Identification of onco-miRNAs in hepatocellular carcinoma and analysis of their regulatory network
Identification of onco-miRNAs in hepatocellular carcinoma and analysis of their regulatory network
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肝细胞癌中onco-miRNA的鉴定及其调控网络分析
DOI：
10.12122/j.issn.1673-4254.2022.01.05
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Tianbing Chen
中科院分区：
文献类型：
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作者：
Jingjing Ye;Wenqin Xu;Tianbing Chen
目的：构建肝细胞癌（HCC）中生存相关癌基因miRNAs及其靶基因的调控网络，并验证关键miRNAs与靶基因之间的相互作用。方法：我们在OncomiR和Oncolnc数据库中筛选HCC中与生存相关的miRNAs，使用miRNet预测其靶基因，并分别使用GEPIA 2和Ualcan进行生存和表达分析。进行miRNA-靶基因共表达分析，构建miRNA-靶基因网络。在Enrichr中进行富集分析，在STRING数据库中进行蛋白质-蛋白质相互作用分析。采用CCK 8法检测hsa-miR-1226- 3 p或hsa-miR-221- 5 p的模拟物或抑制剂转染HepG 2细胞后对细胞增殖的影响，并采用RT-qPCR检测靶基因表达的变化。采用Western blotting检测转染hsa-miR-221- 5 p模拟物或抑制剂对靶基因蛋白表达的影响。采用双荧光素酶报告基因检测hsa-miR-221- 5 p与其潜在靶基因GCDH的相互作用。我们进一步检测了单独或联合转染hsa-miR-221- 5 p模拟物和pEGFP N1-GCDH对HepG 2细胞增殖、迁移和侵袭的影响。结果：我们从OncomiR和Oncolnc中分别鉴定了223个和146个生存相关的miRNAs，其中131个miRNAs交叉，48个miRNAs在生存和表达分析后被鉴定为HCC中的癌miRNAs。通过miRNA-mRNA共表达分析，筛选出27个符合条件的靶基因。构建的miRNA-靶基因网络由25个miRNA和27个靶基因组成。最丰富的术语是脂肪酸代谢的目标基因。hsa-miR-1226- 3 p和hsa-miR-221- 5 p的模拟物或抑制剂转染HepG 2细胞后，其靶基因的mRNA和蛋白水平均发生显著变化（P <0. 05）。双荧光素酶报告基因检测结果证实了hsa-miR-221- 5 p与GCDH基因的靶向性关系（P <0. 05）。hsa-miR-221- 5 p模拟物转染可显著抑制HepG 2细胞的增殖、迁移和侵袭能力，而pEGFP N1-GCDH共转染可明显减轻hsa-miR-221- 5 p模拟物转染对HepG 2细胞增殖、迁移和侵袭能力的抑制作用（P <0. 05）。结论：脂肪酸代谢可能是介导oncomiRNA在HCC中作用的最关键途径之一，hsa-miR-221- 5 p/GCDH轴是HCC进展的重要分子机制。
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 oncomiRNAs in HCC, and the hsa-miR-221-5p/GCDH axis is an important molecular mechanism for HCC progression.