摘要
目的探讨环状RNA同源域相互作用蛋白激酶3(circHIPK3)促进三阴乳腺癌(TNBC)细胞增殖和迁移的作用机制。方法采用实时荧光定量聚合酶链反应(qRT-PCR)检测circHIPK3在TNBC细胞系HCC1806、MDA-MB-231、HCC-70和人正常乳腺细胞系MCF-10A中的表达水平。将circHIPK3高表达的TNBC细胞系分为sh-NC组、sh-circHIPK3组,分别转染NC敲减质粒、circHIPK3敲减质粒;将circHIPK3低表达的TNBC细胞系分为oe-NC组、oe-circHIPK3组,分别转染NC过表达质粒、circHIPK3过表达质粒。采用qRT-PCR检测各组细胞中circHIPK3表达水平,通过细胞增殖实验(MTS)检测各组细胞增殖活性,采用Transwell实验检测各组细胞迁移能力,采用TOP/FOP Flash双荧光素酶报告基因检测各组细胞荧光素酶活性,并通过蛋白质印迹法(Western blotting)检测各组细胞中Wnt/β-catenin信号通路相关蛋白Wnt3a、β-连环蛋白(β-catenin)、c-MYC和基质金属蛋白酶2(MMP-2)的表达。结果circHIPK3在3种TNBC细胞系中的表达水平(MDA-MB-231细胞中表达水平最高,HCC-70细胞中表达水平最低)均高于人正常乳腺细胞系MCF-10A,差异有统计学意义(F=30.110,P<0.001)。qRT-PCR结果显示,circHIPK3在sh-circHIPK3组中的相对表达量为(0.36±0.06),低于sh-NC组的(0.98±0.03),差异有统计学意义(t=16.008,P<0.001);circHIPK3在oe-circHIPK3组中的相对表达量为(6.29±0.71),高于oe-NC组的(0.99±0.05),差异有统计学意义(t=12.897,P<0.001)。sh-circHIPK3组MDA-MB-231细胞增殖活性和迁移能力均低于sh-NC组,oe-circHIPK3组HCC-70细胞增殖活性和迁移能力均高于oe-NC组,差异有统计学意义(P<0.05或P<0.001)。sh-circHIPK3组MDA-MB-231细胞荧光素酶活性低于sh-NC组,oe-circHIPK3组HCC-70细胞荧光素酶活性高于oe-NC组,差异有统计学意义(P<0.001)。sh-circHIPK3组MDA-MB-231细胞Wnt3a、β-catenin、c-MYC和MMP-2蛋白表达均低于sh-NC组,oe-circHIPK3组HCC-70细胞Wnt3a、β-catenin、c-MYC和MMP-2蛋白表达均高于oe-NC组,差异有统计学意义(P<0.05)。结论circHIPK3通过调控Wnt/β-catenin信号通路促进TNBC细胞的增殖和迁移。
Objective To investigate the mechanism of circular RNA homeodomain interacting protein kinase 3(circHIPK3)in promotion of proliferation and migration of triple-negative breast cancer(TNBC)cells.Methods Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression levels of circHIPK3 in TNBC cell lines HCC1806,MDA-MB-231,HCC-70 and human normal breast cell line MCF-10A.The TNBC cell lines with high expression of circHIPK3 were divided into sh-NC group(transfected NC knockdown plasmid),sh-circHIPK3 group(transfected circHIPK3 knockdown plasmid),and the TNBC cell lines with low expression were divided into oe-NC group(transfected NC overexpressed plasmid)and oe-circHIPK3 group(transfected circHIPK3 overexpressed plasmid).The expression levels of circHIPK3 in cells of each group were detected by qRT-PCR;cell proliferation assay(MTS)was used to detect the cell proliferation activity of each group;transwell assay was used to detect the migrated ability of cells in each group;TOP/FOP flash luciferase reporter assay was used to detect the luciferase activity of cells in each group;the expressions of Wnt/β-catenin signaling pathway-related proteins Wnt3a,β-catenin,c-MYC and matrix metalloproteinase 2(MMP-2)proteins in the cells of each group were detected by western blotting.Results The expression level of circHIPK3 in TNBC cell lines was higher than that in human normal breast cell line MCF-10A,its expression was the highest in MDA-MB-231 cells,and the lowest expression in HCC-70 cells,the differences were statistically significant differences(P<0.05).qRT-PCR results showed that the relative expression of circHIPK3 in the sh-circHIPK3 group was(0.36±0.06),which was lower than(0.98±0.03)in the sh-NC group,and the difference was statistically significant(t=16.008,P<0.001).The relative expression of circHIPK3 in the oe-circHIPK3 group was(6.29±0.71),which was higher than(0.99±0.05)in the oe-NC group,the difference was statistically significant(t=12.897,P<0.001).Compared with the sh-NC group,the proliferation activity and migration ability of MDA-MB-231 cells in the sh-circHIPK3 group were decreased,and compared with the oe-NC group,the proliferation and metastasis of HCC-70 cells in the oe-circHIPK3 group were higher(P<0.05 or P<0.01).Compared with sh-NC group,the luciferase activity of MDA-MB-231 cells in the sh-circHIPK3 group was decreased,the luciferase activity of HCC-70 cells in the oe-circHIPK3 group was increased compared with the oe-NC group(P<0.001).The protein expressions of Wnt3a,β-catenin,c-MYC and MMP-2 in MDA-MB-231 cells in the sh-circHIPK3 group were lower than those in the sh-NC group,while were higher in the oe-circHIPK3 group than those in the oe-NC group(P<0.05).Conclusion CircHIPK3 can promote the proliferation and migration of TNBC cells by regulating Wnt/β-catenin signal pathway.
作者
丛佳
石杨
刘远远
CONG Jia;SHI Yang;LIU Yuanyuan(Breast Department,Dalian Women and Children's Medical Center(Group),Dalian,Liaoning,116033;Reproductive Laboratory,Dalian Women and Children's Medical Center(Group),Dalian,Liaoning,116033;Department of Pathology,Dalian Women and Children's Medical Center(Group),Dalian,Liaoning,116033)
出处
《实用临床医药杂志》
CAS
2022年第13期93-99,共7页
Journal of Clinical Medicine in Practice
基金
辽宁省自然基金资助计划项目(2019-MS-071)。