该研究旨在探讨富含半胱氨酸的酸性分泌蛋白基因(secreted protein acidic and rich in cysteine gene,SPARC)过表达对卵巢癌淋巴结高转移细胞(SKOV3-PM4)生物学特性的影响。构建SPARC基因的慢病毒表达载体并转染SKOV3-PM4细胞,Real-time PCR和Western blot验证转染后的表达效率,激光共聚焦免疫荧光进行蛋白的细胞定位,细胞计数法和集落形成实验测定细胞增殖能力,流式细胞仪检测细胞周期,Transwell小室实验测定细胞体外侵袭、迁移能力。实验结果显示,SPARC蛋白存在于核周及胞质;过表达SPARC基因后,SKOV3-PM4细胞增殖受到明显抑制(P<0.05);细胞周期检测结果显示,各期改变无明显差异;体外侵袭、迁移实验结果显示,SKOV3-PM4细胞侵袭、迁移能力显著降低(P<0.05)。实验结果表明,SPARC基因在卵巢癌淋巴结转移中可能发挥抑癌基因的生物学作用。
Objective:To study explores the effect of HLEC on the secreted proteins of epithelial ovarian cancer(EOC)cells(SKOV3-PM4)with directional highly lymphatic metastasis.Methods:Supernatants of four groups of cultured cells,namely,SKOV3(A),SKOV3+HLEC(B),SKOV3-PM4(C),SKOV3-PM4+HLEC(D),were collected,and their proteins were detected by antibody arrays and iTRAQ-2D-LC-MALDITOF/TOF/MS.Significantly differential proteins were further analyzed via bioinformatics and validated in human serums and cell media via ELISA.Results:Results of antibody arrays and mass spectrometry demonstrated that GRN and VEGFA were upregulated in group C(compared with group A),whereas IGFBP7 and SPARC were downregulated in group D(compared with group C).Comprehensive bioinformatics analysis results showed that IGFBP7 and VEGFA were closely linked to each other.Further validation with serums showed statistical significance in VEGFA and IGFBP7 levels among groups of patients with ovarian cancers,benign tumors,and control groups.Two proteins were upegulated in the first group.VEGFA in the control group was downregulated.For IGFBP,upregulation in the control group and down-regulation in the first group were also observed.Conclusion:The HLEC microenvironment is closely associated with directional metastasis to lymph nodes and with differential proteins including cell stromal proteins and adhesion factors.The upregulation of VEGFA and GRN and the downregulation of SPARC and IGFBP7 are closely associated with directional metastasis to lymph nodes in EOC cells.
Xin-Ying ZhangFu-Qiang YinLi LiuTing GaoHe-Yun RuanXiao GuanYing-Xin LuDan-Rong Li
