关键词: 乳腺肿瘤, 周期蛋白依赖性蛋白样激酶1, 磷酸酶和张力蛋白同源物, 蛋白激酶, 哺乳动物雷帕霉素靶蛋白, 细胞增殖, 细胞迁移, 细胞侵袭

Abstract:

Objective To discuss the effect of cell division cyclin-dependentkinase like-1 （CDKL1） gene silencing on the proliferation， invasion， and metastasis of the breast cancer MCF-7 cells， and to clarify its possible mechanism. Methods The breast cancer MCF-7 cells were used as the research subjects， and the small interfering RNA （siRNA） technology was used to silence the expression of the CDKL1 gene. The intervention was performed by using 1 μmol·L^－1 phosphatase and tension homolog （PTEN） inhibitor BpV or 10 μmol·L^－1 protein kinase B（Akt） agonist SC79. The MCF-7 cells at logarithmic growth phase were divided into control group （MCF-7 cells without any treatment）， transfection control （siRNA-NC） group （MCF-7 cells transfected with siRNA-NC plasmid）， siRNA-CDKL1 group （MCF-7 cells transfected with siRNA-CDKL1 plasmid）， siRNA-CDKL1+BpV group （MCF-7 cells transfected with siRNA-CDKL1 and treated with 1 μmol·L^－1 PTEN inhibitor BpV for 2 h）， and siRNA-CDKL1+SC79 group （MCF-7 cells transfected with siRNA-CDKL1 and treated with 10 μmol·L^－1 Akt agonist SC79 for 2 h）. Real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods were used to detect the expression levels of CDKL1 mRNA and protein in the breast cancer MCF-7 cells in various groups； CCK-8 assay was used to detect the proliferation activities of the breast cancer MCF-7 cells in various groups；EdU assay was used to detect the rates of EdU positive cells in the breast cancer MCF-7 cells in various groups；cell scratch healing experiment was used to detect the scratch healing rates of the breast cancer MCF-7 cells in various groups； Transwell chamber assay was used to detect the numbers of migration and invasion breast cancer MCF-7 cells in various groups；Western blotting method was used to detect the expression levels of matrix metalloproteinase-2 （MMP-2）， matrix metalloproteinase-9 （MMP-9）， PTEN， Akt， mammalian target of rapamycin （mTOR）， phosphorylated Akt （p-Akt）， and phosphorylated mTOR （p-mTOR） proteins in the breast cancer MCF-7 cells in various groups. Results Compared with siRNA-NC group， the expression levels of CDKL1 mRNA and protein in the breast cancer MCF-7 cells in siRNA-CDKL1 group were decreased （P<0.01）， the proliferation activity was decreased（P<0.01），the rate of EdU positive cells was decreased（P<0.01），and the numbers of migration and invasion cells were decreased （P<0.01）， the expression levels of MMP-2， MMP-9， p-Akt， and p-mTOR proteins in the cells were decreased （P<0.01）， and the expression level of PTEN protein was increased （P<0.01）. Compared with siRNA-CDKL1 group， the proliferation activities of the breast cancer MCF-7 cells in siRNA-CDKL1+BpV group and siRNA-CDKL1+SC79 group were increased （P<0.01），the rate of EdU-positive cells was increased（P<0.01）， the numbers of migration and invasion cells were increased（P<0.01）， the expression levels of MMP-2， MMP-9， p-Akt， and p-mTOR proteins in the cells were increased（P<0.01），and the expression level of PTEN protein in the cells was decreased （P<0.01）. Conclusion Silencing CDKL1 gene can inhibit the proliferation， invasion，and metastasis abilities of the breast cancer MCF-7 cells， and its mechanism may be related to the regulation of the PTEN/Akt/mTOR signaling pathway.

Key words: Breast neoplasm, Cyclin-dependentkinase like-1, Phosphatase and tension homolog, Protein kinase, Mammalian target of rapamycin, Cell proliferation, Cell migration, Cell invasion