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目的:构建pCDNA3.1-ENDO-VEGI151/survivin-shRNA(pEV/si-survivin)双功能表达质粒,观察其对乳腺癌细胞MDA-MB-231和人脐静脉血管内皮细胞(human umbilical vein endothelial cell,HUEVC)增殖和凋亡的影响,探讨其治疗肿瘤的可行性。方法:利用MDA-MB-231细胞筛选获得survivin的高效siRNA序列,构建pEV/si-survivin表达质粒并分别转染MDA-MB-231和HUEVC,以real-time PCR和Western blotting检测转染细胞中ENDO-VEGI151和survivin的表达;MTT法检测细胞增殖抑制情况,流式细胞术检测细胞周期和细胞凋亡。结果:成功构建pEV/si-survivin双功能表达质粒,并能在MDA-MB-231和HUEVC中正确表达相应基因产物。该质粒可明显抑制MDA-MB-231细胞内survivin的表达,并抑制细胞增殖[48、72 h的抑制率为(39.36±4.16)%、(48.43±3.49)%],促进MDA-MB-231细胞凋亡[(18.33±1.48)%vs(4.80±1.01)%,P<0.01)和细胞周期阻滞(P<0.05);该质粒也明显抑制HUEVC的增殖[(48、72 h的抑制率为(38.16±3.37)%、(53.75±4.53)%),并促进HUEVC凋亡和细胞周期阻滞(P<0.05)。结论:成功构建的pEV/si-survivin双功能表达质粒可以同时发挥抑制新生血管生成和促肿瘤细胞凋亡的作用,提高抗肿瘤的效果。
OBJECTIVE: To construct a bi-functional expression plasmid pCDNA3.1-ENDO-VEGI151 / survivin-shRNA (pEV / si-survivin) and observe its effect on breast cancer cells MDA-MB-231 and human umbilical vein endothelial cells , HUEVC) proliferation and apoptosis, to explore the feasibility of its treatment of cancer. Methods: The efficient siRNA sequence of survivin was screened by MDA-MB-231 cells. The pEV / si-survivin expression plasmid was constructed and transfected into MDA-MB-231 and HUEVC respectively. The transfected cells were detected by real-time PCR and Western blotting ENDO-VEGI151 and survivin. MTT assay was used to detect the cell proliferation inhibition. Cell cycle and apoptosis were detected by flow cytometry. Results: The recombinant plasmid pEV / si-survivin was successfully constructed and its corresponding gene product was correctly expressed in MDA-MB-231 and HUEVC. The plasmids could significantly inhibit the expression of survivin and inhibit the proliferation of MDA-MB-231 cells [(39.36 ± 4.16)% and (48.43 ± 3.49)%, 48 and 72 h] (18.33 ± 1.48)% vs (4.80 ± 1.01)%, P <0.01) and cell cycle arrest (P <0.05). The plasmid also significantly inhibited the proliferation of HUEVC [(48 and 72 h inhibition rate (38.16 ± 3.37)%, (53.75 ± 4.53)% respectively), and promoted the apoptosis and cell cycle arrest of HUEVC (P <0.05). Conclusion: The successfully constructed pEV / si-survivin dual-expression plasmid can simultaneously inhibit angiogenesis and promote tumor cell apoptosis, and improve antitumor effect.