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目的:探讨HAP1基因过表达对人乳腺癌细胞株MDA-MB-231增殖、体外迁移侵袭和细胞凋亡的影响及其可能机制。方法:使用实时荧光定量PCR检测30例女性乳腺癌患者肿瘤组织与癌旁组织中HAP1的表达情况。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MDA-MB-231,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小室法检测细胞侵袭和迁移,流式细胞仪检测细胞凋亡和周期,荧光定量PCR检测凋亡相关基因表达情况。结果:荧光定量结果显示,约75%正常组织中HAP1表达比相应癌组织表达高3倍以上。成功构建稳定表达pBabe-HAP1的MDA-MB-231-pBabe-puro-HAP1细胞模型。CCK-8检测72h的细胞存活率MDA-MB-231-pBabe-puro-HAP1为(74.9±6.0)%,低于MDA-MB-231细胞(100.0±0.0)%,P=0.019,也低于MDA-MB-231-pBabe-puro细胞(99.8±6.0)%,P=0.007;细胞克隆形成实验结果显示,MDA-MB-231-pBabe-puro-HAP1组的克隆形成率为(9.3±1.9)%,明显低于MDA-MB-231细胞组(16.0±2.6)%,P=0.024,也低于MDA-MB-231-pBabe-puro细胞组(15.8±2.5)%,P=0.022;Transwell小室侵袭和迁移实验表明,MDA-MB-231-pBabe-puro-HAP1组的侵袭(F=0.339,P=0.725)和迁移(F=0.844,P=0.475)能力无明显改变;流式细胞仪检测细胞凋亡结果显示,MDA-MB-231-pBabe-puro-HAP1凋亡率与MDA-MB-231和MDA-MB-231-pBabe-puro细胞组差异无统计学意义,F=0.564,P=0.596;细胞周期结果发现,HAP1组细胞周期中G2M期所占比例为(14.84±0.90)%,与pBabe组(11.76±0.93)%和MDA-MB-231组(11.19±0.94)%相比明显增加。3组细胞中凋亡相关蛋白Caspase-3、Caspase-8、Caspase-9、Bcl-2、Bax和Survivin的表达差异无统计学意义,F=0.564,P=0.596。结论:HAP1可能通过细胞周期阻滞抑制乳腺癌细胞的生长,其降低或缺失可能引起乳腺癌的发生发展。
Objective: To investigate the effect of HAP1 overexpression on the proliferation, migration, invasion and apoptosis of human breast cancer cell line MDA-MB-231 and its possible mechanism. Methods: Real-time fluorescent quantitative PCR was used to detect the expression of HAP1 in tumor tissues and adjacent tissues of 30 female breast cancer patients. The retroviral pBabe-puro (puromycin) HAP1 plasmid and the pBabe-puro plasmid were transfected into the human breast cancer cell line MDA-MB-231 by transfection. Puromycin was used to screen the cell lines stably expressing the two plasmids. Fluorescence quantitative PCR and Western blotting were used to identify the successful construction of HAP1 overexpression cell line. Cell proliferation and toxicity assay kit (CCK-8) and clonogenic assay were used to detect cell proliferation and proliferation. Transwell assay was used to detect cell invasion and migration. Flow cytometry Apoptosis and cycle were detected. Fluorescence quantitative PCR was used to detect the expression of apoptosis related genes. Results: Fluorescence quantitative results showed that HAP1 expression in about 75% of normal tissues was more than 3 times higher than that in corresponding cancerous tissues. The MDA-MB-231-pBabe-puro-HAP1 cell model stably expressing pBabe-HAP1 was successfully constructed. The cell survival rate of MDA-MB-231-pBabe-puro-HAP1 assayed by CCK-8 at 72h was (74.9 ± 6.0)% lower than that of MDA-MB-231 cells The results of colony formation assay showed that the clonogenic rate of MDA-MB-231-pBabe-puro-puro cells was (9.3 ± 1.9) %, Which was significantly lower than that of MDA-MB-231 cells (16.0 ± 2.6)%, P = 0.024, and also lower than that of MDA-MB-231-pBabe-puro cells The invasion and migration experiments showed that there was no significant change in the ability of invasion (F = 0.339, P = 0.725) and migration (F = 0.844, P = 0.475) in MDA-MB-231-pBabe-puro-HAP1 group. The results of apoptosis showed that the apoptosis rate of MDA-MB-231-pBabe-puro-HAP1 was not significantly different from that of MDA-MB-231 and MDA-MB-231-pBabe- 0.596. The cell cycle showed that the proportion of G2M phase in HAP1 group was (14.84 ± 0.90)%, which was significantly higher than that in pBabe group (11.76 ± 0.93)% and MDA-MB-231 group (11.19 ± 0.94)% increase. The expressions of Caspase-3, Caspase-8, Caspase-9, Bcl-2, Bax and Survivin in the three groups of cells were not significantly different (F = 0.564, P = 0.596). CONCLUSION: HAP1 may inhibit the growth of breast cancer cells through cell cycle arrest. The decrease or deletion of HAP1 may cause the development of breast cancer.