盐诱导激酶2通过调控转化生长因子-β/上皮-间充质转化通路抑制三阴性乳腺癌迁移和侵袭的机制

Molecular mechanism of inhibitory effect of salt-induced kinase 2 on migration and invasion of triple-negative breast cancer by regulating the transforming growth factor-β/epithelial-mesenchymal transition pathway

目的分析和验证盐诱导激酶2(SIK2)在三阴性乳腺癌(TNBC)中的表达并探究其对TNBC细胞迁移侵袭能力的影响及机制。方法利用Ualcan在线分析工具探索SIK2在TNBC中表达情况,结合8对TNBC组织标本运用荧光定量PCR技术进行验证。构建SIK2重组过表达质粒(GV703-SIK2)并包装重组慢病毒。分别感染TNBC细胞株MDA-MB-231和HCC1937,经嘌呤霉素筛选构建SIK2稳定过表达细胞株和阴性对照细胞株。用蛋白质印迹法(Western blot)检测转染后TNBC细胞株中SIK2表达水平,通过划痕实验和Transwell小室实验观察SIK2过表达对TNBC细胞迁移侵袭能力的影响;采用Western blot分析SIK2过表达后对上皮型钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)、锌指转录因子SNAI1蛋白(Snail)、锌指转录因子SNAI2(Slug)、磷酸化SMAD家族成员2(p-SMAD2)蛋白、磷酸化SMAD家族成员3(p-SMAD3)蛋白表达水平的影响。组间比较采用t检验。结果SIK2 mRNA在TNBC组织中表达明显低于其配对癌旁组织(t=2.199,P<0.05)。SIK2过表达组MDA-MB-231、HCC1937细胞SIK2蛋白表达水平(2.105±0.148、0.942±0.204)明显高于其对照组(0.053±0.007、0.024±0.007),差异有统计学意义(t=24.030、7.792,P<0.01)。划痕实验结果显示SIK2过表达组MDA-MB-231、HCC1937细胞划痕愈合率[(61.700±5.126)%、(58.360±14.380)%]均明显低于其对照组[(87.870±1.097)%、(91.410±7.486)%],差异有统计学意义(t=8.645、3.530,P<0.05)。Transwell小室实验结果显示SIK2过表达组MDA-MB-231、HCC1937细胞穿过小室数量[(111.700±15.310)、(101.700±14.220)个]明显低于其对照组[(183.000±19.970)、(187.000±15.100)个],差异有统计学意义(t=4.909、7.125,P<0.01)。SIK2过表达组E-cadherin表达水平(0.754±0.186)明显高于对照组(0.298±0.066),差异有统计学意义(t=3.995,P<0.05);而Vimentin、Snail、Slug、p-SMAD2蛋白、p-SMAD3蛋白的表达水平(0.595±0.144、0.429±0.179、0.609±0.125、0.198±0.090、0.659±0.088)均明显低于对照组(1.112±0.201、0.959±0.198、0.920±0.140、0.497±0.059、1.248±0.148),差异有统计学意义(t=3.624、3.439、2.872、4.804、5.926,P<0.05)。结论SIK2在TNBC中呈低表达,推测SIK2是通过调控转化生长因子-β(TGF-β)/上皮-间充质转化(EMT)通路抑制TNBC细胞的EMT、迁移和侵袭过程。

Objective To analyze and verify the expression of salt-induced kinase 2(SIK2)in triple negative breast cancer(TNBC),and explore its influence and molecular mechanism on migration and invasion ability of TNBC cells.Methods The expression of SIK2 mRNA in TNBC was explored by Ualcan,and verified by fluorescence quantitative PCR through the 8 pairs of TNBC tissue samples.The recombinant SIK2 overexpressed vector GV703-SIK2 was constructed and the recombinant lentiviruses were packaged and infected into TNBC cells MDA-MB-231 and HCC1937.The SIK2 stable overexpressed and negative control cell lines were constructed by puromycin screening.The expression level of SIK2 protein in the infected cells was detected by Western blotting.Wound healing and Transwell chamber assays were used to observe the effects of SIK2 up-regulation on the migration and invasion ability of TNBC cells.The influence of SIK2 overexpression on the expression levels of E-cadherin,Vimentin,Snail,Slug,p-SMAD2 and p-SMAD3 proteins was analyzed by Western blotting.The comparison between groups was carried out by t-test.Results The expression of SIK2 mRNA in TNBC tissues was significantly lower than that in their normal counterparts(t=2.199,P<0.05).The protein expression levels of SIK2 in stable overexpression cell lines(MDA-MB-231,HCC1937)(2.105±0.148,0.942±0.204)were significantly higher than those in their control(0.053±0.007,0.024±0.007,t=24.030,7.792,P<0.01).The wound healing rates of MDA-MB-231 and HCC1937 cells in SIK2 overexpression group[(61.700±5.126)%,(58.360±14.380)%]were significantly lower than those in their controls[(87.870±1.097)%,(91.410±7.486)%,t=8.645,3.530,P<0.05].The number of invasive cells of MDA-MB-231 and HCC1937 cells in SIK2 overexpression group(111.700±15.310,101.700±14.220)was significantly less than that in their controls(183.000±19.970,187.000±15.100,t=4.909,7.125,P<0.01).The expression level of E-cadherin in SIK2 overexpression group(0.754±0.186)was significantly higher than that in their controls(0.298±0.066,t=3.995,P<0.05).The expression level of Vimentin,Snail,Slug,p-smad2,p-smad3(0.595±0.144,0.429±0.179,0.609±0.125,0.198±0.090,0.659±0.088)was significantly lower than in their controls(1.112±0.201,0.959±0.198,0.920±0.140,0.497±0.059,1.248±0.148,t=3.624,3.439,2.872,4.804,5.926,P<0.05).Conclusion SIK2 is downregulated in TNBC,which is supposed to inhibit the epithelial-mesenchymal transition(EMT),migration and invasion of TNBC cells through regulating transforming growth factor-β(TGF-β)/EMT pathway.