核因子白细胞介素3调节因子促进乳腺癌细胞增殖和侵袭能力研究

Nuclear factor interleukin 3-regulated promotes the proliferation and metastasis of breast cancer cells

目的探讨核因子白细胞介素3调节因子(NFIL3)对人乳腺癌细胞增殖和侵袭能力的影响。方法采用慢病毒载体构建过表达及敲减NFIL3乳腺癌细胞系BT549和Hs578T(中国科学院上海细胞库),以荧光定量聚合酶链反应(PCR)及蛋白印迹实验(Western blot)进行验证。通过细胞计数试剂盒(MTT)检测细胞增殖、小室细胞迁移实验(Transwell)和侵袭实验以及动物模型裸鼠皮下荷瘤和鼠尾静脉肺转移实验,验证NFIL3对乳腺癌细胞增殖和侵袭能力。组间比较采用t检验及One-way ANOVA检验。结果MTT细胞增殖实验证实,过表达NFIL3组与对照组相比促进细胞增殖(BT549-对照4.37±0.27比BT549-NFIL35.98±0.31,t=6.783,P<0.01;Hs578T-对照4.75±0.32比Hs578T-NFIL36.05±0.39,t=4.463,P<0.05),敲减NFIL3抑制细胞增殖(BT549-PLKO 4.17±0.20比BT549-sh12.83±0.29,t=6.557,P<0.01;BT549-PLKO 4.17±0.20比BT549-sh23.11±0.17,t=4.943,P<0.01;Hs578T-PLKO 4.11±0.31比Hs578T-sh13.11±0.21,t=12.540,P<0.01;Hs578T-PLKO 4.11±0.31比Hs578T-sh23.01±0.23,t=10.930,P<0.01);Transwell细胞迁移证实,过表达NFIL3组与对照组比较促进细胞迁移[BT549-对照(198.00±28.03)个比BT549-NFIL3(397.00±52.10)个,t=10.640,P<0.01;Hs578T-对照(294.00±32.01)个比Hs578T-NFIL3(598.00±46.11)个,t=17.130,P<0.01],敲减NFIL3抑制细胞迁移[BT549-PLKO(178.00±23.11)个比BT549-sh1(41.01±15.12)个,t=9.820,P<0.01;BT549-PLKO(178.00±23.11)个比BT549-sh2(56.10±24.27)个,t=10.430,P<0.01;Hs578T-PLKO(197.60±21.13)个比Hs578T-sh1(69.80±19.96)个,t=8.403,P<0.01;Hs578T-PLKO(197.60±21.13)个比Hs578T-sh2(58.00±14.26)个,t=8.042,P<0.01];Transwell细胞侵袭证实,过表达NFIL3组与对照组比较促进细胞侵袭[BT549-对照(74.6±23.14)个比BT549-NFIL3(142.50±28.30)个,t=5.874,P<0.01;Hs578T-对照(103.20±28.14)个比Hs578T-NFIL3(183.70±22.14)个,t=7.110,P<0.01],敲减NFIL3抑制细胞侵袭[BT549-PLKO(89.13±19.86)个比BT549-sh1(37.45±15.63)个,t=9.062,P<0.01;BT549-PLKO(89.13±19.86)个比BT549-sh2(50.17±17.12)个,t=8.067,P<0.01;Hs578T-PLKO(135.10±16.52)个比Hs578T-sh1(47.10±16.30)个,t=6.747,P<0.01;Hs578T-PLKO(135.10±16.52)个比Hs578T-sh2(58.45±14.13)个,t=6.598,P<0.01]。过表达NFIL3组与对照组比较促进裸鼠皮下肿瘤生长[Hs578T-对照(0.126±0.017)g比Hs578T-NFIL3(0.301±0.098)g,t=3.934,P<0.01],敲减NFIL3组抑制裸鼠皮下肿瘤生长[Hs578T-PLKO(0.190±0.078)g比Hs578T-sh(0.040±0.019)g,t=4.168,P<0.01];过表达NFIL3组与对照组比较,增加鼠尾静脉肺转移瘤形成数量[Hs578T-对照(1.45±0.98)个比Hs578T-NFIL3(4.12±1.79)个,t=4.137,P<0.01],敲减NFIL3组减少鼠尾静脉肺转移瘤形成数量[Hs578T-PLKO(1.53±1.21)个比Hs578T-sh(0.34±0.27)个,t=3.035,P<0.05]。结论NFIL3在乳腺癌细胞系中具有促进癌细胞增殖、迁移和侵袭的能力。

