肌钙蛋白相关蛋白对肺腺癌细胞增殖与迁移的影响及与患者预后的关系

Effect of troponin-related proteins on proliferation and migration of lung adenocarcinoma cells and its relationship with prognosis of patients

目的探讨肌钙蛋白相关蛋白(TROAP)对肺腺癌细胞生物学功能和免疫细胞浸润的影响及与患者预后的关系。方法选取2014-01-01-2016-06-30在河北省胸科医院接受手术治疗并经病理确诊的68例肺腺癌患者癌及癌旁正常组织(距肿瘤边缘>5 cm)标本为研究对象。通过癌症基因组图谱(TCGA)数据库和基因表达综合数据库(GEO)分析TROAP mRNA在肺腺癌中的表达及与临床病理特征和预后的关系;采用实时荧光定量聚合酶链式反应(qRT-PCR)、蛋白质印迹法和免疫组织化学方法检测TROAP在肺腺癌组织和细胞株中的表达;构建TROAP干扰序列并将其转染入A549细胞中,将其分为空白对照组(Blank组)、阴性对照组(si-NC)、转染靶向TROAP的siRNA组(si-TROAP-1组和si-TROAP-2组);细胞计数盒8(CCK8)、克隆形成、Transwell和划痕实验检测细胞增殖、迁移和侵袭能力;通过GO功能和KEGG通路富集分析TROAP在肺腺癌中的生物学功能;采用单样本基因集富集分析(ssGSEA)探索TROAP表达水平与肿瘤免疫浸润的关系。结果TROAP在TCGA、GSE19804和GSE43458数据集中肺腺癌的表达水平均高于肺正常组织,t值分别为12.260、5.547和6.848,均P<0.001。TROAP表达与TNM分期(χ^(2)=11.708,P<0.001)、肿瘤大小(χ^(2)=5.968,P=0.015)和淋巴结转移(χ^(2)=18.634,P<0.001)有关联,差异均有统计学意义。肺腺癌患者预后显示,TROAP表达与患者总生存期(OS)和无进展生存期(PFS)均有关联,χ^(2)值分别为13.310和10.040,P值分别为<0.001和0.002,且TROAP高表达(HR=4.724,95%CI:1.307~17.071,P=0.018)和肿瘤远处转移(HR=4.906,95%CI:1.234~19.508,P=0.024)可作为肺腺癌的独立预后因子。CCK8增殖实验结果显示,72 h时si-NC组、si-TROAP-1组和si-TROAP-2组细胞生长率分别为0.897±0.060、0.653±0.021和0.513±0.045,差异有统计学意义,F=55.510,P<0.001。克隆形成实验结果显示,si-NC组、si-TROAP-1组和si-TROAP-2组细胞克隆形成数量分别为59.330±4.040、17.330±2.517和21.000±5.292,差异有统计学意义,F=96.130,P<0.001。细胞凋亡结果显示,si-NC组、si-TROAP-1组和si-TROAP-2组细胞凋亡率分别为(12.390±1.072)%、(37.850±6.914)%和(33.520±2.275)%,差异有统计学意义,F=30.860,P<0.001。细胞划痕实验结果显示,24 h时si-NC组、si-TROAP-1组和si-TROAP-2组细胞愈合度分别为(61.790±1.524)%、(23.320±0.386)%和(23.520±1.781)%,差异有统计学意义,F=782.500,P<0.001。Transwell侵袭实验结果显示,si-NC组、si-TROAP-1组和si-TROAP-2组穿过小室的细胞数量分别为为(100.000±4.847)、(43.850±3.026)和(39.540±6.482)个,差异有统计学意义,F=137.200,P<0.001。TROAP可能参与细胞周期和DNA复制通路等过程,错误发现率<0.05,P<0.05。结论TROAP在肺腺癌中高表达与患者的不良预后相关;沉默TROAP可抑制肺腺癌细胞A549增殖、侵袭和迁移;TROAP表达可能与肿瘤免疫浸润细胞相关,表明TROAP可能是肺腺癌患者预后标志物。

