脂质体介导的VEGF-C siRNA治疗肺癌体外实验研究
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摘要
目的:肺癌是我国高发的呼吸系统恶性肿瘤,通常与体内某些基因突变导致细胞周期改变和凋亡抑制有关。近年来的很多实验研究证明了VEGF-C在肿瘤的发生发展中起重要作用。本研究探讨通过体外合成靶向VEGF-C mRNA的小分子干扰RNA(short interfering RNA,siRNA)经脂质体介导转染人肺癌A-549细胞株,观察对VEGF-C mRNA和VEGF-C蛋白表达的影响及对细胞生长和凋亡的作用。
方法:设计合成VEGF-C编码基因序列特异小分子干扰RNA通过脂质体介导转染肺癌A-549细胞来沉默VEGF-C的表达。用荧光显微镜及倒置显微镜观察转染效率及转染后细胞形态学的变化;通过MTT实验方法,检测实验浓度的单纯siRNA及脂质体对A-549细胞的抑制作用,观察靶向的VEGF-C siRNA经脂质体介导体外感染肺腺癌A-549细胞生长抑制作用,并绘制细胞生长曲线。用半定量逆转录聚合酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)和蛋白免疫印迹法(western blotting)测定VEGF-C mRNA和蛋白的表达。用流式细胞术检测VEGF-C siRNA对A-549细胞凋亡的影响。应用透射电镜观察外源性VEGF-C siRNA转染A-549细胞后细胞超微结构的变化。
结果:脂质体介导siRNA能有效转染肺腺癌A-549细胞。实验浓度的单纯siRNA及脂质体对A-549细胞无明显毒性。脂质体介导VEGF-C siRNA转染肺癌细胞48h后,实验组细胞贴壁浸润生长能力下降,部分细胞脱落皱缩,细胞形态变圆,体积缩小,折光性增强,空白对照组及实验对照组细胞正常生长。VEGF-C编码基因序列特异siRNA能有效抑制肺腺癌A-549细胞中VEGF-C mRNA和蛋白的表达,与空白对照组及实验对照组比较差异均有统计学意义(P<0.05)。下调VEGF-C表达可以在体外显著抑制A-549细胞的生长及增殖,一定浓度范围内抑制率有浓度依赖性,下调VEGF-C能诱导A-549细胞凋亡增加,转染48h后实验组生长抑制率(38.87%)、细胞凋亡率(19.31±2.21)%显著高于对照组(P<0.05),且在透射电镜下发现转染siRNA后出现典型凋亡细胞,核染色质边集,分布不均匀。
结论:RNA干扰技术能有效抑制VEGF-C mRNA和VEGF-C蛋白的表达,降低肺腺癌A-549细胞增殖能力,诱导其凋亡增加,为靶向VEGF-C siRNA治疗肺癌提供理论支持,RNA干扰作为一种治疗肺癌的手段值得进一步研究及推广。
Objectives: Lung cancer is one of respiratory system malignancies and occurs at a higher incidence in China. Lung cancer usually connects with changes in cell cycle and apoptosis mechanisms caused by some gene mutation. Recently many experiments have shown that VEGF-C plays a very important role in tumor occurring and progress. Our object is to evaluate the application of RNA interference in the study of VEGF-C gene in Lung cancer cell line A-549. Observe the effects of VEGF-C silencing on Lung cancer cell line A-549 growth suppression, apoptosis and expression of VEGF-C and VEGF-C mRNA.
Methods:Designed the specific small interference RNA (siRNA) of the gene sequence of VEGF-C and transfected into A-549 lung cancer cells using liposomes LipofectamineTM2000. The efficiency of infection of A-549 cells transfected with VEGF-C siRNA in vitro was observed by the fluorescence microscope. The change of cell morphology was observed by invert microscope. The toxicity and inhibition effect of VEGF-C siRNA for A-549 cells proliferation was determined by MTT assay. The curve of growth. was drawn . mRNA and protein levels of VEGF-C were detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. Flow cytometry was used to study apoptosis after transfected VEGF-C siRNA. The ultrastructure changes of A549 cells transfected with VEGF-C siRNA was observed by the electron microscopy.
Results: The specific siRNA of the gene sequence of VEGF-C can transfect A-549 cells efficiently. The density of siRNA and liposomes for experiments have no toxicity for A-549 cells. The invert light microscopy demonstrated that A-549 cells transfected with VEGF-C siRNA appeared inhibitory effects including float growth, volum lessen and high photonasty. The VEGF-C siRNA can suppress the VEGF-C mRNA and protein expression efficiently and stably. The results also revealed that down regulation of VEGF-C resulted in significantly inhibition growth and proliferation of A-549 cells in vitro. At 48h, after the transfection the rates of growth inhibitory(38.87%) and apoptotic cells[(19.31±2.21)% ]were significantly higher in specific VEGF-C siRNA group than those in the control. The electron microscopy demonstrated that A-549 cells transfected with VEGF-C siRNA appeared typical morphological characteristics of apoptosis including the compaction and margination of the chromosomes, nuclear fragments and formation of apoptotic bodies.
