基于TCGA数据库筛选宫颈鳞状细胞癌潜在的预后标志物及验证

颜怡君; 杨宏英; 张红平; 李政; 贾岳; 赵敏

doi:10.12259/j.issn.2095-610X.S20230808

基于TCGA数据库筛选宫颈鳞状细胞癌潜在的预后标志物及验证

doi: 10.12259/j.issn.2095-610X.S20230808

颜怡君^1,,
杨宏英²,
张红平²,
李政²,
贾岳²,
赵敏^3, ,

1.
云南大学附属医院核医学科，云南昆明　650021
2.
昆明医科大学第三附属医院/云南省肿瘤医院妇科，云南昆明　650118
3.
医务部，云南昆明　650118

基金项目: 云南省科技厅-昆明医科大学应用基础研究联合专项基金资助项目（202201AY070001-167）；云南省教育厅科学研究基金资助项目（2022J0144）；云南省妇科肿瘤精准防治研究和应用推广创新团队项目（202305AS350020）

详细信息

作者简介:
颜怡君（1989～），女，云南施甸人，硕士，主管技师，主要从事临床检验诊断学

通讯作者:
赵敏，E-mail：573532996@qq.com

中图分类号: R446.1
计量
- 文章访问数: 1788
- HTML全文浏览量: 1321
- PDF下载量: 13
- 被引次数: 0
出版历程
- 收稿日期: 2023-05-17
- 网络出版日期: 2023-09-01
- 刊出日期: 2023-08-30

Screening and Validation of Potential Prognostic Markers of Cervical Squamous Cell Carcinoma Based on The Cancer Genome Atlas Database

1.
Dept. of Nuclear Medicine，Affiliated Hospital of Yunnan University，Kunming Yunnan 650021
2.
Dept. of Gynecology
3.
Dept. of Medical Administration，The Third Affiliated Hospital of Kunming Medical University，Kunming Yunnan 650118，China

摘要

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。
- 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.
- Cervical squamous cell carcinoma /
- Prognostic markers /
- miR-505-5p /
- TBL1XR1

HTML全文

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

图 3 9个与CESC患者生存相关miRNAs的生存曲线

Figure 3. Survival curves of 9 miRNAs related to CESC

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

图 8 RT-PCR验证转染效率

Figure 8. Transfection efficiency verified by RT-PCR

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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×）

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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。

下载: 导出CSV

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。

下载: 导出CSV

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
指标	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。

下载: 导出CSV

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

下载: 导出CSV

参考文献(22)

