shRNA沉默VEGF—C基因对大肠癌淋巴管生成和淋巴结转移的影响

Inhibitory effect of RNA interference targeting vascular epithelial growth factor-C on proliferation of human colon cancer cell line LOVO in vitro and In vivo

目的 观察RNA干扰（RNA interference,RNAi）技术抑制大肠癌细胞株Lovo中VEGF-C基因的表达,探讨抑制shRNA-VEGF-C对人大肠癌细胞Lovo生物学特性的影响.方法 构建表达ShRNA-VEGF-C重组质粒转染Lovo细胞,72 h后用实时RT-PCR（real-time polymerase chain recation）检测VEGF-C mRNA表达;建立Lovo细胞皮下移植瘤裸鼠模型,注射shRNA-VEGF-C,动态观察肿瘤体积并于4周后处死裸鼠称取瘤重、计算局部淋巴结转移率,免疫组化法检测大肠癌组织微淋巴管密度（microlymphatic density,MLD）.结果 转染shRNA-VEGF-C后,Lovo细胞VEGF-C mRNA表达下调;体内实验结果显示,4周后shRNA-VEGF-C组移植瘤体积[（324.9±64.8）mm3]明显小于空质粒组[（553.5±90.1）mm3]和生理盐水对照组[（570.1±85.4）mm3]（P〈0.01）;shRNA-VEGF-C组瘤重[（3.01±0.55）g]也低于空质粒组[（4.65±0.65）g]和生理盐水对照组[（4.75±0.55）g]（P〈0.01）;shRNA-VEGF-C组微淋巴管密度LMVD（15.5±6.90）明显低于HK组（24.18±6.45）和生理盐水对照组（29.59±8.21）（P〈0.01）;shRNA-VEGF-C组局部淋巴结转移率（30.1%）明显低于空质粒组（50.2%）和生理盐水组（53.1%）.结论 shRNA-VEGF-C可影响VEGF-C诱导的淋巴管生成并抑制大肠癌淋巴结转移.

Objective This study was to explore the inhibitory effect of shRNA-VEGF - C on growth of human colon cancer cell line Lovo in vitro and vivo. Methods Recombinant VEGF-C short hairpin RNA （shRNA） plasmid was constructed and transfected into Lovo cells. The expression of VEGF-C was detected at mRNA and protein levels by real-time reverse transcription-polymerase chain reaction （RT-PCR）. In vivo study, xenograft tumors were established by injecting LOVO cells into nude mice, then shR-NA-VEGF-C were injected into the tumors, the tumor volume and weight and the incidences of lymph node metastasis were detected. Immunohistochemical staining was used to detect the lymphatic microvessel density of colon cancer tissues. Results After transfection of shRNA-VEGF-C, the mRNA of VEGF-C in Lovo cells were down-regulated. Four weeks after injection, the tumor volume and tumor weight in VEGF-C-shR-NA group were significantly smaller than that in empty plasmid group and NS group [（324. 9 ± 64. 8 ） mm3 vs. （553.5±90. 1）mm3 and （570. 1±85.4）mm3; （3.01 ±0.55）g vs （4.65 ±0.65）g and （4.75 ±0. 75）g]. The incidences of lymph node metastasis （30. 1% ） were significantly inhibited compared with empty plasmid group （50. 2% ） and normal saline group （53. 1% ）. In shRNA-VEGF-C group, and microlymphatic density （15.5 ± 6. 90） was also decreased compared with empty plasmid group （24. 18 ±6. 45 ）, and normal saline group （29. 59 ± 8. 21 ） （ all P 〈0. 01 ）. Conclusion shRNA-VEGF-C can inhibit the growth of LOVO cells in vitro and vivo. VEGF-C may inhibit the lymph node metastasis of colon cancer by suppressing lymphangiogenesis.