树突表达特异性7跨膜蛋白促进甲状腺癌细胞增殖、迁移和侵袭的机制

Mechanism of dendritic-cell specific 7 transmembrane protein promoting proliferation,migration,and invasion of thyroid papillary carcinoma cells

目的观察树突表达特异性7跨膜蛋白(DCSTAMP)对甲状腺乳头状癌细胞增殖、迁移和侵袭的影响, 并探讨其机制。方法在基因表达谱交互式分析平台(GEPIA)中分析DCSTAMP在甲状腺乳头状癌组织中的表达;采用实时荧光定量聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)检测甲状腺乳头状癌细胞系KTC-1、FTC-133及甲状腺正常细胞Nthy-ori 3-1中DCSTAMP表达的差异;在KTC-1和FTC-133细胞中分别转染过表达和敲低质粒及其对照, 构建DCSTAMP过表达和敲低细胞模型;过表达组通过细胞增殖/毒性检测试剂盒(CCK-8)、5-乙炔基-2’-脱氧尿嘧啶核苷(EdU)细胞增殖检测试剂盒检测DCSTAMP基因过表达后对增殖的影响;蛋白印迹法验证过表达后对细胞核增殖抗原(Ki-67)蛋白、丝裂原活化蛋白激酶(MAPK)通路的影响;敲低组通过细胞划痕、侵袭测定实验(Transwell)实验检测DCSTAMP基因敲低后对迁移、侵袭的影响;蛋白印迹法验证敲低后对神经钙黏着蛋白(NCAD)、磷酸化蛋白激酶B蛋白(p-Akt)、总蛋白激酶B蛋白(t-Akt)表达的影响;两样本均数比较采用成组配对t检验分析。结果 RT-qPCR和Western blot结果均显示DCSTAMP基因在甲状腺乳头状癌细胞KTC-1、FTC-133中的表达高于甲状腺正常细胞Nthy-ori 3-1(RT-qPCR:DCSTAMP基因在Nthy-ori 3-1、KTC-1、FTC-133细胞中mRNA相对表达量分别为1.020±0.202、17.350±3.326、8.684±0.030, Nthy-ori 3-1与KTC-1比较:t=4.901, P<0.05;Nthy-ori 3-1与FTC-133比较:t=37.460, P<0.01;Western blot:DCSTAMP基因在Nthy-ori 3-1、KTC-1、FTC-133细胞中蛋白相对表达量分别为1.000±0.009、3.227±0.444、3.367±0.073, Nthy-ori 3-1与KTC-1比较:t=9.628, P<0.01;Nthy-ori 3-1与FTC-133比较:t=55.580, P<0.01);KTC-1、FTC-133细胞中DCSTAMP过表达组细胞增殖速度高于对照组(KTC-1对照组与过表达组第3天相对增殖速度:0.215±0.010:0.397±0.029, t=6.198, P<0.01;FTC-133对照组与过表达组第3天相对增殖速度:0.350±0.364:0.432±0.025, t=33.640, P<0.01), 过表达组增殖能力高于对照组[KTC-1对照组比过表达组:(8.382±1.400)%比(91.253±1.699)%, t=65.220, P<0.01;FTC-133对照组与过表达组:(14.385±2.441)%比(91.332±1.159)%, t=49.320, P<0.01], 过表达组Ki-67表达高于对照组(KTC-1对照组与过表达组蛋白相对表达量:1.000±0.038比1.282±0.134, t=9.968, P<0.01;FTC-133对照组与过表达组蛋白相对表达量:1.000±0.011比1.382±0.221, t=40.920, P<0.01), 过表达组MAPK表达高于对照组(KTC-1对照组与过表达组:1.000±0.012比1.088±0.057, t=10.720, P<0.01;FTC-133对照组与过表达组:1.000±0.039比1.220±0.123, t=9.404, P<0.01), 过表达组MAPK通路亚家族细胞外信号调节激酶(ERK)蛋白表达高于对照组:(1.000±0.005比7.047±0.088, t=68.650, P<0.01;FTC-133对照组与过表达组:1.000±0.003比1.983±0.029, t=34.000, P<0.01);而在KTC-1、FTC-133中敲低DCSTAMP基因后, 对照组细胞迁移速度高于敲低组(KTC-1对照组与敲低组:(72.545±0.704)%比(45.016±2.156)%, t=21.020, P<0.01;FTC-133对照组与敲低组:(82.422±0.196)%比(49.824±5.402)%, t=10.450, P<0.01), 对照组侵袭能力高于敲低组(KTC-1对照组与敲低组:(320.667±13.013)%比(172.333±11.240)%, t=14.940, P<0.01;FTC-133对照组与敲低组侵袭细胞数:302.667±24.111比228.000±14.422, t=4.603, P<0.01), p-Akt表达对照组高于敲低组(KTC-1对照组与敲低组:1.000±0.029比0.376±0.355, t=21.980, P<0.01;FTC-133对照组与敲低组:1.000±0.010比0.809±0.110, t=35.900, P<0.01)。结论 DCSTAMP基因通过激活MAPK通路亚家族ERK及Akt磷酸化在甲状腺乳头状癌细胞系中可以促进肿瘤细胞增殖、迁移和侵袭。

