LINC00649调节微小RNA-524-5p/UHRF1轴对乳腺癌细胞增殖、迁移和侵袭的影响

Effects of LINC00649 on proliferation,migration and invasion of breast cancer cells by regulating microRNA-524-5p/UHRF1 axis

目的 探究长链非编码RNA LINC00649对乳腺癌细胞增殖、迁移和侵袭及微小RNA-524-5p/环指域蛋白1(miR-524-5p/UHRF1)轴的影响.方法 采用实时荧光定量聚合酶链反应(qRT-PCR)法检测各乳腺细胞中LINC00649,miR-524-5p和UHRF1 mRNA的表达水平;将人乳腺癌细胞MCF-7分为NC组(未转染质粒)、si-NC组(转染LINC00649阴性对照)、si-LINC00649组(转染si-LINC00649)、si-LINC00649+inhibitor-NC 组(共转染 si-LINC00649 和 miR-524-5p 抑制剂阴性对照)和 si-LINC00649+miR-524-5p inhibitor 组(共转染 si-LINC00649 和 miR-524-5p inhibitor),转染培养48 h后检测各转染组细胞中LINC00649,miR-524-5p和UHRF1 mRNA表达水平;采用CCK-8法和克隆形成实验检测各转染组细胞的存活率及克隆形成能力;采用Transwell实验检测各组细胞的迁移及侵袭能力;采用蛋白质印迹法(Western blot)检测各组转染细胞中UHRF1表达水平;双荧光素酶报告基因实验验证miR-524-5p与LINC00649,UHRF1之间的靶向关系.多组间比较采用单因素方差分析,两两组间多重比较采用Tukey's事后检验.结果 乳腺癌细胞系中LINC00649(1.57±0.12 比 0.98±0.02,t=11.879,P<0.01)和 UHRF1 mRNA(1.63±0.14 比 0.99±0.01,t=11.169,P<0.01)表达水平高于正常乳腺上皮细胞,miR-524-5p表达水平则低于正常乳腺上皮细胞(0.39±0.02 比 1.00±0.01,t=66.822,P<0.01);si-LINC00649 组细胞存活率[(69.36±1.86)%比(98.33±2.55)%,t=22.483,P<0.01]、细胞克隆形成数量[(162.35±6.02)个 比(207.27±8.21)个,t=10.808,P<0.01]、迁移细胞数[(105.22±7.26)个比(178.57±6.20)个,t=18.819,P<0.01]、侵袭细胞数[(98.27±3.77)个比(152.05±5.17)个,t=20.588,P<0.01]以及细胞中LINC00649(0.57±0.04 比 0.96±0.05,t=14.919,P<0.01)、UHRF1(0.37±0.02 比 0.95±0.10,t=13.931,P<0.01)和 UHRF1 mRNA(0.63±0.05 比 0.97±0.04,t=11.859,P<0.01 表达水平低于 si-NC 组,而 miR-524-5p(1.37±0.09 比 0.97±0.03,t=10.328,P<0.01)表达水平高于 si-NC组;回补实验结果显示,si-LINC00649+miR-524-5p inhibitor 组细胞中 UHRF1 mRNA(0.83±0.07 比0.65±0.05,t=5.125,P<0.01)和 UHRF1(0.65±0.04 比 0.41±0.02,t=13.145,P<0.01)表达水平、细胞存活率[(82.11±2.29)%比(65.36±1.53)%,t=14.897,P<0.01]、克隆形成数量[(183.60±7.22)个比(158.35±5.36)个,t=40.429,P<0.01]、发生迁移[(160.50±5.89)个比(110.22±6.59)个,t=13.934,P<0.01]及侵袭[(139.55±4.60)个比(100.02±3.71)个,t=16.385,P<0.01]的细胞数高于 si-LINC00649+inhibitor-NC 组,而 miR-524-5p 表达水平低于si-LINC00649+inhibitor-NC 组(1.15±0.08 比 1.33±0.10,t=3.443,P<0.05);双荧光素酶活性验证miR-524-5p 和 LINC00649、UHRF1 均存在靶向结合位点,且 LINC00649-WT 或 UHRF1-WT 和miR-524-5p mimic共转染后细胞相对荧光素酶活性低于共转染mimic-NC的细胞(0.44±0.03比1.01±0.02、0.39±0.02 比 1.00±0.05,t=38.724、27.746,P<0.01).结论 敲低 LINC00649 能够通过上调miR-524-5p,下调UHRF1的表达来抑制乳腺癌细胞的增殖、迁移及侵袭.

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.