[1]	Bray F,Ferlay J,Soerjomataram I,et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin,2018,68(6):394-424. doi: 10.3322/caac.21492
[2]	Yang M,Du J,Lu H,et al. Global trends and age-specific incidence and mortality of cervical cancer from 1990 to 2019: an international comparative study based on the Global Burden of Disease[J]. BMJ Open,2022,12(7):e055470. doi: 10.1136/bmjopen-2021-055470
[3]	FENG C,DONG J,CHANG W,et al. The progress of methylation regulation in gene expression of cervical cancer[J]. Int J Genomics,2018,2018:8260652. doi: 10.1155/2018/8260652
[4]	LV X J,TANG Q,TU Y Q,et al. Long noncoding RNA PCAT6 regulates cell growth and metastasis via Wnt/β-catenin pathway and is a prognosis marker in cervical cancer[J]. Eur Rev Med Pharmacol Sci,2019,23(5):1947-1956.
[5]	Sung H,Feriay J,Siegel R L,et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin,2021,71(3):209-249.
[6]	Xie M,Nie F Q,Sun M,et al. Decreased long noncoding RNA SPRY4-IT1 contributing to gastric cancer cell metastasis partly via affecting epithelialmesenchymal transition[J]. J Transl Med,2015,13:250. doi: 10.1186/s12967-015-0595-9
[7]	WangY,Luo J,Zhang H,et al. microRNAs in the same clusters evolve to coordinately regulate functionally related genes[J]. Mol Biol Evol,2016,33(9):2232-2247. doi: 10.1093/molbev/msw089
[8]	Shen J,Hung M C. Signaling-mediated regulation of MicroRNA processing[J]. Cancer Res,2015,75(5):783-791. doi: 10.1158/0008-5472.CAN-14-2568
[9]	Svoronos A A,Engelman D M,Slack F J. OncomiR or tumor suppressor? The duplicity of microRNAs in cancer[J]. Cancer Res,2016,76(13):3666-3670. doi: 10.1158/0008-5472.CAN-16-0359
[10]	Tornesello M L,Faraonio R,Buonaguro L T,et al. The Role of microRNAs,Long Non-coding RNAs,and Circular RNAs in Cervical Cancer[J]. Front Oncol,2020,10:150. doi: 10.3389/fonc.2020.00150
[11]	张勇,张哲,朱育焱等. miR-505-5p在膀胱癌细胞中靶向调控PLK1抑制细胞增殖和迁移[J]. 实用肿瘤杂志,2019,34(5):402-409. doi: 10.13267/j.cnki.syzlzz.2019.05.004 ZHANG Y,ZHANG Z,ZHU Y Y,et al. miR-505-5p inhibits proliferation and migration of human bladder cancer cells by regulating PLK1[J]. Journal of Practical Oncology,2019,34(5):402-409. doi: 10.13267/j.cnki.syzlzz.2019.05.004
[12]	Qin Z,He W,Tang J,et al. MicroRNAs provide feedback regulation of epithelial-mesenchymal transition induced by growth factors[J]. J Cell Physiol,2016,231(1):120-129. doi: 10.1002/jcp.25060
[13]	Perissi V,Scafoglio C,Zhang J,et al. TBL1 and TBLR1 phosphorylation on regulated gene promoters overcomes dual CtBP and NCoR/SMRT transcriptional repression checkpoints[J]. Mol Cell,2008,29(6):755-766. doi: 10.1016/j.molcel.2008.01.020
[14]	Xi X,Wu Q,Bao Y,et al. Overexpression of TBL1XR1 confers tumorigenic capability and promotes recurrence of osteosarcoma[J]. Eur J Pharmacol,2019,844:259-267. doi: 10.1016/j.ejphar.2018.12.013
[15]	Pan Q,Shao Z G,Zhang Y J,et al. MicroRNA-1178-3p suppresses the growth of hepatocellular carcinoma by regulating transducin (beta)-like 1 X-linked receptor 1[J]. Hum Cell,2021,34(5):1466-1477. doi: 10.1007/s13577-021-00565-5
[16]	Lai W Q, Yue Y H, Zeng GH. MicroRNA-34c-5p Reduces Malignant Properties of Lung Cancer Cells through Regulation of TBL1XR1/Wnt/β-catenin Signaling. CURRENT MOLECULAR MEDICINE. 2023. doi: 10.2174/1566524023666230330083819.
[17]	Venturutti L,Teater M,Zhai A,et al. TBL1XR1 mutations drive extranodal lymphoma by inducing a pro-tumorigenic memory fate[J]. Cell,2020,182(2):297-316. doi: 10.1016/j.cell.2020.05.049
[18]	张银鹏,孙金兵,宗洋等. 干扰TBL1XR1表达调控c -Met /PI3K/Akt通路对胰腺癌细胞生物学行为影响的实验研究[J]. 临床肿瘤学杂志,2021,26(2):97-103.
[19]	Zhang T,Liu C,Yu Y,et al. TBL1XR1 is involved in c-Met-medi- ated tumorigenesis of human nonsmall cell lung cancer[J]. Cancer Gene Ther,2020,27:136-146. doi: 10.1038/s41417-019-0111-0
[20]	Lu J,Bang H J,Su S M,et al. Lymphatic metastasis-related TBL1XR1 enhances stemness and metastasis in gastric cancer stem-like cells by activating ERK1/2-SOX2 signaling[J]. Oncogene,2020,40(5):922-936.
[21]	Wang X F,Xu X Y,Cai W Z,et al. TBL1XR1 mutation predicts poor outcome in primary testicular diffuse large B-cell lymphoma patients[J]. Biomark Res,2020,8:10. doi: 10.1186/s40364-020-00189-1
[22]	Wang J,Ou J,Guo Y,et al. TBLR1 is a novel prognostic marker and promotes epithelial-mesenchymal transition in cervical cancer[J]. Br J Cancer,2014,111(1):112-124. doi: 10.1038/bjc.2014.278