Conclusion: These results provided solid testimony that VEGF-C gene was vulnerable to RNA interference and, selective inhibition of VEGF-C had anti-proliferation effect on A-549 cells. Inhibition of the expression of VEGF-C can increase apoptosis of A-549 cells. RNA interference could be a powerful tool in further investigations of VEGF-C and a novel therapeutic strategy for lung cancer patients.
方法:设计合成VEGF-C编码基因序列特异小分子干扰RNA通过脂质体介导转染肺癌A-549细胞来沉默VEGF-C的表达。用荧光显微镜及倒置显微镜观察转染效率及转染后细胞形态学的变化;通过MTT实验方法,检测实验浓度的单纯siRNA及脂质体对A-549细胞的抑制作用,观察靶向的VEGF-C siRNA经脂质体介导体外感染肺腺癌A-549细胞生长抑制作用,并绘制细胞生长曲线。用半定量逆转录聚合酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)和蛋白免疫印迹法(western blotting)测定VEGF-C mRNA和蛋白的表达。用流式细胞术检测VEGF-C siRNA对A-549细胞凋亡的影响。应用透射电镜观察外源性VEGF-C siRNA转染A-549细胞后细胞超微结构的变化。
结果:脂质体介导siRNA能有效转染肺腺癌A-549细胞。实验浓度的单纯siRNA及脂质体对A-549细胞无明显毒性。脂质体介导VEGF-C siRNA转染肺癌细胞48h后,实验组细胞贴壁浸润生长能力下降,部分细胞脱落皱缩,细胞形态变圆,体积缩小,折光性增强,空白对照组及实验对照组细胞正常生长。VEGF-C编码基因序列特异siRNA能有效抑制肺腺癌A-549细胞中VEGF-C mRNA和蛋白的表达,与空白对照组及实验对照组比较差异均有统计学意义(P<0.05)。下调VEGF-C表达可以在体外显著抑制A-549细胞的生长及增殖,一定浓度范围内抑制率有浓度依赖性,下调VEGF-C能诱导A-549细胞凋亡增加,转染48h后实验组生长抑制率(38.87%)、细胞凋亡率(19.31±2.21)%显著高于对照组(P<0.05),且在透射电镜下发现转染siRNA后出现典型凋亡细胞,核染色质边集,分布不均匀。
结论:RNA干扰技术能有效抑制VEGF-C mRNA和VEGF-C蛋白的表达,降低肺腺癌A-549细胞增殖能力,诱导其凋亡增加,为靶向VEGF-C siRNA治疗肺癌提供理论支持,RNA干扰作为一种治疗肺癌的手段值得进一步研究及推广。
Objectives: Lung cancer is one of respiratory system malignancies and occurs at a higher incidence in China. Lung cancer usually connects with changes in cell cycle and apoptosis mechanisms caused by some gene mutation. Recently many experiments have shown that VEGF-C plays a very important role in tumor occurring and progress. Our object is to evaluate the application of RNA interference in the study of VEGF-C gene in Lung cancer cell line A-549. Observe the effects of VEGF-C silencing on Lung cancer cell line A-549 growth suppression, apoptosis and expression of VEGF-C and VEGF-C mRNA.
Methods:Designed the specific small interference RNA (siRNA) of the gene sequence of VEGF-C and transfected into A-549 lung cancer cells using liposomes LipofectamineTM2000. The efficiency of infection of A-549 cells transfected with VEGF-C siRNA in vitro was observed by the fluorescence microscope. The change of cell morphology was observed by invert microscope. The toxicity and inhibition effect of VEGF-C siRNA for A-549 cells proliferation was determined by MTT assay. The curve of growth. was drawn . mRNA and protein levels of VEGF-C were detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot. Flow cytometry was used to study apoptosis after transfected VEGF-C siRNA. The ultrastructure changes of A549 cells transfected with VEGF-C siRNA was observed by the electron microscopy.
Results: The specific siRNA of the gene sequence of VEGF-C can transfect A-549 cells efficiently. The density of siRNA and liposomes for experiments have no toxicity for A-549 cells. The invert light microscopy demonstrated that A-549 cells transfected with VEGF-C siRNA appeared inhibitory effects including float growth, volum lessen and high photonasty. The VEGF-C siRNA can suppress the VEGF-C mRNA and protein expression efficiently and stably. The results also revealed that down regulation of VEGF-C resulted in significantly inhibition growth and proliferation of A-549 cells in vitro. At 48h, after the transfection the rates of growth inhibitory(38.87%) and apoptotic cells[(19.31±2.21)% ]were significantly higher in specific VEGF-C siRNA group than those in the control. The electron microscopy demonstrated that A-549 cells transfected with VEGF-C siRNA appeared typical morphological characteristics of apoptosis including the compaction and margination of the chromosomes, nuclear fragments and formation of apoptotic bodies.
Conclusion: These results provided solid testimony that VEGF-C gene was vulnerable to RNA interference and, selective inhibition of VEGF-C had anti-proliferation effect on A-549 cells. Inhibition of the expression of VEGF-C can increase apoptosis of A-549 cells. RNA interference could be a powerful tool in further investigations of VEGF-C and a novel therapeutic strategy for lung cancer patients.
引文
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