Objective To investigate the effect and mechanism of dendritic-cell specific 7 trans-membrane protein(DCSTAMP)on the proliferation,migration,and invasion of thyroid papillary carcinoma cells.Methods The expression of DCSTAMP in thyroid papillary carcinoma was analyzed in GEPIA data-base.The expression of DCSTAMP between thyroid papillary carcinoma cell lines KTC-1,FTC-133 and thyroid normal cell line Nthy-ori 3-1 was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blotting.The knockdown and overexpression plasmids and their controls were transfected respectively into KTC-1 and FTC-133 cells to construct DCSTAMP overexpression and knockdown cell models.In the knockdown group,the effect of DCSTAMP gene knockdown on migration and invasion was detected by cell scratch and Transwell test.Western blotting was used to verify the effect of knockdown on the expression of NCAD,phosphorylated protein kinase B(p-Akt)and total protein ki-nase B(t-Akt)proteins.In the overexpression group,the effect of overexpression of DCSTAMP gene on proliferation was detected using the cell counting kit-8(CCK-8)and the EdU cell proliferation test kit(DAB).Western blotting was used to verify the effect of overexpression on proliferation cell nuclear antigen(Ki-67)protein and mitogen-activated protein kinase(MAPK)pathway.The mean of the two samples was compared by paired t test analysis.Results Both RT-qPCR and Western blotting results showed that the expression of DCSTAMP gene in KTC-1 and FTC-133 was significantly higher than that in Nthy ori 3-1[RT-qPCR(KTC-1:t=4.901,P<0.05;FTC-133:t=37.460,P<0.01);Western bloting(KTC-1:t=9.628,P<0.01;FTC-133:t=55.580,P<0.01)].When the DCSTAMP gene was overexpressed in thy-roid cancer cells KTC-1 and FTC-133,the rate of cell proliferation was increased(KTC-1 on the day 3:t=6.198,P<0.01;FTC-133 on the day 3:t=33.640,P<0.0005),and the proliferation ability was in-creased(KTC-1:t=65.220,P<0.01;FTC-133:t=49.320,P<0.01).The expression of Ki-67 and MAPK was increased(P<0.01).The MAPK pathway subfamily extracellular signal-regulated kinase(ERK)showed varying degrees of elevation(KTC-1:t=68.650,P<0.01;FTC-133:t=34.000,P<O.01).After knockdown of DCSTAMP gene in KTC-1 and FTC-133,the cell migration rate slowed down(KTC-1:t=21.020,P<0.01;FTC-133:t=10.450,P<0.01).The invasion ability decreased(KTC-1:t=14.940,P<0.01;FTC-133:t=4.603,P<0.01).The p-Akt expression decreased(KTC-1:t=21.980,P<0.01;FTC-133:t=35.900,P<0.01).Conclusion The increased expression of DCSTAMP gene in thyroid papillary carcinoma cell lines KTC-1 and FTC-133 can promote tumor cell proliferation,mi-gration,and invasion through the activation of MAPK pathway,and Akt phosphorylation.