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目的:探讨亨廷顿相关蛋白1(HAP1)基因过表达对人乳腺癌细胞株MCF-7增殖、体外迁移侵袭和细胞凋亡的影响及其可能机制。方法:通过转染的方法将逆转录病毒pBabe-puro(嘌呤霉素)HAP1质粒和pBabe-puro质粒导入人乳腺癌细胞系MCF-7,用嘌呤霉素筛选稳定表达两质粒的细胞系,荧光定量PCR和蛋白质印迹法鉴定是否成功构建HAP1过表达细胞系;细胞增殖-毒性检测试剂盒(CCK-8)和克隆形成实验检测细胞的生长增殖,Transwell小室法检测细胞的侵袭和迁移,流式细胞仪检测细胞的凋亡。结果:成功构建稳定表达pBabe-HAP1的MCF-7-pBabe-puro-HAP1细胞模型。CCK-8检测72h细胞增殖率,MCF-7-pBabe-puro-HAP1为(75.97±6.76)%,明显低于MCF-7-pBabe-puro细胞(93.98±6.63)%(P=0.03)及MCF-7细胞(100.00±0.00)%,P=0.004;MCF-7-pBabe-puro-HAP1细胞克隆形成率为(22.67±1.26)%,明显低于MCF-7(35.00±0.50)%(P=0.000)和MCF-7-pBabe-puro细胞(33.83±0.76)%,P=0.000;Transwell小室侵袭和迁移实验表明,MCF-7-pBabe-puro-HAP1组的侵袭(3.33±0.58,P=0.000)和迁移(50.00±3.61,P<0.01)能力明显降低;流式细胞仪检测细胞凋亡,MCF-7-pBabe-puro-HAP1凋亡率为(8.03±0.15)%,高于MCF-7-pBabe-puro(3.13±0.25)%(P=0.000)和MCF-7细胞(3.33±0.35)%,P=0.000。结论:HAP1基因能够抑制肿瘤细胞增殖和迁移侵袭,并能诱导细胞凋亡,其可能作为一个抑癌基因在肿瘤发生发展中发挥重要作用。
Objective: To investigate the effects of over-expression of Huntington’s protein-1 (HAP1) gene on the proliferation, migration, invasion and apoptosis of human breast cancer cell line MCF-7 and its possible mechanism. Methods: The retroviral pBabe-puro (puromycin) HAP1 and pBabe-puro plasmids were transfected into human breast cancer cell line MCF-7 by transfection. Puromycin was used to screen stable expression plasmids of two plasmids. Fluorescence Quantitative PCR and Western blotting were used to identify whether HAP1 overexpression lines were successfully constructed. Cell proliferation and toxicity assay kit (CCK-8) and clonogenic assay were used to detect cell proliferation and proliferation. Transwell chamber assay was used to detect cell invasion and migration. Flow cytometry Cytometry detects cell apoptosis. Results: The MCF-7-pBabe-puro-HAP1 cell model stably expressing pBabe-HAP1 was successfully constructed. The proliferation rate of MCF-7-pBabe-puro-HAP1 was significantly lower than that of MCF-7-pBabe-puro cells (93.98 ± 6.63)% 7 cells (100.00 ± 0.00)%, P = 0.004. The colony formation rate of MCF-7-pBabe-puro-HAP1 cells was significantly lower than that of MCF-7 cells (22.67 ± 1.26% The invasion and migration of transwell cells showed that the invasion of MCF-7-pBabe-puro-HAP1 group was significantly higher than that of MCF-7-pBabe-puro-HAP1 group (3.33 ± 0.58, P = ) And migration (50.00 ± 3.61, P <0.01). The apoptotic rate of MCF-7-pBabe-puro-HAP1 was (8.03 ± 0.15)% higher than that of MCF-7 -pBabe-puro (3.13 ± 0.25)% (P = 0.000) and MCF-7 cells (3.33 ± 0.35)%, P = 0.000. Conclusion: The HAP1 gene can inhibit the proliferation, migration and invasion of tumor cells and induce apoptosis. It may play an important role as a tumor suppressor gene in tumor development.