相关文章(20)

[1]	谭博文, 梁云, 陈环, 雷毅, 王昱涵, 田衍. 术前外周血炎性指标联合肿瘤标记物对可切除结肠癌患者的预后价值, 昆明医科大学学报.
[2]	李妍平, 董小林, 李青芸, 李红梅, 魏欢, 曾毅. miR-21-5p通过抑制STAT3缓解OGD诱导的HT22细胞炎症和凋亡并促进增殖, 昆明医科大学学报.
[3]	郭小兵, 李晓文, 李恒希, 曹艳, 李坪. miR-212-3p靶向调控NAP1L1抑制胶质瘤细胞增殖、迁移和上皮-间充质转化, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20241104
[4]	张梁, 王保全, 雷喜锋, 王旭, 柯阳, 张玮. miR-29c-3p/IGF1分子轴对肝星状细胞活化，增殖和凋亡的作用机制, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230926
[5]	李婷, 郭维华. 糖酵解重编程在口腔鳞状细胞癌中的研究进展, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230611
[6]	冯毅, 王小峰, 白西民, 姚胜, 党俊涛, 赵云洁, 蔡冰. miR-149-5p通过MSH5/Wnt信号通路调控胶质瘤细胞恶性生物学行为, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230823
[7]	王东, 高必波, 孙会英, 冷登辉, 冉小平, 林文. miR-216b-5p通过靶向NCOA3促进胶质母细胞瘤细胞铁死亡, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230805
[8]	周婷, 何影, 董晓函, 习杨彦彬, 陈波, 佟钧, 毛瑞. miR-219-5p靶向SOX5在口腔癌中的作用初探, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20230201
[9]	李诗洲, 何晓光, 林雁, 李玉晓, 杨曦, 王晓雨, 杨声豪. 原发灶不明颈部淋巴结转移鳞状细胞癌的临床分析, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220710
[10]	赵斌, 段元鹏, 张国颖, 毕城伟, 杨李波, 施致裕, 杨勇, 张建朋, 高婷. CircRNA EZH2通过调控miR-30c-5p促进前列腺癌细胞增殖和迁移, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220731
[11]	刘演龙, 光雪峰, 尹小龙, 戴海龙. miR-125a-3p对动脉粥样硬化斑块及M1/M2巨噬细胞、MMP-9和VEGF的影响, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220915
[12]	张玮, 王保全, 雷喜锋, 王旭, 张梁. miR-125b-5p调控HK2抑制胆囊癌细胞增殖和糖酵解, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20221206
[13]	王峰, 杨伟, 钱佃伦, 梅松, 王文杰, 冯科翔, 白向锋. miR-582-5p调控Notch1减轻心肌缺血再灌注损伤的研究, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20221207
[14]	王保全, 张伟, 田园, 雷喜锋, 王旭. miR-142-5p通过CCND1调控胆囊癌细胞的增殖和转移, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20220223
[15]	戴海龙, 张星, 潘云, 冯晓岚, 卢祥婷, 光雪峰, 尹小龙. 冠心病患者外周血单核细胞中miR-125a-5p、miR-193b的表达, 昆明医科大学学报.
[16]	吴琪燕, 边革元, 程绘珺, 李娇霞, 王书廷, 黄照略. 肺腺癌预后因素及血清肿瘤标记物的诊断效能, 昆明医科大学学报.
[17]	刘冲, 曹慧, 杨彩彩, 王震. 柚皮苷调控miR-199a-5p/ECE1分子轴促进骨损伤修复, 昆明医科大学学报.
[18]	徐永琴, 梅雪, 罗汝琼, 李伟, 张海, 张方芳. HPV DNA含量及血清、分泌物MMP在宫颈癌患者中的表达及对预后的反应价值, 昆明医科大学学报.
[19]	李建娇, 代晓明. 组蛋白去乙酰化酶2在人舌鳞状细胞癌中的表达, 昆明医科大学学报.
[20]	胡滔, 洪颖, 陈红兰, 常业飞, 姜水, 习杨彦彬, 檀雅欣, 李珊, 刘光彩, 吴晓虹. miR-1对HR HPV 16~+/18~+宫颈癌细胞周期相关蛋白的调控, 昆明医科大学学报.