RRS1对乳腺癌细胞增殖和迁移的影响及其作用机制

EFFECT OF REGULATOR OF RIBOSOME SYNTHESIS 1 ON THE PROLIFERATION AND MIGRATION OF BREAST CANCER CELLS AND ITS MECHANISM OF ACTION

目的探讨核糖体合成调控因子1(RRS1)基因对乳腺癌BT549细胞增殖和迁移的影响及其作用机制。方法通过Western Blot实验检测人乳腺癌细胞系(MDA-MB-231、MDA-MB-468、BT549、MCF-7细胞)和人正常乳腺上皮MCF-10A细胞RRS1蛋白的表达量。通过免疫荧光检测RRS1在BT549细胞中的定位。慢病毒感染法建立RRS1敲低的BT549细胞系,采用实时荧光定量PCR(RT-qPCR)和Western Blot实验检测感染阴性对照慢病毒细胞(Con组)和感染shRNA-RRS1慢病毒细胞(sh-RRS1组)中RRS1 mRNA和蛋白相对表达量。采用CCK8实验和集落形成实验检测RRS1对BT549细胞增殖能力的影响,通过细胞划痕实验、Transwell实验检测RRS1对BT549细胞迁移能力的影响。采用Western Blot实验检测两组细胞AKT-mTOR相关信号通路蛋白及其下游缺氧诱导因子-1α(HIF-1α)蛋白、血管内皮生长因子(VEGF)蛋白相对表达量。结果MDA-MB-231、MDA-MB-468、BT549、MCF-7乳腺癌细胞系中RRS1相对表达量与人正常乳腺上皮MCF-10A细胞相比均显著增高(F=48.92,P<0.05)。免疫荧光染色显示,RRS1主要在BT549细胞的细胞核和核仁表达,细胞质也有少量表达。与Con组进行比较,sh-RRS1组RRS1 mRNA和蛋白表达量显著降低,增殖和迁移能力均明显减弱,p-AKT、p-mTOR、HIF-1α、VEGF蛋白表达量均显著降低(t=6.29~32.04,F=1368.00、2699.00,P<0.05)。结论RRS1可能通过AKT-mTOR通路促进乳腺癌细胞的增殖和迁移。

Objective To investigate the effect of the regulator of ribosome synthesis 1(RRS 1)gene on the proliferation and migration of breast cancer BT549 cells and its mechanism of action.Methods Western blot was used to measure the protein expression of RRS1 in human breast cancer cell lines(MDA-MB-231,MDA-MB-468,BT549,and MCF-7 cells)and normal human breast epithelial MCF-10A cells,and immunofluorescence assay was used to observe the localization of RRS1 in BT549 cells.The lentivirus infection method was used to establish an RRS1-knockdown BT549 cell line,and quantitative real-time PCR and Western blot were used to measure the relative mRNA and protein expression levels of RRS1 in the Con group transfected with negative control lentivirus and the sh-RRS1 group transfected with shRNA-RRS1 lentivirus.CCK8 assay and colony-forming assay were used to observe the effect of RRS1 on the proliferation ability of BT549 cells,and wound healing assay and Transwell assay were used to observe the effect of RRS1 on the migration ability of BT549 cells.Western blot was used to measure the relative expression levels of AKT-mTOR signaling pathway proteins and their downstream proteins hypoxia-inducible factor 1α(HIF-1α)and vascular endothelial growth factor(VEGF)in the two groups.Results The relative expression level of RRS1 in breast cancer cell lines MDA-MB-231,MDA-MB-468,BT549,and MCF-7 was significantly higher than that in normal human breast epithelial MCF-10A cells(F=48.92,P<0.05).Immunofluorescent staining showed that RRS1 was mainly expressed in the nucleus and nucleolus of BT549 cells,with a low expression level in the cytoplasm.Compared with the Con group,the sh-RRS1 group had significant reductions in the mRNA and protein expression levels of RRS 1,the proliferation and migration abilities of cells,and the protein expression le-vels of p-AKT,p-mTOR,HIF-1α,and VEGF(t=6.29-32.04,F=1368.00,2699.00,P<0.05).Conclusion RRS1 may promote the proliferation and migration of breast cancer cells through the AKT-mTOR pathway.