Screening and Validation of Potential Prognostic Markers of Cervical Squamous Cell Carcinoma Based on The Cancer Genome Atlas Database
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摘要:
目的 筛选并验证宫颈鳞状细胞癌（cervical squamous cell carcinoma，CESC）潜在的miRNA预后标志物，初步探究CESC发生的分子机制。 方法 利用TCGA数据库中宫颈鳞状细胞癌组织和癌旁组织的miRNA测序信息及患者临床数据，通过生物信息学分析，构建了由4个miRNAs （miR-505-5p、miR-142-3p、miR-532-5p、miR-218-1-3p）组成的预测宫颈鳞状细胞癌患者预后的风险模型并进行诊断效果评价，同时应用逆转录-聚合酶链反应（RT-PCR）检测21对CESC组织及癌旁正常组织中该4个miRNAs的表达水平，以差异表达显著的miR-505-5p作为研究对象，分析其表达水平与CESC患者各临床病理特征间的关系。在CESC细胞中转染miR-505-5p mimics后通过细胞实验探究miR-505-5p对CESC细胞功能的影响；最后通过生物信息法预测miR-505-5p的可能靶基因，并通过RT-PCR及Western blot实验进行验证。 结果 通过对TCGA数据库中宫颈鳞状细胞癌相关数据的分析，构建了由4个miRNAs（miR-505-5p、miR-142-3p、miR-532-5p、miR-218-1-3p）组成的预后风险模型，预后风险模型能有效地将CESC患者分为预后不良的高风险组和低风险组；miR-505-5p在收集到的21对CESC组织中的表达低于癌旁组织（P < 0.05），其表达量与CESC患者的临床分期和侵袭程度负相关；过表达miR-505-5p后抑制了宫颈鳞状细胞癌SiHa细胞的增殖（P < 0.05）、迁移，促进了凋亡（P < 0.05）；TBL1XR1作为miR-505-5p的最佳预测靶基因，过表达miR-505-5p后TBL1XR1的RNA水平及蛋白水平下降（P < 0.05）。 结论 miR-505-5p可作为CESC患者的潜在预后标志物，在CESC中miR-505-5p可能通过靶向TBL1XR1发挥抑癌作用。 -
关键词:
- 宫颈鳞状细胞癌 /
- 预后标记物 /
- miR-505-5p /
- TBL1XR1
Abstract:Objective To screen and verify the potential prognostic markers of miRNA in cervical squamous cell carcinoma （CESC）, and to explore the molecular mechanism of CESC. Methods Using miRNA sequencing data of cervical squamous cell carcinoma tissues and adjacent tissues in the TCGA database, as well as patient clinical data, a risk model for predicting the prognosis of cervical squamous cell carcinoma patients was constructed using bioinformatics analysis. The model consists of four miRNAs （miR-505-5p, miR-142-3p, miR-532-5p, miR-218-1-3p）. The diagnostic performance of the model was evaluated. Reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the expression levels of these four miRNAs in 21 pairs of cervical squamous cell carcinoma tissues and adjacent normal tissues. MiR-505-5p, which showed significant differential expression, was selected for further analysis to examine its relationship with various clinical pathological features of cervical squamous cell carcinoma patients. Transfection of miR-505-5p mimics into cervical squamous cell carcinoma cells was performed to investigate the impact of miR-505-5p on the cellular functions of cervical squamous cell carcinoma. Finally, potential target genes of miR-505-5p were predicted using bioinformatics methods and validated through RT-PCR and Western blot experiments. Results By analyzing the cervical squamous cell carcinoma （CESC） related data from the TCGA database, a prognostic risk model composed of 4 miRNAs （miR-505-5p, miR-142-3p, miR-532-5p, miR-218-1-3p） was constructed. The prognostic risk model effectively stratifies CESC patients into high-risk and low-risk groups for adverse prognosis. The expression of miR-505-5p in the collected 21 pairs of CESC tissues was lower than that in adjacent tissues （P < 0.05）, and its expression level was negatively correlated with the clinical stage and invasion degree of CESC patients. Overexpression of miR-505-5p inhibited the proliferation （P < 0.05） and migration of cervical squamous cell carcinoma SiHa cells, and promoted apoptosis （P < 0.05）. TBL1XR1 was identified as the best predicted target gene of miR-505-5p, and overexpression of miR-505-5p decreased the RNA and protein levels of TBL1XR1 （P < 0.05）. Conclusion miR-505-5p can be used as a potential prognostic marker in patients with CESC, and miR-505-5p may play a tumor suppressive role in CESC by targeting TBL1XR1. -
