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乳腺癌转移抑制基因1对卵巢癌细胞血管生成的影响及其机制探讨

Effects of Breast Cancer Metastasis Suppressor 1 Gene on Angiogenesis of Human Ovarian Cancer Cell and Its Mechanism

  • 摘要: 目的 探讨乳腺癌转移抑制基因1(breast cancer metastasis suppressor 1,BRMS1)对人卵巢癌细胞OVCAR3诱导血管内皮细胞形成管腔能力的影响及可能的机制。方法应用脂质体介导的方法将BRMS1 shRNA重组质粒转染人卵巢癌细胞OVCAR3,经G418筛选获得稳定转染株。荧光定量PCR和Western blot法检测BRMS1 mRNA和蛋白的表达水平;体外血管形成实验检测OVCAR3诱导人脐静脉内皮细胞(HUVECs)的血管形成能力;Western blot法检测生长抑制因子4(ING4)和白细胞介素-6(IL-6)的蛋白表达情况。结果稳定转染BRMS1 shRNA后,OVCAR3细胞BRMS1的表达在 mRNA和蛋白水平均受到明显抑制。体外血管形成实验提示,BRMS1表达下调后能显著促进卵巢癌细胞诱导的HUVECs形成管腔样结构的能力;Western blot法显示干扰组中ING4蛋白表达量较对照组下降30%,而IL-6蛋白表达上调,是空白对照组的1.5倍,差异有统计学意义(P<0.05)。结论干扰BRMS1基因的表达可促进卵巢癌细胞诱导血管形成能力,其机制可能与调节下游基因ING4和IL-6的表达有关。

     

    Abstract: Objective To investigate the effects and mechanism of breast cancer metastasis suppressor 1(BRMS1) on angiogenesis in human ovarian cancer cell line OVCAR3. Methods Vector containing a short hairpin RNA(shRNA) against BRMS1 was constructed and the positive clone was named pGPU6/GFP/Neo-BRMS1.The BRMS1 shRNA or a non-specific sequence(as the negative control) was transfected into OVCAR3 cells by LipofectamineTM2000 and stably transfected cells were obtained by screening with G418.The expressions of BRMS1 mRNA and protein in OVCAR3 cells were performed using real time PCR and Western blot,respectively.Angiogenesis capacity of human umbilical venous endothelial cells(HUVECs) co-cultured with OVCAR3 cells was determined by tube formation assay.Furthermore,the expressions of inhibitor of growth 4(ING4) and Interleukin-6(IL-6) were detected by Western blot. Results The recombinant plasmid could be successfully transferred into OVCAR3 cells.Real time PCR and Western blot analyses demonstrated that BRMS1 expression was efficiently down-regulated.Stable suppression of BRMS1 in human ovarian cancer cells significantly promoted the ability of HUVECs to form tubular structures in the co-culture,and the number of tubules was markedly increased in transfection of BRMS1 shRNA than that in the blank control group or negative control group.Moreover,the protein level of ING4 in interference group was decreased by 30%,while BRMS1 knockdown led to 1.5 fold increase of IL-6 protein expression(P<0.05). Conclusion Knockdown of BRMS1 may play a critical role in promoting the angiogenesis-inducing ability of ovarian cancer cells,and BRMS1 regulates metastatic potential at least in part through the regulation of ING4 and IL-6.

     

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