Objective To investigate the effects of Nuclear factor interleukin 3regulator(NFIL3)on proliferation and invasion of human breast cancer cells.Methods we successfully established cell models of NFIL3 over-expression and knockdown in breast cancer cell lines BT549 and Hs578T respectively(Shanghai cell bank of Chinese Academy of Sciences)thought lentiviral vectorsand validated by fluorescence quantitative polymerase chain reaction and Western blotting.Methyl thiazolyl tetrazolium assay(MTT)test cell proliferation,Transwell assays,Subcutaneous tumor bearing and tail vein lung metastasis in nude mice,were verified the proliferation and invasion of NFIL3 in breast cancer cells.Data between sample groups expressed by mean±standard deviation,and comparison between groups performed by T-test and One-way ANOVA test.Results MTT cell proliferation assay demonstrated that NFIL3 over-expression significantly promoted cell proliferation(BT549-control 4.37±0.27 vs.BT549-NFIL35.98±0.31,t=6.783,P<0.01;Hs578T-control 4.75±0.32 vs.Hs578T-NFIL36.05±0.39,t=4.463,P<0.05),and NFIL3 knockdown significantly inhibited cell proliferation(BT549-PLKO 4.17±0.20 vs.BT549-sh12.83±0.29,t=6.557,P<0.01;BT549-PLKO 4.17±0.20 vs.BT549-sh23.11±0.17,t=4.943,P<0.01;Hs578T-PLKO 4.11±0.31 vs.Hs578T-sh13.11±0.21,t=12.540,P<0.01;Hs578T-PLKO 4.11±0.31 vs.Hs578T-sh23.01±0.23,t=10.930,P<0.01).Transwell assay showed that NFIL3 over-expression significantly promoted cell migration(BT549-control 198.00±28.03 vs.BT549-NFIL3397.0±52.1,t=10.640,P<0.01;Hs578T-control 294±32.01 vs.Hs578T-NFIL3598±46.11,t=17.130,P<0.01),and oNFIL3 knockdown significantly inhibited cell migration(BT549-PLKO 178±23.11 vs.BT549-sh141.01±15.12,t=9.820,P<0.01;BT549-PLKO 178±23.11 vs.BT549-sh256.1±24.27,t=10.430,P<0.01;Hs578T-PLKO 197.6±21.13 vs.Hs578T-sh169.8±19.96,t=8.403,P<0.01;Hs578T-PLKO 197.6±21.13 vs.Hs578T-sh258.0±14.26,t=8.042,P<0.01).Transwell assay showed that NFIL3 over-expression significantly promoted cell invasion(BT549-control 74.6±23.14 vs.BT549-NFIL3142.5±28.3,t=5.874,P<0.01;Hs578T-control 103.2±28.14 vs.Hs578T-NFIL3183.7±22.14,t=7.110,P<0.01),and oNFIL3 knockdown significantly inhibited cell invasion(BT549-PLKO 89.13±19.86 vs.BT549-sh137.45±15.63,t=9.062,P<0.01;BT549-PLKO 89.13±19.86 vs.BT549-sh250.17±17.12,t=8.067,P<0.01;Hs578T-PLKO 135.1±16.52 vs.Hs578T-sh147.1±16.3,t=6.747,P<0.01;Hs578T-PLKO 135.1±16.52 vs.Hs578T-sh258.45±14.13,t=6.598,P<0.01).NFIL3 over-expression significantly promoted subcutaneous tumor growth in nude mice[Hs578T-control(0.126±0.017)g vs.Hs578T-NFIL3(0.301±0.098)g,t=3.934,P<0.01],and NFIL3 knockdown significantly inhibited subcutaneous tumor growth in nude mice[Hs578T-PLKO(0.19±0.078)g vs.Hs578T-sh(0.04±0.019)g,t=4.168,P<0.01].NFIL3 over-expression increased the number of lung metastases in nude mice(Hs578T-control 1.45±0.98 vs.Hs578T-NFIL34.12±1.79,t=4.137,P<0.01),while NFIL3 knockdown reduced the number of lung metastases in nude mice(Hs578T-PLKO 1.53±1.21 vs.Hs578T-sh 0.34±0.27,t=3.035,P<0.05).Conclusion NFIL3 promotes proliferationand invasion ofbreast cancer cells.