Effects of LINC00649 on proliferation,migration and invasion of breast cancer cells by regulating microRNA-524-5p/UHRF1 axis
Objective To investigate the effects of long non-coding RNA LINC00649 on the prolif-eration,migration and invasion of breast cancer cells and the microRNA-524-5p/ubiquitin-like with PHD and ring finger domain 1(miR-524-5p/UHRF1)axis.Methods Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the mRNA expression levels of LINC00649,miR-524-5p and UHRF1 in breast cells.Human breast cancer cells MCF-7 were divided into NC group(untransfected plas-mid),si-NC group(transfected with LINC00649 negative control),si-LINC00649 group(transfected with si-LINC00649),si-LINC00649+inhibitor-NC group(co-transfected with si-LINC00649 and miR-524-5p inhibitor negative control)and si-LINC00649+miR-524-5p inhibitor group(co-transfected with si-LINC00649 and miR-524-5p inhibitor).After 48 h of transfection,the mRNA expression levels of miR-524-5p and UHRF1 were detected in LINC00649 cells of each transfection group.The CCK-8 method and clone formation assay were used to measure the viability and colony formation of cells in each transfec-tion group.Transwell assay was used to measure the migration and invasion of cells in each group.Western blotting was used to detect UHRF1 expression in transfected cells.Dual luciferase reporter assays were used to verify the targeting relationship between miR-524-5p and LINC00649,UHRF1.Single factor analy-sis of variance was used for inter group comparisons,and Tukey's post hoc tests were used for pairwise multiple comparisons.Results The expression levels of LINC00649(1.57±0.12 vs.0.98±0.02,t=11.879,P<0.01)and UHRF1 mRNA(1.63±0.14 vs.0.99±0.01,t=11.169,P<0.01)in breast cancer cell lines were higher than those in normal breast epithelial cells,while the expression level of miR-524-5p was lower than that in normal breast epithelial cells(0.39±0.02 vs.1.00±0.01,t=66.822,P<0.01),the cell survival rate of si-LINC00649 group was[(69.36±1.86)%vs.(98.33±2.55)%,t=22.483,P<0.01],the number of cell clones formed[(162.35±6.02)cells vs.(207.27±8.21)cells,t=10.808,P<0.01],the number of migrating cells[(105.22±7.26)cells vs.(178.57±6.20)cells,t=18.819,P<0.01],the number of invasive cells[(98.27±3.77)cells vs.(152.05±5.17)cells,t=20.588,P<0.01]and the expression levels of LINC00649(0.57±0.04 vs.0.96±0.05,t=14.919,P<0.01),UHRF1(0.37±0.02 vs.0.95±0.10,t=13.931,P<0.01)and UHRF1 mRNA(0.63±0.05 vs.0.97±0.04,t=11.859,P<0.01)in the cells were lower than those in the si NC group,and the expression level of miR-524-5p(1.37±0.09 vs.0.97±0.03,t=10.328,P<0.01)was higher than that of the si NC group.The results of the complementation experiment showed that the expression levels of UHRF1 mRNA(0.83±0.07 vs.0.65±0.05,t=5.125,P<0.01)and UHRF1(0.65±0.04 vs.0.41±0.02,t=13.145,P<0.01),as well as the cell survival rate[(82.11±2.29)%vs.(65.36±1.53)%,t=14.897,P<0.01]in the si-LINC00649+miR-524-5p in-hibitor group cells were significantly reduced,t=14.897,P<0.01),the number of clones formed[(183.60±7.22)cells vs.(158.35±5.36)cells,t=40.429,P<0.01],the number of cells undergo-ing migration[(160.50±5.89)cells vs.(110.22±6.59)cells,t=13.934,P<0.01]and invasion[(139.55±4.60)cells vs.(100.02±3.71)cells,t=16.385,P<0.01]was higher in the si-LINC00649+inhibitor NC group than in the si-LINC00649+inhibitor NC group,and the expression level of miR-524-5p was lower in the si-LINC00649+inhibitor NC group(1.15±0.08 vs.1.33±0.10,t=3.443,P<0.05);Dual luciferase activity verified the existence of targeted binding sites for miR-524-5p and LINC00649,UHRF1,and the relative luciferase activity of cells co transfected with LINC00649-WT or UHRF1-WT and miR-524-5p mimic was lower than that of cells co transfected with mimic NC(0.44±0.03 vs.1.01±0.02,0.39±0.02 vs.1.00±0.05,t=38.724,27.746,P<0.01).Conclusion Knockdown of LINC00649 can inhibit the proliferation,migration and invasion of breast cancer cells by up-regulating miR-524-5p and down-regulating the expression of UHRF1.
Breast cancerLong non coding RNAProliferationMigrationInvasion