高通量测序方法鉴定肝癌中的 lncRNA 生物标志物，并揭示 ZFAS1/miR-150-5p 对肝癌进展的影响。
High-throughput sequencing approach for the identification of lncRNA biomarkers in hepatocellular carcinoma and revealing the effect of ZFAS1/miR-150-5p on hepatocellular carcinoma progression.
机构信息
Department of Hepatic Surgery (III), The Third Affiliated Hospital of Naval Medical University, Shanghai, China.
School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, Zhongshan, Guangdong, China.
出版信息
PeerJ. 2023 Feb 23;11:e14891. doi: 10.7717/peerj.14891. eCollection 2023.
AIMS
To screen abnormal lncRNAs and diagnostic biomarkers in the progression of hepatocellular carcinoma through high-throughput sequencing and explore the underlying mechanisms of abnormal lncRNAs in the progression of hepatocellular carcinoma.
METHODS
The transcriptome sequencing was used to analyze the RNA expression profile and identify differentially expressed RNAs. Hub lncRNAs were screened by combining (WGCNA, ceRNA regulatory network, PPI, GO and KEGG analyses, Kaplan-Meier curve analysis, Cox analysis, risk model construction and qPCR). Thereafter, the correlation between the expression of hub lncRNAs and tumor clinicopathological parameters was analyzed, and the hub lncRNAs were analyzed by GSEA. Finally, the effects of hub RNAs on the proliferation, migration and invasion of HepG2 cells were investigated .
RESULTS
Compared with the control group, a total of 610 lncRNAs, 2,593 mRNAs and 26 miRNAs were screened in patients with hepatocellular carcinoma. Through miRNA target prediction and WGCNA, a ceRNA was constructed, comprising 324 nodes and 621 edges. Enrichment analysis showed that mRNAs in ceRNA were involved mainly in cancer development progression. Then, the ZFAS1/miR-150-5p interaction pair was screened out by Kaplan Meier curve analysis, Cox analysis and qPCR analysis. Its expression was related to tumor stage, TNM stage and patient age. ROC curve analysis showed that it has a good predictive value for the risk of hepatocellular carcinoma. GSEA showed that ZFAS1 was also enriched in the regulation of immune response, cell differentiation and proliferation. Loss-of-function experiments revealed that ZFAS1 inhibition could remarkably suppress HepG2 cell proliferation, migration and invasion . Bioinformatic analysis and luciferase reporter assays revealed that ZFAS1 directly interacted with miR-150-5p. Rescue experiments showed that a miR-150-5p inhibitor reversed the cell proliferation, migration and invasion functions of ZFAS1 knockdown .
CONCLUSION
ZFAS1 is associated with the malignant status and prognosis of patients with hepatocellular carcinoma, and the ZFAS1/miR-150-5p axis is involved in hepatocellular carcinoma progression.
目的
通过高通量测序筛选肝细胞癌进展过程中的异常长链非编码 RNA 和诊断生物标志物，并探讨异常长链非编码 RNA 在肝细胞癌进展中的潜在机制。
方法
采用转录组测序分析 RNA 表达谱，筛选差异表达 RNA。通过 WGCNA、ceRNA 调控网络、PPI、GO 和 KEGG 分析、Kaplan-Meier 曲线分析、Cox 分析、风险模型构建和 qPCR 等方法筛选出关键长链非编码 RNA。然后，分析关键长链非编码 RNA 与肿瘤临床病理参数之间的相关性，并通过 GSEA 进行分析。最后，研究关键 RNA 对 HepG2 细胞增殖、迁移和侵袭的影响。
结果
与对照组相比，肝癌患者共筛选出 610 个长链非编码 RNA、2593 个 mRNA 和 26 个 miRNA。通过 miRNA 靶基因预测和 WGCNA 构建了 ceRNA，包含 324 个节点和 621 个边缘。富集分析显示，ceRNA 中的 mRNAs 主要参与癌症的发生发展。然后，通过 Kaplan-Meier 曲线分析、Cox 分析和 qPCR 分析筛选出 ZFAS1/miR-150-5p 相互作用对。其表达与肿瘤分期、TNM 分期和患者年龄有关。ROC 曲线分析表明，其对肝细胞癌的风险具有良好的预测价值。GSEA 显示 ZFAS1 还富集在免疫反应、细胞分化和增殖的调控中。功能丧失实验表明，ZFAS1 抑制可显著抑制 HepG2 细胞的增殖、迁移和侵袭。生物信息学分析和荧光素酶报告基因实验表明，ZFAS1 可直接与 miR-150-5p 相互作用。挽救实验表明，miR-150-5p 抑制剂可逆转 ZFAS1 敲低引起的细胞增殖、迁移和侵袭功能。
结论
ZFAS1 与肝细胞癌患者的恶性状态和预后相关，ZFAS1/miR-150-5p 轴参与肝细胞癌的进展。
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