沉默血管生成素-2表达对人肺腺癌细胞生物学特性的影响

The Influence of Inhibiting Angiopoietin-2 on the Biological Characteristics of Bronchogenic Adenocarcinoma

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。

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 tumorj 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 efficien- cy proliferation rate, apoptosis, and microvessel density ofA549 cell line were analyzed. Results In vitro experiment indicated that the Ang-2 expression level （P〈0.001） and growth rate （P〈0.001） ofA549 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 ofAAV-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 volumn of tumorrelative to two control groups. In addition, the apoptosis index （AI） ofAAV-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 M （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. Conclusion AAV-mediated expression of shRNA significantly reduces concentration of Ang-2 in A549 cell line, lowers prolif- eration and growth rate and induce .apoptosis ofA549 cell line.