| 肝细胞癌中促癌miRNA调控网络分析与验证 |
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| 引用本文： | 叶静静,徐文琴,陈天兵. 肝细胞癌中促癌miRNA调控网络分析与验证[J]. 南方医科大学学报, 2022, 42(1): 45-54. DOI: 10.12122/j.issn.1673-4254.2022.01.05 |
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| 作者姓名： | 叶静静 徐文琴 陈天兵 |
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| 作者单位： | 皖南医学院弋矶山医院中心实验室，重大疾病非编码 RNA 转化研究安徽普通高校重点实验室，安徽 芜湖 241001 |
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| 摘 要： | 目的 构建肝细胞癌中生存相关的促癌miRNA与其靶基因的调控网络并对关键miRNA-靶基因进行实验验证。方法 利 用OncomiR和Oncolnc获取肝癌中生存相关的miRNA，并进行表达分析和生存分析；利用miRNet预测miRNA的靶基因，通过GEPIA2、Ualcan分别对靶基因进行生存和表达分析，通过Starbase进行miRNA-靶基因共表达分析，利用Cytoscape软件构建miRNA-靶基因网络，通过Enrichr进行靶基因富集分析，利用String数据库进行蛋白互作网络构建。将NC，hsa-miR-1226-3p的mimic或inhibitor，hsa-miR-221-5p的mimic或inhibitor分别转染HepG2细胞，利用CCK8检测转染后细胞活力的变化情况，另取转染后48 h样品利用Q-PCR检测靶基因表达情况；将NC，hsa-miR-221-5p的mimic或inhibitor分别转染HepG2细胞，48 h后用 Werstern blot检测靶基因蛋白表达情况；将 NC+p-GCDH-WT质粒，NC+p-GCDH-MUT质粒，hsa-miR-221-5p mimic+p-GCDH-WT质粒，hsa-miR-221-5p mimic+p-GCDH-MUT质粒分别转染HepG2细胞，48 h后利用多功能酶标仪检测荧光素酶活性；将NC，hsa-miR-221-5p mimic，pEGFP N1-GCDH质粒，hsa-miR-221-5p mimic+pEGFP N1-GCDH质粒分别转染HepG2细胞，利用CCK8检测转染后细胞活力的变化情况，transwell 实验检测细胞迁移和侵袭能力。结果 从OncomiR和Oncolnc数据库中分别得到223个（P<0.05）和146个（P<0.05）在肝癌中与生存相关的miRNA，两者交集为131个；结合表达分析和生存分析的结果共鉴定出 48 个促癌 miRNA；miRnet 共预测出 10 278 个靶基因，通过生存和表达分析符合预期的为 44 个，miRNA-mRNA共表达分析后的结果显示 27 个靶基因符合预期。通过 Cytoscape软件构建的 miRNA-靶基因网络包含了 25个促癌miRNA和27个抑癌基因。富集分析结果显示靶基因集中作用于脂肪酸代谢通路。验证实验显示，转染hsa-miR-1226-3p的mimic和inhibitor，hsa-miR-221-5p的mimic和inhibitor后，其对应的靶基因mRNA或蛋白水平均显著改变（P<0.05）；hsa-miR-221-5p mimic 和 p-GCDH-WT 质粒共转可显著降低其荧光素酶的活性（P<0.05）；pEGFP N1-GCDH 质粒和 hsa-miR-221-5pmimic共转可显著恢复其对细胞活力，以及细胞迁移、侵袭能力的影响（P<0.05）。结论 通过综合分析鉴定了肝癌中的促癌miRNA并构建了其调控网络；脂肪酸代谢可能是肝细胞癌中促癌miRNA发挥作用的重要的靶标信号通路之一。hsa-miR-221- 5p/GCDH轴是肝癌进展的重要分子机制。
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| 关 键 词： | 肝癌；促癌miRNA；调控网络；生信分析 |
Identification of onco-miRNAs in hepatocellular carcinoma and analysis of their regulatory network |
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| YE Jingjing,XU Wenqin,CHEN Tianbing. Identification of onco-miRNAs in hepatocellular carcinoma and analysis of their regulatory network[J]. Journal of Southern Medical University, 2022, 42(1): 45-54. DOI: 10.12122/j.issn.1673-4254.2022.01.05 |
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| Authors: | YE Jingjing XU Wenqin CHEN Tianbing |
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| Affiliation: | Central Laboratory, Yijishan Hospital, Key Laboratory of Non-coding RNA Transformation Research of Anhui Higher Education Institution, Wannan Medical College, Wuhu 241001, China |
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| Abstract: | Objective To construct the regulatory network of survival-related onco-miRNAs and their target genes in hepatocellular carcinoma (HCC) and verify the interactions between the key miRNAs and their targets. Methods We screened survival-related miRNAs in HCC in OncomiR and Oncolnc databases, predicted their target genes using miRNet, and conducted survival and expression analysis using GEPIA2 and Ualcan, respectively. The miRNA-target gene co-expression analysis was performed and the miRNA-target network was constructed. Enrichment analysis was performed in Enrichr and protein-protein interaction analysis in STRING database. We tested the effects of transfection with the mimic or inhibitor of hsa-miR-1226-3p or hsa-miR-221-5p on proliferation of HepG2 cells using CCK8 assay and examined the changes in the expressions of the target genes using RT-qPCR. The effect of transfection with hsa-miR-221-5p mimic or inhibitor on protein expressions of the target genes was examined using Western blotting in. A dual luciferase reporter assay was used to test the interaction between hsa-miR-221-5p and its potential target gene GCDH. We further examined the effect of transfection with hsa-miR-221-5p mimic and pEGFP N1-GCDH, alone or in combination, on proliferation, migration and invasion of HepG2 cells. Results We identified 223 survival-related miRNAs in HCC from OncomiR and 146 miRNAs from Oncolnc with an intersection of 131 miRNAs, and 48 miRNAs were identified as onco-miRNAs in HCC after survival and expression analysis. Twenty-seven eligible target genes were identified after miRNA-mRNA co-expression analysis. The constructed miRNA-target gene network consisted of 25 miRNAs and 27 target genes. The most enriched term was fatty acid metabolism for the target genes. In HepG2 cells, transfection with the mimic or inhibitor of hsa-miR-1226-3p or hsa-miR-221-5p caused significant changes of the mRNA and protein levels of their respective target genes (P<0.05). The results of dual luciferase reporter assay confirmed the targeting relationship between hsa-miR-221-5p and GCDH gene (P<0.05). Transfection with hsa-miR-221-5p mimic significantly suppressed the proliferation, migration and invasion of HepG2 cells, but this effect was obviously relieved by co-transformation with pEGFP N1-GCDH (P<0.05). Conclusion Fatty acid metabolism might be one of the most crucial pathways that mediate the effect of the onco-miRNAs in HCC, and the hsa-miR-221-5p/GCDH axis is an important molecular mechanism for HCC progression. |
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| Keywords: | hepatocellular carcinoma onco-miRNA regulatory network bioinformatic analysis, |
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