Table_5_A TIMM17A Regulatory Network Contributing to Breast Cancer.DOCX

BackgroundTranslocase of inner mitochondrial membrane 17A (TIMM17A) is overexpressed in breast cancer (BRCA), and upregulation can increase the aggressiveness of BRCA cells. This study examined the influence of the TIMM17A gene network on BRCA outcome.
MethodsExpression levels of TIMM17A were compared between normal and tumor tissues from the OncomineTM database, and the association with patient survival was analyzed using Kaplan–Meier Plotter. Clinical factors influencing TIMM17A expression were studied by UALCAN. cBioPotal was then used to identify genes interacting with TIMM17A, and network relationships were assessed using the R clusterProfiler package. The association between TIMM17A mutation and mRNA expression in BRCA was examined using the LinkFinder application in LinkedOmics, and coexpressed genes were assessed for functional enrichment using the LinkInterpreter application. Furthermore, TIMM17A expression correlation with cell cycle phase distribution was performed by flow cytometry. Finally, the target networks of kinases, microRNAs (miRNAs), and transcription factors were identified using GeneMANIA. The expression and correlation of potential miRNAs and targets were further validated in BRCA cell lines by qRT-PCR.
ResultsExpression of TIMM17A was significantly elevated in BRCA compared with normal tissue (p < 0.05), and overexpression was associated with both poor overall survival (OS) and shorter distant metastasis-free survival (DMFS) (p < 0.05). Expression of TIMM17A was not associated with age, sex, BRCA subclass, clinical stage, or patient ethnicity. The coexpressed TIMM17A network was enriched in genes targeted by cell cycle regulators such as CDK1, miR-331, and E2F family transcription factors (FDR < 0.001). Furthermore, flow cytometry revealed a strong association between higher TIMM17A expression and faster cell cycle progression in these BRCA cell lines. In addition, expression of TIMM17A protein was correlated with CDK1 protein expression in BRCA cell lines as measured by western blotting.
ConclusionElevated TIMM17A expression accelerates the progression of BRCA, thereby reducing OS and DMFS. The TIMM17A-associated networks identified here provide clues to the molecular pathogenesis of BRCA and potential targets for BRCA treatment.

背景 线粒体内膜转位酶17A（TIMM17A）在乳腺癌（BRCA）中呈过表达状态，其表达上调可增强乳腺癌细胞的侵袭能力。本研究探讨了TIMM17A基因调控网络对乳腺癌预后的影响。方法 本研究从Oncomine™数据库中提取正常组织与肿瘤组织的TIMM17A表达水平，通过卡普兰-迈耶绘图仪（Kaplan–Meier Plotter）分析其与患者生存的相关性；利用UALCAN数据库探究影响TIMM17A表达的临床因素。随后通过cBioPortal数据库筛选与TIMM17A存在相互作用的基因，并采用R语言clusterProfiler包分析基因网络调控关系。借助LinkedOmics数据库中的LinkFinder工具分析BRCA中TIMM17A突变与mRNA表达的关联，并通过LinkInterpreter工具对共表达基因进行功能富集分析。此外，采用流式细胞术检测TIMM17A表达与细胞周期时相分布的相关性。最后，利用GeneMANIA数据库鉴定激酶、微小RNA（microRNAs，miRNAs）及转录因子的靶向调控网络，并通过实时定量聚合酶链反应（quantitative real-time polymerase chain reaction，qRT-PCR）在BRCA细胞系中验证潜在miRNAs及其靶基因的表达与相关性。结果 与正常组织相比，BRCA组织中TIMM17A的表达水平显著升高（p < 0.05）；过表达TIMM17A与患者较差的总生存期（overall survival，OS）及较短的远处无转移生存期（distant metastasis-free survival，DMFS）均显著相关（p < 0.05）。TIMM17A的表达水平与患者年龄、性别、乳腺癌亚型、临床分期及种族均无显著关联。TIMM17A共表达基因网络显著富集于细胞周期调控因子（如细胞周期蛋白依赖性激酶1（cyclin-dependent kinase 1，CDK1）、miR-331及E2F家族转录因子）的靶基因集合中（错误发现率（false discovery rate，FDR）< 0.001）。流式细胞术结果显示，在BRCA细胞系中，TIMM17A高表达与更快的细胞周期进程显著相关。此外，蛋白质印迹（western blotting）实验结果表明，BRCA细胞系中TIMM17A蛋白的表达与CDK1蛋白表达呈正相关。结论 TIMM17A过表达可加速BRCA的疾病进展，进而缩短患者的OS与DMFS。本研究鉴定的TIMM17A相关调控网络为乳腺癌的分子发病机制提供了新的研究线索，同时也为乳腺癌治疗提供了潜在的药物靶点。