Li Bailing, Zhang Guanxin, Hou Xiaolei, Yuan Yang, Liu Xiaohong, Gong Dejun, Huang Shengdong
Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
Zhongguo Fei Ai Za Zhi. 2011 Jul;14(7):561-7. doi: 10.3779/j.issn.1009-3419.2011.07.01.
It is well-known that angiopoietin-2 (Ang-2) plays an important role in the formation of the blood vascular system. Angiopoietin is involved in many diseases about angiogenesis such as tumor, so may have great prospects for the treatment of these diseases. The aim of this study is to evaluate the influence of inhibiting Ang-2 via adeno-associated virus induced RNA interference (RNAi) on the biological characteristics of bronchogenic adenocarcinoma.
AAV-Ang-2shRNA driven by H1 promoter was constructed to transfect A549 cell line. Normal and AAV-Null cell line were utilized in the control groups. The influence of RNAi on Ang-2 expression as well as the growth rate, tumorigenic efficiency, proliferation rate, apoptosis, and microvessel density of A549 cell line were analyzed.
In vitro experiment indicated that the Ang-2 expression level (P<0.001) and growth rate (P<0.001) of A549 cell line 48 h transfected with AAV-Ang-2shRNA were significantly lower than those in the normal and AAV-Null cell lines. Cell cycle analysis showed the proliferation index (PI) of normal, AAV-Null, and AAV-Ang-2shRNA transfected A549 cell line were 0.51± 0.43, 0.48 ± 0.29, and 0.26 ± 0.31, respectively, which indicated the PI of AAV-Ang-2shRNA transfected cell line was significantly lower, compared with the normal and AAV-Null cell lines. In vivo experiment exhibited that AAV-Ang-2shRNA transfected cell line possessed a lower mass and volume of tumor relative to two control groups. In addition, the apoptosis index (AI) of AAV-Ang-2shRNA transfected, normal, and AAV-Null cell lines were (5.98 ± 3.11)%, (7.51 ± 4.42)% and (17.06 ± 7.43)% respectively, which manifested that AAV-Ang-2shRNA transfected cell line possessed a higher AI (P=0.005, P=0.007). A lower percentage of PCNA-positive cell was observed in AAV-Ang-2shRNA transfected cell line (92.75 ± 9.7)% as well, compared with the normal (85.8 ± 11.8)% and AAV-Null (69.8 ± 16.5)% cell lines.
AAV-mediated expression of shRNA significantly reduces concentration of Ang-2 in A549 cell line, lowers proliferation and growth rate and induce .apoptosis of A549 cell line.
众所周知，血管生成素-2（Ang-2）在血管系统形成中起重要作用。血管生成素参与了许多与血管生成有关的疾病，如肿瘤，因此在这些疾病的治疗中可能具有广阔前景。本研究旨在评估通过腺相关病毒介导的RNA干扰（RNAi）抑制Ang-2对支气管腺癌生物学特性的影响。
构建由H1启动子驱动的AAV-Ang-2shRNA转染A549细胞系。正常细胞系和AAV-Null细胞系作为对照组。分析RNAi对Ang-2表达以及A549细胞系的生长速率、致瘤效率、增殖率、凋亡和微血管密度的影响。
体外实验表明，转染AAV-Ang-2shRNA的A549细胞系48小时后的Ang-2表达水平（P<0.001）和生长速率（P<0.001）显著低于正常细胞系和AAV-Null细胞系。细胞周期分析显示，正常、AAV-Null和转染AAV-Ang-2shRNA的A549细胞系的增殖指数（PI）分别为0.51±0.43、0.48±0.29和0.26±0.31，这表明转染AAV-Ang-2shRNA的细胞系的PI显著低于正常细胞系和AAV-Null细胞系。体内实验显示，相对于两个对照组，转染AAV-Ang-2shRNA的细胞系形成的肿瘤质量和体积更低。此外，转染AAV-Ang-2shRNA、正常和AAV-Null细胞系的凋亡指数（AI）分别为（5.98±3.11）%、（7.51±4.42）%和（17.06±7.43）%，这表明转染AAV-Ang-2shRNA的细胞系具有更高的AI（P=0.005，P=0.007）。与正常细胞系（8个5.8±11.8）%和AAV-Null细胞系（69.8±16.5）%相比，转染AAV-Ang-2shRNA的细胞系中PCNA阳性细胞的百分比也更低（92.75±9.7）%。
AAV介导的shRNA表达显著降低A549细胞系中Ang-2的浓度，降低其增殖和生长速率，并诱导A549细胞系凋亡。
