病毒滴度测定。重组慢病毒转染宿主细胞A549、A549/DDP两天后,流式细胞仪检测转染效率、荧光定量PCR和Western Blot检测MDR1、MRP在mRNA水平和蛋白水平上的干扰效率。(3)三种化疗药物顺铂(DDP)、阿霉素(ADM)、健择(Gemzer)分别作用A549-Si-MDR1及A549/DDP-Si-MDR1细胞24小时后,荧光显微镜观察EGFP阳性细胞形态变化。(4)PTM、PTM-Si-MDR1、PTM-Si-MRP在体外分别感染A549及A549DDP细胞(MOI=10)1周后,加入不同浓度的化疗药物:Gemzer、ADM、DDP,作用48小时后CCK-8试剂盒检测细胞凋亡情况。(5)建立PTM、PTM-Si-MDR1、PTM-Si-MR稳定转染的A549及A549/DDP细胞株,在BALB/c裸鼠体内建立原位肺癌异种移植模型,给予药物后观察动物体重、肿瘤生长及多处转移情况,全面评价MDR表型。
结果(1)成功设计MDR1、MRP基因的SiRNA干扰片段,并构建MDR1、MRP SiRNA筛选质粒,pSUPER-Si-MDR1、pSUPER-Si-MRP筛选有效干扰位点。(2)成功将构建的筛选质粒转染到人肺癌细胞A549及其耐顺铂细胞株A549/DDP中,转染48小时后,荧光显微镜检测绿色荧光蛋白确定转染效率约47.9%。(3)采用荧光定量PCR检测转染48小时后A549细胞中MDR1、MRP在mRNA水平上的表达情况。结果显示,MDR1、MRP基因得到特异高效的沉默,证明pSUPER-Si-MDR1, pSUPER-Si-MRP质粒构建成功,是有效的SiRNA表达质粒。(4)筛选出的有效干扰位点序列用于慢病毒包装。转染293T细胞进行扩增,荧光显微镜及流式细胞仪检测转染效率,结果证明包装好的慢病毒载体能高效转染293T细胞,转染效率85%,并能稳定表达SiRNA。(5)重组慢病毒转染Hela细胞进行滴度测定,为5×108TU/ml。(6)PTM-Si-MDR1、PTM-Si-MRP均以MOI=10转染A549两天后,流式细胞仪检测转染效率,PTM-Si-MDR1转染效率为77.3%,PTM-Si-MRP转染效率80.8%。(7) Real-time PCR、Western Blot检测转染两天的A549细胞MDR1、MRP在mRNA水平和蛋白水平上的表达,计算MDR1抑制效率为72%,MRP抑制效率为69%,抑制效果明显。(8)化疗药物DDP(50ug/ml)、ADM(1ug/ml)、Gemzer(200ug/ml)作用于A549-Si-MDR1细胞(转染一周)24小时后,明场及荧光显微镜下观察细胞形态,发现细胞皱缩,形态发生变化,出现凋亡现象。(9)不同浓度的DDP、ADM、Gemzer分别作用于RNAi后的细胞A549 ( A549-PTM、A549-PTM-Si-MRP、A549-PTM-Si-MDR1)和A549/DDP(A549/DDP-PTM、A549/DDP-PTM-Si-MRP、A549/DDP-PTM-Si-MDR1),48小时后CCK-8法检测细胞活性,结果显示RNAi后,A549/DDP细胞中,三组药物的Si-MRP、Si-MDR干扰组和对照组相比较,其敏感性有明显提高;A549细胞中MRP、MDR干扰组和对照组对药物的敏感性无明显差异。(10)在肺癌原位异种移植裸鼠模型体内,与PTM空载体转染的A549/DDP耐药株相比,稳定转染PTM-Si-MDR1、PTM-Si-MRP至A549/DDP耐药株,可显著降低原发肿瘤大小及对侧肺转移、远处皮下转移率,完全逆转其MDR表型。
结论我们首次把慢病毒介导的RNAi技术用于人肺癌中多药耐药性基因的研究,并证明它能持久抑制人肺癌细胞A549及其耐顺铂细胞株A549/DDP中的MDR1、MRP基因表达,在细胞、基因及蛋白多个水平,通过体外和体内实验多方面证实以MDR1、MRP作为RNAi的靶基因可完全逆转A549/DDP的MDR表型。本研究为肿瘤尤其是NSCLC的临床化疗提供了实验依据和新的思路,结果提示对于继发性耐药的肿瘤病人,应用RNAi等基因治疗方法辅助化疗药物,极有可能在临床上提高缓解率、增强肿瘤细胞对药物的敏感性,延长化疗后患者的生存期。
Objective Multi-drug resistance gene MDR1 and multi-drug resistance related protein gene MRP are two factors generating multi-drug resistance, which were selected in this research to study the effects of multi-drug resistance in non small cell lung cancer (NSCLC). Using RNA interference, we constructed two recombinants mediated by lentivirus vector PTM-Si-MDR1 and PTM-Si-MRP to express Si-MDR1 and Si-MRP. After transfected these recombinants to human lung cancer cell A549 and DDP-resistant cell A549/DDP, we studied their resistance to Gemzer, ADM, and DDP. We expected to find a novel way to cure NSCLC by our research. This research involved three parts: (1) Screening of efficient interfering sites of Si-MDR1 and Si-MRP. (2) Construction of Si-MDR1 and Si-MRP recombinant lentivirus. (3) Drug resistance of A549-Si-MDR1 and A549-Si-MRP cells.
