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. 2011 Jul;14(7):561-7.
doi: 10.3779/j.issn.1009-3419.2011.07.01.

[The influence of inhibiting angiopoietin-2 on the biological characteristics of bronchogenic adenocarcinoma]

[Article in Chinese]
Bailing Li  1 , Guanxin Zhang, Xiaolei Hou, Yang Yuan, Xiaohong Liu, Dejun Gong, Shengdong Huang
Affiliations

[The influence of inhibiting angiopoietin-2 on the biological characteristics of bronchogenic adenocarcinoma]

[Article in Chinese]
Bailing Li et al. Zhongguo Fei Ai Za Zhi. 2011 Jul.

Abstract
in English, Chinese

Background and objective: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

背景与目的: 促血管生成素-2（Ang-2）是一类调节血管生成的重要细胞因子，与肿瘤的发生发展有密切的联系。本研究通过腺相关病毒（adeno-associated virus, AAV）介导的RNA干扰（RNA interference, RNAi）抑制肺腺癌细胞A549 Ang-2的表达，观察其对癌细胞生物学特性的影响。

方法: 构建H1启动子驱动的表达针对Ang-2的小干扰RNA（siRNA）重组腺相关病毒（AAV-Ang-2shRNA），转染A549细胞，同时以正常A549细胞及其转染空病毒（AAV-Null）的A549细胞作为对照，观察RNAi沉默Ang-2表达在体外及体内对A549细胞生长、致瘤、增殖、凋亡及肿瘤微血管密度的影响。

结果: 体外实验结果表明重组腺相关病毒转染A549细胞48 h后Ang-2蛋白表达比对照组明显降低（P < 0.001）；细胞生长明显减慢（P < 0.001）。RNA干扰后A549细胞细胞周期分析结果提示A549细胞对照组、AAV-Null对照组和AAV-Ang-2shRNA实验组的增殖指数（proliferation index, PI）分别为0.51±0.43、0.48±0.29和0.26±0.31。AAV-Ang-2shRNA实验组与对照组比较，PI明显减少（P=0.001)。体内实验结果表明，AAV-Ang-2shRNA实验组在胸腺缺陷小鼠成瘤体积、瘤质量均明显低于两对照组（P < 0.01）。各组微血管密度分析结果分别为46.4±13.1、44.2±13.6和34.9 ±12.8；AAV-Ang-2shRNA实验组肿瘤组织内新生血管数目明显低于对照组（P < 0.001）。各组凋亡指数（apoptosis index, AI）分别为（5.98±3.11）%、（7.51±4.42）%及（17.06±7.43）%。AAV-Ang-2shRNA组比PBS组、AAV-Null组凋亡百分率明显增高（P=0.005, P=0.007）。各组细胞增殖核抗原（proliferating cell nuclear antigen, PCNA）阳性表达率分别为（92.75±9.7）%、（85.8±11.8）%、（69.8±16.5）%；AAV-Ang-2shRNA组PCNA指数低于两对照组（P=0.014, P=0.021）。

结论: 腺相关病毒介导的siRNA表达能明显抑制A549细胞中Ang-2的表达，减慢肿瘤细胞的增殖，促进肿瘤细胞凋亡，抑制肿瘤生长。

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Figures

1
1
Western blot检测Ang-2蛋白的表达 Western blot detected the expression of Ang-2 protein
2
2
流式细胞检测RNA干扰后细胞周期变化。A：A549组；B：AAV-Null组；C：AAV-Ang-2shRNA组。 Flow cytometry cell cycle after RNA interference. A: A549 group; B: AAV-Null group; C: AAV-Ang-2shRNA group.
3
3
Ang-2靶向RNA干扰A549细胞后免疫组化分析（×400）。MVD计数：FVⅢ因子染色定位于血管内皮细胞，呈棕黄色；TNNEL染色：TdT酶介导的原位末端标记法，凋亡细胞核呈棕黄色；PCNA计数：阳性表达定位于细胞核，显示为棕黄色。Ang-2蛋白阳性染色均定位于胞浆内，呈现粗细颗粒状棕黄色着色。VEGF定位于肿瘤细胞的细胞浆或细胞膜；阳性物质均呈棕黄色。 Ang-2 RNA interference targeting A549 cells immunohistochemical analysis (×400). MVD count: FVⅢ factor was localized in vascular endothelial cells, brownish yellow; TNNEL staining: TdT-mediated in situ end labeling of apoptotic nuclei showed brown; PCNA count: expression located in the nucleus, appears as brown. Ang-2 protein positive staining were localized in the cytoplasm, showing the thickness of granular brown staining. VEGF localized in the cytoplasm of tumor cells or the cell membrane; positive material showed a brown-yellow.
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