伪基因和长链非编码RNA在肾透明细胞癌患者血浆中的表达与临床意义

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摘要目的: 探讨肾透明细胞癌（简称肾癌）患者血浆中伪基因和长链非编码RNA(lncRNA)的表达及临床价值。方法: 收集50例肾癌患者和26例体检健康者的血浆样本，采用qRT-PCR检测血浆样本中伪基因和lncRNA的表达水平；运用cBioPortal、GEPIA、oncolnc、UALCAN、KaplanMeier plotter等数据库分析差异表达的伪基因和lncRNA与患者预后的关系；应用MPH-dCase9介导转录调控上调目的基因在肾癌细胞系786-O中的表达，qRT-PCR验证其表达水平，CCK8检测目的基因上调后细胞增殖活性；运用StarBase数据库预测目的基因的miRNA靶标并分析其在肾癌中的表达，通过Kaplan-Meier plotter数据库分析miRNA靶标与患者预后的关系。结果：伪基因PLEKHA8P1、CXCR2P1、KLRAP1、FER1L4、PDXDC2P和lncRNA TLX1NB、DIO3OS在肾癌患者血浆中的表达水平均明显高于体检健康者，而LINC00482的表达明显低于体检健康者。伪基因PLEKHA8P1、CXCR2P1、FER1L4和lncRNA TLX1NB、LINC00482、DIO3OS在肾癌患者中发生不同频率的突变，且与患者总体生存期相关，其中，CXCR2P1、FER1L4和TLX1NB还与患者无病生存期相关；另外，PLEKHA8P1、KLRAP1、PDXDC2P、FER1L4和 DIO3OS高表达与患者不良预后相关，而LINC00482表达与患者总体生存期无关，TLX1NB低表达与不良预后相关，与预期相反。对TCGA数据库的肾癌样本进行分析发现，与正常组织比较，FER1L4和DIO3OS在肾癌组织中高表达，体外实验结果表明FER1L4和DIO3OS上调可促进786-O细胞增殖。通过StarBase数据库预测，分别获得24、13个FER1L4和DIO3OS的miRNA靶标，其中hsa-miR-556-5p和hsa-miR-660-5p在肾癌中的表达分别与FER1L4和DIO3OS表达呈负相关，并与肾癌患者不良预后相关。结论：伪基因PLEKHA8P1、CXCR2P1、KLRAP1、FER1L4、PDXDC2P和lncRNA DIO3OS可作为肾癌潜在的早期诊断及预后生物标志物，其中FER1L4和DIO3OS可能通过调节hsamiR5565p和hsa-miR-660-5p参与肾癌发展。

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关键词 ：伪基因, 长链非编码RNA, 肾透明细胞癌, 血浆, 生物标志物

Abstract：Objective: To investigate the expression and clinical significance of pseudogenes and long non-coding RNAs (lncRNAs) in the plasma of patients with renal clear cell carcinoma (RCC). Methods: Plasma samples were collected from 50 patients with RCC and 26 healthy subjects. qRT-PCR assay was used to detect the expression level of pseudogenes and lncRNAs in the plasma samples.The relationship between differentially expressed pseudogenes and lncRNAs and the prognosis of patients was analyzed based on cBioPortal, GEPIA, oncolnc, UALCAN, and Kaplan-Meier plotter databases. Target genes were up-regulated by MPH-dCase9-mediated transcription regulation in RCC cell line 786-O. Then their expression levels were validated by qRT-PCR, and CCK-8 assay was performed to measure the cell proliferation activity after the up-regulation of target genes. StarBase database was used to predict the miRNA targets of the target genes and analyze their expression in RCC. The relationship between miRNA targets and the prognosis of patients was analyzed in the KaplanMeier plotter database. Results: The expression levels of pseudogene PLEKHA8P1, CXCR2P1, KLRAP1, FER1L4, PDXDC2P and lncRNA TLX1NB, DIO3OS in the plasma of patients with RCC were obviously higher than that of healthy subjects, while the expression of LINC00482 displayed the opposite result. Pseudogenes PLEKHA8P1, CXCR2P1, FER1L4, and lncRNA TLX1NB, LINC00482 as well as DIO3OS were mutated at different frequencies in RCC and were relevant to the overall survival of patients. Among them, CXCR2P1, FER1L4, and TLX1NB were also related to the disease-free survival of patients. In addition, high expression of PLEKHA8P1, KLRAP1, PDXDC2P, FER1L4, and DIO3OS were associated with poor prognosis of RCC patients, while the expression of LINC00482 was no significance in overall survival. In addition low expression of TLX1NB was related to poor prognosis, which was contrary to the expectation. Analysis of RCC samples from TCGA database demonstrated that FER1L4 and DIO3OS were high expression in RCC tissues when compared with normal tissues. The results of in vitro experiment indicated that FER1L4 and DIO3OS up-regulation could promote the proliferation of 786-O cells. We identified 24 and 13 predicted miRNA targets for FER1L4 and DIO3OS from StarBase database, respectively. Of which the expression of hsa-miR-556-5p and hsa-miR-660-5p were negatively correlated with FER1L4 and DIO3OS, respectively. Additionally, they were also associated with poor prognosis of patients with RCC. Conclusion: The pseudogene PLEKHA8P1, CXCR2P1, KLRAP1, FER1L4, PDXDC2P and lncRNA DIO3OS may serve as potential biological indicators for early diagnosis and prognosis of RCC, of which FER1L4 and DIO3OS may be involved in the development of RCC via regulating hsa-miR-556-5p and hsa-miR-660-5p.

收稿日期: 2020-02-10

基金资助:国家自然科学基金资助项目(81402100)

通讯作者: 陈兵海(通讯作者)，副教授，硕士生导师，E-mail: chenbhny@163.com

作者简介: 邹元章(1994—)，男，硕士研究生

引用本文:

邹元章，何宜宸，陈兵海. 伪基因和长链非编码RNA在肾透明细胞癌患者血浆中的表达与临床意义[J]. 江苏大学学报:医学版, 2020, 30(04): 312-319. ZOU Yuan-zhang, HE Yi-chen, CHEN Bing-hai.

Expression and clinical significance of pseudogenes and long non-coding RNAs in the plasma of patients with renal clear cell carcinoma

. Journal of Jiangsu University(Medicine Edition), 2020, 30(04): 312-319.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2020/V30/I04/312

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