敲减Rab1A表达通过抑制AKT的磷酸化抑制乳腺癌细胞的恶性生物学行为

目的:探究Rab1A基因表达的改变对乳腺癌细胞MDA-MB-231恶性生物学行为的影响。方法:Western blot检测Rab1A在乳腺癌组织及癌旁正常组织的表达和Rab1A在乳腺正常上皮及不同乳腺癌细胞系中的基础表达。设计并构建靶向Rab1A的小干扰RNA(siRNA),Western blot法验证敲减效果;分别采用CCK-8实验、划痕实验、Transwell实验、流式细胞术和Western blot观察Rab1A基因表达改变对乳腺癌MDA-MB-231细胞恶性生物学行为的影响和相关蛋白表达的改变。结果:Rab1A在乳腺正常组织和细胞中低表达,在癌组织及癌细胞中高表达(P<0.05);与对照组相比,干扰Rab1A的表达可以显著抑制乳腺癌MDA-MB-231细胞的活力(P<0.05),诱导凋亡增加(P<0.05),G_2/M期细胞比例增加(P<0.05),迁移与侵袭能力减弱(P<0.05),p53、Bax、cleaved caspase-3和PTEN的蛋白水平升高(P<0.05),Bcl-2、cyclin D1、cyclin B1、基质金属蛋白酶2(MMP2)、p-AKT和mTOR的蛋白水平降低(P<0.05)。结论:Rab1A具有调控乳腺癌细胞生长、迁移、侵袭、细胞周期和凋亡的能力,并调控增殖、周期和凋亡相关蛋白的表达。干扰Rab1A可通过抑制AKT通路的磷酸化从而抑制乳腺癌的演进与发展;Rab1A可能是基因治疗乳腺癌的一个潜在靶点。

Rab1A knockdown inhibits malignant biological behaviors of breast carcinoma cells by inhibiting phosphorylation of AKT

AIM:To investigate the role of Rab1A gene in the malignant biological behaviors of breast carcinoma cells.METHODS:The expression levels of Rab1A in breast carcinoma tissues and normal adjacent tissues,and the basic expression level of Rab1A in different breast carcinoma cell lines were measured by Western blot.Small interfering RNA(siRNA)targeting Rab1A was designed,synthetized and transfected into the breast carcinoma MDA-MB-231 cells.After validation of efficiency of Rab1A gene expression knock-down,the malignant biological behaviors of the MDA-MB-231 cells were measured by CCK-8 assay,wound healing assay,Transwell assay and flow cytometry.The protein levels were determined by Western blot.RESULTS:Rab1A was expressed in normal breast tissue and cells at low level,and at high level in the cancer tissues and cancer cells(P<0.05).Compare with control group,after knock-down of Rab1A expression,the viability of MDA-MB-231 cells was significantly inhibited(P<0.05),the abilities of migration and invasion were reduced(P<0.05),the apoptosis was decreased(P<0.05),the percentage of G 2/M phase was increased,the protein levels of p53,Bax,cleaved caspase-3 and PTEN were significantly increased(P<0.05),and the protein levels of Bcl-2,cyclin D1,cyclin B1,matrix metalloproteinase 2(MMP2),p-AKT and mTOR were significantly decreased(P<0.05).CONCLUSION:Rab1A modulates the breast carcinoma cell viability,inhibits the migration and invasion abilities,induces G2 arrest and effectively regulates the cell growth-,cell cycle-and apoptosis-related proteins.Knock-down of Rab1A expression inhibits the evolution and development of breast cancer by inhibiting the phosphorylation of AKT pathway,and Rab1A may function as a potential target in breast carcinoma treatment.