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目的 通过生物信息学方法筛选影响宫颈癌辐射敏感性的差异基因并进行表达验证以及免疫相关性分析.方法 从GEO数据库下载包含放疗前后宫颈癌组织、正常宫颈组织、宫颈癌组织的数据集,使用R软件筛选其中的差异基因,使用韦恩图取交集筛选宫颈癌辐射敏感性差异基因.从TCGA数据库及GTEx数据库收集宫颈癌组织样本、正常宫颈组织样本的表达数据及患者临床资料、随访数据,通过GEPIA2数据库对筛选出的宫颈癌辐射敏感性差异基因进行表达分析,验证所选差异基因是否可靠.通过GEPIA2数据库进行生存分析,筛选不同基因表达水平对患者预后有影响的差异基因为宫颈癌辐射敏感性关键差异基因;通过R软件对患者临床资料及预后情况进行单因素、多因素COX回归分析,验证筛选出的关键差异基因是否为宫颈癌患者预后的影响因素;通过HPA数据库比较筛选出的关键差异基因在宫颈癌以及正常宫颈组织中的蛋白表达.通过基因集富集分析对关键差异基因的GO功能及KEGG信号通路进行富集分析,通过TIMER数据库分析关键差异基因在宫颈癌中的表达与免疫细胞浸润水平的关系,通过TCGA数据库数据观察免疫检查点相关基因的表达情况,并对关键差异基因与宫颈癌高度相关的免疫检查点基因进行Spearman相关性分析.结果 共筛选出宫颈癌辐射敏感性差异基因10个,分别为HELLS、TOP2A、KIF23、TAP1、WDR76、LMNB1、RFC4、TFRC、IFI44L、IFIT3;10个差异基因均在宫颈癌组织中高表达.生存分析结果显示,TFRC高表达的宫颈癌患者总生存率及无病生存率低于TFRC低表达的患者,故选择TFRC为宫颈癌辐射敏感性关键差异基因.单因素COX回归分析结果显示,TFRC及宫颈癌TNM分期是宫颈癌患者预后的影响因素;多因素COX回归分析结果显示,TFRC及宫颈癌TNM分期是宫颈癌患者预后的独立影响因素.通过HPA数据库验证TFRC在宫颈癌组织和正常宫颈组织中的蛋白表达,结果显示TFRC在正常宫颈组织中未检测到,在宫颈癌组织中呈高表达.GO功能分析显示,TFRC主要涉及染色体分离、RNA定位、适应性免疫反应等生物过程,染色体区、核糖前体、免疫突触等细胞成分,解旋酶活性、组蛋白结合、抗原结合等分子功能.KEGG信号通路富集分析显示,TFRC主要与RNA转运、细胞周期、同种异体移植物排斥反应等信号通路有关.TIMER数据库分析结果显示,宫颈癌中TFRC表达与细胞纯度呈正相关,与CD8+ T淋巴细胞呈负相关,TFRC表达影响记忆B淋巴细胞、CD8+ T淋巴细胞、巨噬细胞M0、静息肥大细胞的浸润程度.Spearman相关性分析显示,TFRC表达与大部分宫颈癌免疫检测点相关基因呈负相关.结论 宫颈癌辐射敏感性差异基因为TFRC,其可能与适应性免疫反应、宫颈癌相关的免疫检测点相关;这提示其可在宫颈癌的放射免疫治疗中发挥作用,从而影响宫颈癌的进展和预后.
Objective To screen the differentially expressed genes(DEGs)affecting radiosensitivity of cervical can-cer and to conduct the expression validation and immune correlation analysis by bioinformatics methods.Methods The datasets of cervical cancer tissues before or after radiotherapy,normal cervical tissues and cervical cancer tissues were downloaded from Gene Expression Database(GEO),and the DEGs of cervical cancer were screened by Rstudio software,and Venn diagram was used to screen DEGs for radiosensitivity of cervical cancer.The expression data of cervical cancer tissue samples and normal cervical tissue samples,and clinical information and follow-up data of the patients were collect-ed from TCGA database and GTEx database.DEGs for radiosensitivity of cervical cancer were screened and their expres-sion levels were analyzed from GEPIA2 database,and then the reliability of selected DEGs were verified.The key DEGs for radiosensitivity of cervical cancer were screened through the effects of different gene expression levels on patients'prog-nosis by survival analyses from GEPIA2 database.The clinical data and prognosis of patients were porformed by single fac-tor and multi-factor COX regression analyses to verify the screened key DEGs as the influencing factors on the prognosis of cervical cancer patients by Rstudio software.The protein expression of screened key DEGs between cervical cancer and normal cervical tissues was compared through HPA database.The gene enrichment analysis was set for GO function and KEGG signaling pathways of key DEGs.The relationship between the expression of key DEGs in cervical cancer and the in-filtration level of immune cells was analyzed through TIMER database.The expression of immune checkpoint-related genes were observed through TCGA database.Spearman correlation analysis was conducted on the key DEGs and immune check-point genes highly associated with cervical cancer.Results Ten DEGs with cervical cancer radiosensitivity were screened out,which were HELLS,TOP2A,KIF23,TAP1,WDR76,LMNB1,RFC4,TFRC,IFI44L and IFIT3.All 10 DEGs were highly expressed in the cervical cancer tissues.Survival analysis results showed that the overall survival and disease-free survival of cervical cancer patients with high TFRC expression were lower than those of patients with low TFRC expression,so TFRC was selected as the key DEG for radiosensitivity of cervical cancer.TFRC and TNM stage of cervical cancer were both the influential factors for the prognosis of cervical cancer through the single factor COX regression analy-sis,but were the independent risk factors through the multi-factor COX regression analysis.The protein expression of TFRC in cervical cancer tissues and normal cervical tissues was verified through HPA database,and the results showed that TFRC was highly expressed in cervical cancer tissue,but not detected in normal cervical tissues.GO functional analy-sis showed that TFRC was mainly involved in the biological processes including chromosome segregation,RNA localization and adaptive immune response,the cellular components containing chromosomal regions,ribosomal precursors,and im-mune synapses,and the molecular functions such as deconjugating enzyme activity,histone binding,and antigen binding,etc.KEGG signaling pathway enrichment analysis showed that TFRC was mainly associated with the signaling pathways such as RNA transporters,cell cycle,and allogeneic graft rejection response,etc.TIMER database analysis showed that TFRC expression in cervical cancer was positively correlated with cell purity and was negatively correlated with CD8+ T lymphocytes,and it could affect the infiltration degree of memory B lymphocytes,CD8+ T lymphocytes,macrophage M0 and resting mast cells.Spearman correlation analysis showed that TFRC expression was negatively correlated with most of immune checkpoint-related genes for cervical cancer.Conclusion TFRC is DEG for radiosensitivity of cervical cancer,which may be associated with adaptive immune responses and cervical cancer-related immune checkpoint,suggesting that it may play a role in the radioimmunotherapy for cervical cancer and influence its progression and prognosis.