Key words:
- Cervical squamous cell carcinoma /
- Prognostic markers /
- miR-505-5p /
- TBL1XR1
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图 1 CESC中115个差异表达的miRNAs火山图
Figure 1. Volcano map of 115 differentially expressed miRNAs in CESC heatmap
图 2 CESC组织中差异表达最显著的前20个miRNAs热图
C：正常对照组，T：肿瘤组。
Figure 2. Heat map of the top 20 miRNAs with the most significant differential expression in CESC tissues
图 4 Train组、Test组及所有样本的生存曲线
Figure 4. Survival curves of Train group，Test group and all samples
图 5 Train组、Test组及所有样本的ROC曲线
Figure 5. ROC curves of Train group，Test group and all samples
图 6 miR-505-5p在CESC组织及癌旁组织中的差异表达
Figure 6. Differential expression of miR-505-5p in CESC tissues and adjacent tissues
图 7 miR-505-5p相对表达量与临床分期、宫颈侵袭程度的相关性
Figure 7. Correlation between the relative expression of Mir-505-5p and clinical stage and cervical invasion degree
图 9 CCK8检测miR-505-5p对SiHa细胞增殖的影响
Figure 9. Effect of miR-505-5p on the proliferation of SiHa cells detected by CCK8
图 10 划痕实验检测miR-505-5p对SiHa细胞迁移的影响（100×）
Figure 10. Effect of miR-505-5p on the migration of SiHa cells detected by scratch test（100×）
图 11 流式细胞仪检测miR-505-5p对SiHa细胞凋亡的影响
Figure 11. Effect of miR-505-5p on apoptosis of SiHa cells detected by flow cytometry
图 12 3个数据库中miR-505-5p靶基因预测结果
Figure 12. Prediction results of miR-505-5p target genes in three databases
图 13 14个靶基因GO及KEGG通路富集
Figure 13. Enrichment of GO and KEGG pathways of 14 target genes
图 14 转染miR-505-5p后细胞中TBL1XRI RNA及蛋白水平的变化
Figure 14. Changes of TBL1XRI RNA and protein levels in cells transfected with miR505-5p
表 1 多因素COX回归分析
Table 1. Multivariate COX regression analysis
ID coef HR HR.95L HR.95H P hsa-miR-505-5p −0.41441 0.660731 0.400047 1.091285 0.105504 hsa-miR-142-3p −0.4848 0.615818 0.441054 0.859831 0.004417* hsa-miR-532-5p −0.81274 0.443642 0.255144 0.7714 0.003982* hsa-miR-218-1-3p −0.4772 0.620519 0.393131 0.979428 0.04044* *P < 0.05。 
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表 2 预后风险模型中4个miRNAs在CESC肿瘤组织及癌旁组织中的表达
Table 2. Expression of 4 miRNAs in CESC tumor tissues and adjacent tissues in the prognostic risk model
指标 实验组（n = 21） 对照组（n = 21） Z P miR-505-5p 0.2535（0.1071，0.9268） 0.6417（0.1430，5.9848） −1.987 0.047* miR-142-3p 0.2382（0.0088，0.3490） 0.0326（0.0002，0.3573） −1.220 0.222 miR-532-5p 0.5070（0.1346，1.6147） 0.3869（0.0818，6.4232） −0.604 0.546 miR-218-1-3p 0.1029（0.0094，0.3314） 0.3164 （0.0494，0.8123） −1.434 0.152 *P < 0.05。
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表 3 miR-505-5p相对表达量与各临床资料相关性分析
Table 3. Correlation between the relative expression of miR-505-5p and clinical data
指标 hsa-miR-505-5p r P 年龄 −0.408 0.067 CA125 −0.09 0.698 肿瘤直径 −0.064 0.784 临床分期 −0.638 0.002* HPV 感染 −0.166 0.472 淋巴结转移 0.018 0.937 分化程度 0.136 0.568 宫颈癌侵袭程度 −0.545 0.016* 绝经状态 −0.063 0.786 *P < 0.05。
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表 4 TCGA数据库CESC组织中靶基因的表达
Table 4. Expression of target genes in CESC tissues from TCGA database
基因 logFC logCPM P FDR ZFP91 0.075391 5.913616 0.92952 1 TP53RK −0.26862 4.747168 0.592504 0.869575 CDKN1A −0.68114 8.690444 0.189145 0.49348 DDX3X −0.29412 7.243068 0.37913 0.710946 HMGN2 0.641482 8.918896 0.190775 0.494806 LDHA 0.964102 8.983097 0.146179 0.430027 OCRL −0.46166 4.947949 0.255322 0.579773 TBL1XR1 1.033012 6.195864 0.09947 0.345268 KXD1 −0.38121 5.861561 0.221704 0.534162
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