miR-21在肺癌血管生成中的作用及培美曲塞对其影响

Regulatory role of miR-21 in and inhibitory effect of pemetrexed on human lung cancer angiogenesis

目的肺癌死亡率居肿瘤相关疾病死因的首位。肿瘤组织中血管异常增生,与肿瘤的分级、转移和预后不良密切相关。文中利用体内外血管生成模型评价肺癌细胞不同miR-21表达水平肺癌细胞在肿瘤血管生成中的作用及其可能机制,并探讨培美曲塞对其影响。方法采用划痕实验研究不同miR-21表达水平肺癌细胞培养上清对血管内皮细胞增殖能力的影响。采用Luminex检测血管内皮生长因子(vascular endothelial growth factor,VEGF)在不同miR-21表达水平肺癌细胞培养上清中的表达水平。采用免疫组化法检测CD31(亦称血小板内皮细胞黏附分子-1)、血管内皮生长因子受体2(Vascularendothelial growth factor receptor 2,VEGFR2)及磷酸化VEGFR2蛋白在不同miR-21表达水平肺癌实体瘤组织的表达水平差异。结果划痕实验表明,miR-21高表达的人肺癌A549(A549-21H)细胞划痕修复能力明显强于miR-21低表达的人肺癌A549(A549-21L)细胞,表现为划痕直径明显缩小。Luminex检测VEGF在不同miR-21表达水平肺癌细胞培养上清的表达水平,实验结果表明,A549-21H细胞培养上清中VEGF含量显著高于A549-21L。采用IHC法检测裸鼠肺癌移植瘤中CD31、VEGFR2及磷酸化VEGFR2蛋白表达变化研究结果表明,A549-21H瘤体CD31表达明显高于A549-21L(P<0.01),A549-21H瘤体VEGFR2表达与A549-21L比较无显著性差异,A549-21H瘤体Tyr951磷酸化VEGFR2表达明显高于A549-21L(P<0.01)。结论 miR-21可能通过调节VEGF表达水平及VEGFR2磷酸化水平参与肿瘤血管生成的调控,培美曲塞对肿瘤血管生成具有一定的抑制作用。

Objective Lung cancer is the leading cause of cancer-related deaths. Angiogenesis in the cancerous tissue is closely related to the severity, metastasis and prognosis of tumors. This study aimed to study the role of miR-21 in human lung cancer angiogenesis and its possible mechanism using different angiogenesis models. Methods The scratch test of HUVEC proliferation was done by treating HUVECs with cell culture supernatants from human lung cancer cells with different expressions of miR-21. The VEGF level in the cell culture supernatant was analyzed by Luminex technology. The expressions of CD31, VEGFR2 and phosphorylated VEGFR2-Tyr951 was determined by immunohistoehemistry. Results The A549 cells with a high expression of miR-21 showed an obviously decreased scratch diameter as compared with those with a low miR-21 expression, indicating a stronger scratch repair capabil- ity of the former. Pemetrexed showed an inhibitory effect on A549 cells with different levels of miR-21 expression. The VEGF concen- tration was higher in the A549 cells with a high miR-21 expression than in those with a low one. Pemetrexed inhibited VEGF secretion from the A549 cells highly expressing miR-21. The expression level of vascular endothelial cell marker CD31 was significantly higher in the A549 cells with a high miR-21 expression than in those with a low one （ P 〈 0. 01 ）, but there were no significant differences in the VEGFR2 expression between the two groups. VEGFR2 phosphoryla- tion in Tyr951 was significantly higher in the former than in the latter （P〈0.01）. Conclusion miR-21 may play an important role in human lung cancer angiogenesis by regulating VEGF expression and VEGFR2 phosphorylation, and pemetrexed has an inhibitory effect on lung cancer angiogenesis.