LINC00662/微小RNA-186-5p/叉头框转录因子D1调控乳腺癌MCF-7细胞生物学行为的研究

LINC00662/microRNA-186-5p/forkhead box transcription factor D1 regulating the biological behavior of breast cancer MCF-7 cells

目的探究LINC00662能否靶向微小RNA(miR)-186-5p/叉头框转录因子D1(FOXD1)轴调控乳腺癌MCF-7细胞增殖、迁移和侵袭。方法实时定量反转录聚合酶链反应(RT-qPCR)检测28例乳腺癌组织和癌旁组织中LINC00662、miR-186-5p和FOXD1 mRNA表达,蛋白质印迹法(Western blot)检测FOXD1蛋白表达。分别转染LINC00662小干扰RNA(si-LINC00662)、共转染si-LINC00662与miR-186-5p抑制剂、si-LINC00662与FOXD1过表达载体至MCF-7细胞,细胞计数试剂盒(CCK-8)和克隆形成实验检测细胞增殖,Transwell检测细胞迁移和侵袭,Western blot检测p21、E-钙黏蛋白(E-cadherin)、基质金属蛋白酶(MMP)-2和FOXD1蛋白表达。双荧光素酶报告系统验证LINC00662与miR-186-5p、miR-186-5p与FOXD1的调控关系,两组间比较采用独立样本t检验。结果乳腺癌组织组中LINC00662(1.01±0.08比2.36±0.09,t=59.324,P<0.01)、FOXD1水平(0.97±0.07比1.93±0.09,t=44.553,P<0.01)高于癌旁组织,miR-186-5p水平(0.96±0.05比0.34±0.04,t=51.236,P<0.01)低于癌旁组织。si-LINC00662组克隆形成数[(114.00±7.93)个比(62.00±5.81)个,t=15.869,P<0.01]、迁移细胞数[(164±10.03)个比(75±7.14)个,t=21.687,P<0.01]、侵袭细胞数[(103.00±7.83)个比(44.00±4.35)个,t=19.761,P<0.01]少于si-NC组,存活率[(100.03±8.12)%比(52.29±5.34)%,t=14.737,P<0.01]、MMP-2(0.71±0.05比0.24±0.03,t=24.181,P<0.01)蛋白水平低于si-NC组,P21(0.15±0.02比0.58±0.04,t=28.845,P<0.01)、E-cadherin(0.23±0.02比0.66±0.04,t=28.845,P<0.01)蛋白水平高于si-NC组。LINC00662靶向负调控miR-186-5p;miR-186-5p靶向负调控FOXD1。si-LINC00662+抗miR-186-5p组克隆形成数[(60.00±±5.62)个比(102.00±7.23)个,t=13.759,P<0.01]、迁移细胞数[(74.00±7.26)个比(131.00±9.91)个,t=13.920,P<0.01]、侵袭细胞数[(45.00±4.41)个比(88.00±5.67)个,t=17.959,P<0.01]多于si-LINC00662+抗miR-NC组,存活率[(52.31±5.35)%比(85.06±6.46)%,t=11.714,P<0.01]、MMP-2(0.23±0.03比0.59±0.04,t=21.600,P<0.01)蛋白水平低于si-LINC00662+抗miR-NC组,P21(0.57±0.04比0.26±0.03,t=17.400,P<0.01)、E-cadherin(0.64±0.04比0.35±0.03,t=12.969,P<0.01)蛋白水平高于si-LINC00662+抗miR-NC组。结论LINC00662通过发挥miR-186-5p分子海绵上调FOXD1促进乳腺癌MCF-7细胞增殖、迁移和侵袭。

Objective To explore whether LINC00662 can target microRNA(miR)-186-5p/forkhead box transcription factor D1(FOXD1)axis to regulate the proliferation,migration and invasion of breast cancer cells.Methods Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the expression of LINC00662,miR-186-5p and FOXD1 mRNA in 28 cases of breast cancer tissues and adjacent tissues,and Western blotting was used to detect the expression of FOXD1 protein.MCF-7 cells were transfected with small interfering RNA(si-LINC00662),or co-transfected with si-LINC00662 and miR-186-5p inhibitor or si-LINC00662 and FOXD1 overexpression vector.The cell proliferation was detected by cell counting kit-8(CCK-8)and clone formation assays.Transwell was used to detect cell migration and invasion,and Western blotting was used to detect the expression of p21,E-cadherin,matrix metalloproteinase(MMP)-2 and FOXD1 protein.The dual-luciferase reporter system verified the regulatory relationship between LINC00662 and miR-186-5p or miR-186-5p and FOXD1.Results LINC00662(1.01±0.08 vs.2.36±0.09,t=59.324,P<0.01)and FOXD1 levels in breast cancer tissue group(0.97±0.07 vs.1.93±0.09,t=44.553,P<0.01)was higher than that in the adjacent tissue,and the level of miR-186-5p(0.96±0.05 vs.0.34±0.04,t=51.236,P<0.01)was lower than that in the adjacent tissue.The number of colonies formed in the si-LINC00662 group(114.00±7.93 vs.62.00±5.81,t=15.869,P<0.01),the number of migrated cells(164.00±10.03 vs.75.00±7.14,t=21.687,P<0.01),the number of invasive cells(103.00±7.83 vs.44.00±4.35,t=19.761,P<0.01)was less than the si-NC group,and the survival rate[(100.03±8.12)%vs.(52.29±5.34)%,t=14.737,P<0.01],MMP-2(0.71±0.05 vs.0.24±0.03,t=24.181,P<0.01)protein levels were low in the si-NC group,p21(0.15±0.02 vs.0.58±0.04,t=28.845,P<0.01),E-cadherin(0.23±0.02 vs.0.66±0.04,t=28.845,P<0.01)protein level was higher than that of si-NC group.LINC00662 could target and negatively regulates miR-186-5p.miR-186-5p could target and negatively regulate FOXD1.The number of colonies formed in the si-LINC00662+anti-miR-186-5p group(60.00±5.62 vs.102.00±7.23,t=13.759,P<0.01),the number of migrated cells(74.00±7.26 vs.131.00±9.91,t=13.920,P<0.01),number of invasive cells(45.00±4.41 vs.88.00±5.67,t=17.959,P<0.01)more than si-LINC00662+anti-miR-NC group,survival rate[(52.31±5.35)%vs.(85.06±6.46)%,t=11.714,P<0.01],MMP-2(0.23±0.03 vs.0.59±0.59±0.04,t=21.600,P<0.01)protein level was lower than si-LINC00662+anti-miR-NC group,p21(0.57±0.04 vs.0.26±0.03,t=17.400,P<0.01),E-cadherin(0.64±0.04 vs.0.35±0.03,t=12.969,P<0.01)protein level was higher than si-LINC00662+anti-miR-NC group.Conclusion LINC00662 promotes the proliferation,migration and invasion of breast cancer MCF-7 cells by acting as a molecular sponge of miR-186-5p to upregulate FOXD1.LINC00662 is a potential molecular target for breast cancer.