长链非编码RNA叉头框蛋白C1基因启动子上游转录体通过Janus激酶信号对乳腺癌细胞增殖、侵袭和迁移的影响

Effect of long non-coding RNA FOXC1 upstream transcript on proliferation,invasion and migration of breast cancer cells by Janus kinase signaling

目的观察长链非编码RNA(lncRNA)叉头框蛋白C1(FOXC1)基因启动子上游转录体(FOXCUT)调控JAK信号在乳腺癌细胞增殖、迁移和侵袭中的作用。方法采用实时定量反转录聚合酶链反应(qRT-PCR)检测lncRNA FOXCUT在乳腺癌细胞株(MCF-7和MDA-MB-231)和人正常乳腺上皮细胞(MCF-10A)中的表达。体外干扰lncRNA-FOXCUT在乳腺癌细胞MCF-7中表达,利用细胞计数试剂盒(CCK-8)法、伤口愈合法和细胞侵袭实验检测lncRNA FOXCUT对乳腺癌细胞增殖、迁移和侵袭的影响。同时,用qRT-PCR和蛋白质印迹法(Western blot)检测B细胞淋巴瘤2(bcl-2)、基质金属蛋白酶-9(MMP-9)和Janus激酶1(JAK1)、信号转导和转录激活因子3(STAT3)的表达。多组间比较采用单因素方差分析,两两比较采用SNK-q检验。结果lncRNA FOXCUT在乳腺癌细胞MCF-7(1.567±0.270,t=5.086,P<0.01)和MDA-MB-231(1.513±0.387,t=3.533,P<0.01)中表达明显高于MCF-10A细胞(1.004±0.193),差异有统计学意义。siRNA-FOXCUT组细胞增殖活力明显低于NC组(48 h时0.790±0.068比0.991±0.165,t=2.516,P<0.05;72 h时0.928±0.135比1.500±0.311,t=3.775,P<0.01),差异有统计学意义。同时,siRNA-FOXCUT组MCF-7细胞的迁移率[(30.163±4.133)%比(79.330±4.952)%,t=13.200,P<0.01]和侵袭[(101.000±13.115)个比(244.667±18.771)个,t=10.870,P<0.01]能力均低于NC组,差异有统计学意义。siRNA-FOXCUT组细胞中bcl-2(0.565±0.058比1.075±0.242,t=3.555,P<0.01)和MMP-9(0.527±0.138比0.982±0.172,t=3.574,P<0.01)在mRNA和蛋白水平均低于NC组,同时p-JAK1(0.185±0.031比1.095±0.134,t=11.460,P<0.01)和p-STAT3(0.298±0.029比0.948±0.235,t=4.755,P<0.01)蛋白表达低于NC组,差异有统计学意义。结论lncRNA FOXCUT可能通过JAK1/STAT3途径参与调控乳腺癌的增殖、迁移和侵袭。

Objective To investigate the role of long non-coding RNA(lncRNA)forkhead box c1 promoter upstream transcript(FOXCUT)in proliferation,migration and invasion of breast cancer cells.Methods The expression of lncRNA FOXCUT in breast cancer cell lines[michigan cancer foundation 7(MCF-7)and:MD anderson metastatic breast cancer 231(MDA-MB-231)]and human normal michigan cancer foundation 10A(MCF-10A)was detected by real-time quantitative reverse transcription-polymerase chain reactions(qRT-PCR).The expression of lncRNA FOXCUT was silenced in MCF-7 cells,and proliferation,migration and invasion abilities were measured using cell counting kit-8(CCK-8),Wound-healing and transwell assays.The expression of B-cell lymphoma-2(bcl-2),matrix metallopeptidase-9(MMP-9)and janus kinase-1(JAK1),signal transducer and activator of transcription-3(STAT3)was detected by qRT-PCR and Western blotting.One-way ANOVA was used for multiple comparisons,and student-Newman-Keuls(SNK)-q test was used for comparison of selected two groups.Results LncRNA FOXCUT was significantly upregulated in breast cancer cells MCF-7(1.567±0.270,t=5.086,P<0.01)and MDA-MB-231(1.513±0.387,t=3.533,P<0.01)as compared with MCF-10A cells(1.004±0.193).The cell proliferation ability was decreased in siRNA-FOXCUT group as compared with normal control(NC)group(0.790±0.068 vs.0.991±0.165,t=2.516,P<0.05 at 48 h;0.928±0.135 vs.1.500±0.311,t=3.775,P<0.01 at 72 h).The down-expression of lncRNA FOXCUT had lower migration[(30.163±4.133)%vs.(79.330±4.952)%,t=13.200,P<0.01]and invasion[(101.000±13.115)cells vs.(244.667±18.771)cells,t=10.870,P<0.01]abilities than NC group.SiRNA-FOXCUT significantly inhibited the bcl-2(0.565±0.058 vs.1.075±0.242,t=3.555,P<0.01)and MMP-9(0.527±0.138 vs.0.982±0.172,t=3.574,P<0.01)on both mRNA and protein levels.Down-expression of lncRNA FOXCUT significantly inhibited the protein level of p-JAK1(0.185±0.031 vs.1.095±0.134,t=11.460,P<0.01)and p-STAT3(0.298±0.029 vs.0.948±0.235,t=4.755,P<0.01).Conclusion LncRNA FOXCUT may be functionally involved in the proliferation,migration and invasion of breast cancer cells through the regulation of JAK1/STAT3 pathway,demonstrating that lncRNA FOXCUT may serve as a novel biomarker and therapeutic target in breast cancer.