非红细胞血影蛋白-β2在肺腺癌组织中的表达及其对肺腺癌细胞迁移、增殖、侵袭的影响

Expression of spectrin beta in lung adenocarcinoma and its effect on migration, proliferation and invasion of lung adenocarcinoma cells

目的探讨非红细胞血影蛋白-β2(SPTBN2)在肺腺癌(LUAD)组织中的表达水平及其对肺腺癌细胞体外迁移、增殖、侵袭的影响。方法通过基因表达数据库(GEO)下载的3个肺腺癌数据集验证SPTBN2在肺腺癌与正常肺组织中的差异表达。通过癌症基因组图谱(TCGA)分析SPTBN2在肺腺癌(LUAD)患者的预后中所起到的作用。收集2019年7月至2020年9月在郑州大学第一附属医院胸外科经手术证实为肺腺癌患者40例,收取癌组织标本及相应的癌旁组织标本,采用实时定量反转录聚合酶链反应(RT-qPCR)检测肺腺癌组织和癌旁组织中SPTBN2的mRNA表达水平。选择人肺腺癌细胞系A549按实验转染方案随机分组,分为空白(Blank)组、沉默SPTBN2 (si-SPTBN2)阴性对照(NC)组、si-SPTBN2组。采用细胞计数试剂盒(CCK-8)法检测细胞增殖能力;采用划痕实验及Transwell小室法检测细胞迁移和侵袭能力。计量资料组间比较采用t检验。结果分析GSE10072(t=7.552,P<0.01),GSE19188(t=8.059,P<0.01)和GSE32863(t=9.196,P<0.01)数据集,SPTBN2在肺腺癌组织中的表达水平明显高于正常肺组织。TCGA数据库KM-PLOTTER生存分析网站进行生物信息学分析,显示SPTBN2高表达是肺腺癌患者预后的危险因素(P<0.01)。实时荧光定量RT-qPCR检测肺腺癌组织中SPTBN2的表达(7.922±0.956)高于正常肺组织(5.726±1.343,t=3.766,P<0.01),差异有统计学意义。CCK-8结果显示敲低si-SPTBN2实验组的细胞增殖能力明显低于si-SPTBN2 NC对照组(24 h:0.439±0.011比0.495±0.007,t=7.974,P<0.01;48 h:0.509±0.022比0.659±0.023,t=9.577,P<0.01;72 h:0.917±0.033比1.687±0.027,t=34.320,P<0.01;96 h:1.294±0.749比2.930±0.059,t=3.660,P<0.01)。划痕实验结果显示,si-SPTBN2的实验组在划痕48 h后相较于对照组迁移距离更短[划痕愈合比:(36.56±3.02)%比(51.94±5.72)%,P<0.01]。Transwell迁移、侵袭实验结果表明,敲低SPTBN2可使肺腺癌细胞的迁移能力显著低于对照组[(362±18)个比(977±25)个,t=35.750,P<0.01],侵袭能力显著低于对照组[(152±9)个比(770±34)个,t=43.920,P<0.01],差异均有统计学意义。结论 SPTBN2在肺腺癌中高表达,与不良预后相关,具有促进肺腺癌细胞增殖、迁移和侵袭的作用。

Objective To explore the expression of the gene encodingβ-Ⅲspectrin beta(SPTBN2)in lung adenocarcinoma(LUAD)and its effect on the migration,proliferation and invasion of LUAD cells in vitro.Methods A total of 3 datasets were downloaded from gene expression omnibus(GEO)database to verify the differential expression of SPTBN2 in LUAD and normal lung tissues.The Cancer Genome Atlas(TCGA)database analysis was used to study the relationship between the expression of SPTBN2 and its role in the prognosis of LUAD patients.Cancer tissue specimens and adjacent normal lung tissue specimens of 40 patients with LUAD who underwent radical surgery in the Department of Thoracic Surgery of The First Affiliated Hospital of Zhengzhou University from July 2019 to September 2020 were collected.The mRNA expression levels of SPTBN2 in LUAD and normal lung tissues were detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).Human lung adenocarcinoma cell line A549 was transfected for the construction of blank group,silencing SPTBN2(si-SPTBN2)negative control(NC)group,si-SPTBN2 group.The proliferation ability was detected by cell counting kit-8(CCK-8)assay.Scratch test and Transwell chamber method were used to detect the effect on the migration and invasion of LUAD cells,respectively.T-test was used for metrology data comparison between groups.Results In GSE10072(t=7.552,P<0.01),GSE19188(t=8.059,P<0.01)and GSE32863(t=9.196,P<0.01),the mRNA expression levels of SPTBN2 in LUAD were significantly higher than those in normal lung tissues.TCGA database KM-PLOTTER analysis revealed that SPTBN2 was a risk factor for the prognosis of LUAD(P<0.01).The result of RT-qPCR showed that the expression levels of SPTBN2 in LUAD and normal lung tissues were(7.922±0.956)and(5.726±1.343),respectively.In si-SPTBN2 knockdown group,cell proliferation ability significantly decreased(24 h:0.439±0.011 vs.0.495±0.007,t=7.974,P<0.01;48 h:0.509±0.022 vs.0.659±0.023,t=9.577,P<0.01;72 h:0.917±0.033 vs.1.687±0.027,t=34.320,P<0.01;96 h:1.294±0.749 vs.2.930±0.059,t=3.660,P<0.01).The scratch experiment showed that in the SPTBN2 knockdown group,migration distance was significantly shorter than in the control group after 48 h of scratching[scratch healing ratio:(36.56±3.02)%vs.(51.94±5.72)%,P<0.01].Transwell assay showed that the number of migrating cells[(362±18)cells vs.(977±25)cells,t=35.750,P<0.01]and number of invasive cells[(152±9)cells vs.(770±34)cells,t=43.920,P<0.01]in SPTBN2 knockdown group were significantly reduced as compared with that in the control group.Conclusion SPTBN2 is highly expressed in LUAD,positively correlated with poor prognosis,and can promote the proliferation,migration and invasion of LUAD cells.