Objective An Investigation into the Expression of Troponin-Related Protein(TROAP)in lung adenocarcinoma and its effects on the biological function of lung adenocarcinoma cells and infiltration of immune cells.Methods This study involved 68 samples of lung adenocarcinoma tissues obtained from patients who received surgical treatment and were pathologically diagnosed at Hebei Chest Hospital between January 1,2014,and June 30,2016.Additionally,20 samples of adjacent normal tissues(located more than 5cm away from the tumour edge)were included as the subjects of this research.An analysis was conducted on the expression of TROAP mRNA in lung adenocarcinoma using the Cancer Genome Atlas(TCGA)database and the Gene Expression Omnibus(GEO)database.The study also examined the link between TROAP mRNA expression and clinicopathological characteristics as well as prognosis,TROAP expression in lung cancer tissues and cell lines was assessed using real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR),Western blotting,and immunohistochemistry techniques.The TROAP interference sequence was created and introduced into A549 cells by transfection.The cells were subsequently categorised into four groups:a control group with no treatment(Blank group),a control group with a non-targeting siRNA(si-NC),and two groups treated with siRNA that specifically targeted TROAP(si-TROAP-1 group and si-TROAP-2 group).The cells ability to multiply,move,and invade other cells was measured using cell counting kit 8(CCK8),colony formation,transwell,and wound healing tests.An analysis was conducted to examine the biological roles of TROAP in lung cancer using GO function and KEGG pathway enrichment.In addition,we investigated the correlation between TROAP expression levels and the extent of immune cell infiltration in tumours using single-sample gene set enrichment analysis(ssGSEA).Results In the TCGA.GSE19804,and GSE43458 datasets,the levels of TROAP expression were found to be higher in lung adenocarcinoma compared to normal lung tissues.The t-values for these comparisons were 12,260,5.547 and 6.848,respectively,all with a significance level of P<0.001.The expression of TROAP showed a strong correlation with TNM staging(χ^(2)=11.708,P<0.001),tumour size(χ^(2)=5.968,P=0.015),and lymph node metastasis(χ^(2)=18.634,P<0.001),indicating substantial differences between the groups.The analysis of patients with lung adenocarcinoma showed that the expression of TROAP was linked to both overall survival and progression-free survival.Theχ^(2)values for overall survival and progression-free survival were 13.310 and 10.040,respectively,with corresponding P-values of<0.001 and 0.002.Moreover,the study found that a high level of TROAP expression(HR=4.724,95%CI:1.307-17.071,P=0.018)and the presence of distant metastasis in the tumour(HR=4.906,95%CI:1.234-19.508,P=0.024)were identified as separate prognostic indicators for lung adenocarcinoma.The CCK8 proliferation assay results indicated that the cell growth rates at72 hours were 0.897±0.060,0.653±0.021,and 0.513±0.045 in the si-NC group,si-TROAP-1 group,and siTROAP-2 group,respectively.A substantial statistical difference was seen between the groups(F=55.510,P<0.001).The clone formation experiment yielded the following results:the si-NC group had 59.330±4.040 cell clones,the si-TROAP-1 group had 17.330±2.517 cell clones,and the si-TROAP-2 group had 21.000±5.292 cell clones.There was a statistically significant difference between the groups(F=96.130,P<0.001).The cell apoptosis data demonstrated that the apoptosis rates in the si-NC group,si-TROAP-1 group,and si-TROAP-2 group were(12.390±1.072)%,(37.850±6.914)%and(33.520±2.275)%,respectively.There was a statistically significant difference in apoptosis rates across the groups(F=30.860,P<0.001).The wound healing assay results indicated that the cell healing rates at24 hours were(61.790±1.524)%in the si-NC group,(23.320±0.386)%in the si-TROAP-1 group,and(23.520±1.781)%in the si-TROAP-2 group.A statistically significant difference was observed between the groups,with a large effect size(F=782.500,P<0.001).The findings of the Trans well invasion experiment showed that the number of cells that entered the chamber in the si-NC group,si-TROAP-1 group,and si-TR,AP-2 group were 100.000±4.847,43.850±3.026,and 39.540±6.482,respectively.Furthermore,there was a notable and meaningful distinction observed between the groups,as indicated by the statistical analysis(F=137.200,P<0.001).TROAP was potentially implicated in cellular processes such as the cell cycle and DNA replication pathways,with a false discovery rate(FDR)below 0.05 and a P value below 0.05.Conclusions Patients with lung adenocarcinoma who have a high expression of TROAP are more likely to have a bad prognosis.Suppressing the expression of TROAP can impede the growth,infiltration,and movement of A549 lung cancer cells.Furthermore,the presence of TROAP in tumor-infiltrating immune cells indicates a potential correlation,implying that TROAP could be utilised as a prognostic indicator for patients with lung adenocarcinoma.