Methods (1) Four interfering sites of MDR1 and MRP gene were designed respectively, then chemosynthesised SiRNA fragments and transfected to human lung cancer cell A549 linked with vector pSUPER. The transfection efficiency was detected by fluorescence microscope; MDR1 and MRP gene expression in A549 cell were detected by real-time PCR 48 hours after transfection. The efficient sites for packing recombinant lentivirus were screened. (2) Using the lentivirus system of PTM, pHelper1.0 and pHelper2.0 to mediate Si-MDR1 and Si-MRP expression. 293T cells were transfected for amplification and Hela cells were transfected for lentivirus titer determination. After host cells A549 and A549/DDP transfected for 2 days, transfection efficiency was determined by FACS; the suppression of MDR1 and MRP gene in mRNA level and protein level were determined by real-time PCR and Western Blot respectively. (3) A549-Si-MDR1 and A549/DDP-Si- MDR1 cell treated with three drugs (DDP, ADM and Gemzer) for 24 hours, then the morphous of EGFP positive cells were detected by fluorescence microscope. (4)One week after PTM, PTM-Si-MDR1, and PTM-Si-MRP transfected into A549 and A549/DDP cells (MOI=10), the cells treated with DDP, ADM and Gemzer with different dose for 48 hours, then cytoactive was determined using CCK-8 kit. (5) Follwing the establishment of stable transfected A549 and A549/DDP cell lines by PTM, PTM-Si-MDR1, and PTM-Si-MRP lentivirus, the primary lung cancer xenograft model was established in BALB/c nude mice. After administrating chemotheraphy drugs, the MDR phenotype was assessed in vivo by primary tumor weight calculation and multi-site metastasis assessment.
Results (1) Designed SiRNA fragments of MDR1 and MRP genes, constructed screening plasmids pSUPER-Si-MDR1 and pSUPER-Si-MRP to select efficient interfering sites. (2) Transfected the recombinant plasmids to human lung cancer cell A549 and DDP-resistant cell A549/DDP. Transfection efficiency was about 47.9%, determined by fluorescence microscope. (3) Determined MDR1 and MRP gene expression in A549 cell in mRNA level by real-time PCR, 48 hours after transfection. MDR1 and MRP were silenced specificly and efficiently. Thus the plasmid pSUPER-Si-MDR1 and pSUPER-Si-MRP were successful to express SiRNA. (4) Packed two lentivirus vectors using the efficient interfering sites, transfected into 293T cell for amplification. Transfection efficiency was high to 85%, determined by fluorescence microscope and FACS. Results proved that the plasmids could transfect 293T with high efficiency and express SiRNA stably. (5) Recombinant lentivirus titers were determined, being about 5×108TU/ml. (6) A549 and A549/DDP were transfected by PTM-Si-MDR1, PTM-Si-MRP and PTM (blank vector), the transfection efficiencies of PTM-Si-MDR1 and PTM-Si-MRP were 77.3% and 80.8% respectively, determined by FACS. (7) The MDR1 and MRP gene expression in A549 were determined by real-time PCR after transfection 2 days. Suppression efficiencies of MDR1 and MRP were 72% and 69%, which were both obvious. (8) Being treated with three chemotherapy drugs: DDP (50ug/ml), ADM(1ug/ml)and Gemzer (200ug/ml) for 24 hours, A549-SiMDR1 cells (one week after transfection) shrinked and lost morphous, which were typical characters of apoptosis. (9) A549 cells (A549-PTM, A549-Si-MRP, A549-Si-MDR1) and A549/DDPcells (A549/DDP-PTM, A549/DDP-Si-MRP, A549/DDP- Si-MDR1) were treated with DDP, ADM and Gemzer with different doses, and determined cytoactive of each cell group after 48 hours. Results showed that, the sensitivity of A549/DDP to 3 drugs were all boosted. (10) As compared with PTM-transfected A549/DDP group, the primary tumer weight and metastasis ratio of contralater lung and distant organs were all decreased significantly in both the stable-transfected PTM-Si-MDR1 and PTM-Si-MRP A549/DDP group. These date indicated that the MDR phenotype was completely reversed by RNAi technology targeting MDR1 and MRP mRNAs.
Conclutions The lentivirus vector-mediated RNA interference can suppress MDR1 and MRP gene expression remarkably in A549 and DDP-resistant A549/DDP cells to last, and can improve the effects on killing cancer cells. Our research offered experimental prove to raise NSCLC chemotherapy effect clinically, and to prolong the survival time of patients after chemotherapy.
引文
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