摘要
背景与目的肿瘤的生长依赖于血管的新生,抑制肿瘤血管生成已经成为肿瘤治疗的一种策略。VEGF通过与血管内皮细胞膜的VEGF受体(VEGFR)结合,触发信号转导,转导级联反应,引起血管内皮细胞有丝分裂,刺激内皮细胞增殖和血管生成。VEGF/VEGFR通路可特异性促进内皮细胞增殖、参与血管形成、增加血管通透性及保护受损神经元等,不仅在胚胎发育、伤口愈合、炎症、肿瘤等多种生理及病理过程中发挥重要作用,而且参与缺血性脑血管病的损伤及抗损伤过程。VEGF具有增加微血管通透性、促进不同来源的内皮细胞分裂增殖和血管构建、促使内皮细胞迁移等多种作用。大多数肿瘤细胞高水平地表达VEGF,且其表达与肿瘤的恶性程度呈正相关,因此,VEGF及其受体成为目前抗肿瘤血管形成治疗最为成熟的靶分子。
RNA干扰(RNA interference,RNAi)是指由双链RNA(double stranded RNA,dsRNA)引发的转录后基因沉默机制,能够高效特异地沉默同源基因的表达,是一种转录后调控。RNAi技术不仅在功能基因组研究中发挥了重要的作用,而且是一种潜在的以特异性基因沉默为方法治疗疾病的手段。尤其在肿瘤的干预中更具有潜在意义。目前,通过RNAi技术干涉VEGF的表达能够抑制胰腺癌、喉鳞癌、大肠癌和肺癌等细胞株的增殖、生长、侵袭和迁移。
本课题,我们利用RNAi干涉技术,沉默肺癌细胞中VEGF的表达,从而达到干预肺癌生长增殖的目的,为肺癌的基因治疗提供新的理论依据。期望能够获得一个持续高效的抑制肺癌细胞中VEGF表达的RNAi片段,从体外、内观察其对肺癌细胞增殖和肺内癌肿生长的影响,并探讨其干预机制。
材料和方法通过筛选获得理想的能够沉默肺癌细胞中VEGF表达的shRNA片段,并将其插入到载体中,制备成pSilencer-VEGF-shRNA;体外利用该质粒对肺癌A549细胞系进行干预后,动态观察A549细胞中VEGF的表达、肺癌细胞的生长及内皮细胞小管结构的形成;用A549和pSilencer-VEGF-A549细胞分别接种裸鼠,观察荷瘤裸鼠体内肿瘤组织的生长及生存期。使用RT-PCR和免疫印迹的方法检测与内皮细胞相互作用后干涉VEGF的A549细胞内p53、Bcl-2和Bax转录和蛋白水平上的变化。
研究结果经RT-PCR和免疫印迹实验检测发现pSilencer-VEGF-2对肺癌细胞中VEGF的抑制率能够达到90%以上;在稳定转染的肺癌细胞株A549细胞中,2个月以后对VEGF的抑制率仍在80%以上;肺癌细胞生长干预实验结果显示,pSilencer-VEGF-2明显抑制了肺癌A549细胞系中VEGF的表达;虽然稳定转染pSilencer-VEGF-2的肺癌A549细胞株体外生长未发生明显变化,但是,它可以显著抑制内皮细胞的血管生成作用。体内荷瘤裸鼠实验结果显示:与接种对照组荷肺癌A549细胞株瘤的裸鼠相比,pSilencer-VEGF-2-A549荷瘤裸鼠体内肿瘤组织的生长显著减慢(P<0.05),生存期明显延长(P<0.05)。pSilencer-VEGF-2干预肺癌增殖生长的机制研究显示,稳定转染的人肺癌A549细胞株中P53,Bcl-2和Bax的表达无明显改变,也未见到明显的细胞凋亡现象。与对照组相比,在人HUVEC细胞系的作用下,稳定转染的人A549细胞中Bax表达无变化,但P53的表达明显增高,Bcl-2的表达下调,且有细胞凋亡现象。
结论我们筛选构建了理想的、能够有效抑制肺癌VEGF表达的shRNA质粒pSilencer-VEGF-2;该质粒在体外和体内均能够明显抑制肺癌的发展。其机制可能是通过shRNA沉默肺癌细胞VEGF的表达,抑制了血管内皮细胞的VEGF/VEGFR通路,从而引起肺癌细胞凋亡而实现的。
Bacground&Objective Angiogenesis is associated with several pathological disorders. Tumor growth and metastasis are dependent on angiogenesis. Components of vascular basement membrane are involved in regulating angiogenesis. The binding of VEGF to VEGFR expressing on the endothelial cells membrane, which triggered the signaling transduction, resulted in the mitosis and proliferation of endothelial cells, and promoted angiogenesis. The VEGF/VEGFR signaling pathway could enhance specially the penetration of blood vessel and protect the damaged neural cells, which plays an important role in the physiological and pathological process, including the embryonic development, inflammation and tumor. Previous studies showed that most solid tumor cloud secret the VEGF. What’s more, the VEGF expression was related with the malignant tumor development. Therefore, VEGF or VEGFR was the most potent target against tumor angiogenesis.
RNA interference (RNAi), which could silence the mRNA transcription specially, was a mechanism of post-transcription gene silence induced by the double stranded RNA. RNAi is not only a technology in study of genome function, but also an effective method to treat disease, especially, in gene therapy of the tumor. Studies demonstrated that the proliferation and invadation in many solid tumors could be inhibited with shRNA silencing the VEGF expression.In this study, to investigate the effects and mechanism of shRNA silencing VEGF gene expression in lung cancer.
Materials&Methods An effective and stable pSilencer-VEGF-shRNA plasmid to silence VEGF expression was screened and constructed successfully. A549 cells were transfected with pSilencer-VEGF-2 to observe its expression of VEGF, proliferation of the non small cell lung cancer and tubule formation of the endothelial cells in vitro. Finally the A549 cells and pSilencer-VEGF-A549 cells were injected subcutaneously into nude mice to observe the tumor growth and survival of the mice in vivo. Identify the changes of p53, Bcl-2 and Bax in VEGF interference A549 cells which interacted with HUVEC by RT-PCR and Western blot.
Results The results showed that the expression of VEGF in stable transfectant A549 cell line was obviously inhibited over 90% by pSilencer-VEGF-2. Two months later the inhibition rate was still above 80%. Although growth of the pSilencer-VEGF-A549 cells was hard inhibitedt, the angiogenesis of endothelial cells was decreased in vitro. Compared with the control, the tumor growth was significantly slow(P<0.05) and the survival was obviously enlonged in the mice injected pSilencer-VEGF-2-A549(P<0.05)in vivo.
In the mechanism study, we found that the P53, Bcl-2 and Bax expression did not change in the stable transfected A549 cell lines. And we did not observe the apoptosis of stable transfected A549 cells. Compared with the control group, the expression of Bax was not changed and the Bcl-2 expression was decreased, however the P53 expression increased with apoptosis in the stable transfected A549 cell lines under the HUVEC.
Conclusion The shRNA vector pSilencer-VEGF which could silence the VEGF expression in lung cancer effectively was successfully constructed. Proliferation of the lung cancer cells and development of the lung cancer were inhibited with RNAi silencing VEGF expression both in vivo and vitro. The possible mechanism might be due to the block of VEGF/VEGFR resulting in apoptosis of the cancer cells.
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