Department of Gastroenterology, The Affiliated Hospital of Medical School of Ningbo University, Ningbo 315020, China.
Department of Gastroenterology, The Affiliated Hospital of Medical School of Ningbo University, Ningbo 315020, China.
Gene. 2018 Nov 30;677:317-323. doi: 10.1016/j.gene.2018.08.052. Epub 2018 Aug 15.
To screen for circular RNAs (circRNAs) that are associated with the activation of hepatic stellate cell (HSC) by monitoring changes in liver circRNA expression in a model of liver fibrosis.
The classic mouse model of CCl-induced liver fibrosis was established and validated by histopathological examination. JS1 cells were activated by TGF-β1 to model HSC activation in vitro. Differentially expressed circRNAs in the fibrotic liver tissues and JS1 cells were determined using circRNA microarray, and some of those circRNAs were verified by RT-qPCR. The target genes of the above circRNAs were then predicted by bioinformatics analysis and summarized into a "circRNA-miRNA-mRNA" network diagram. Constructed plasmid mmu_circ_34116 siRNA was transfected to JS1 cells by Lipo2000, then we detected the expression changes of α-SMA.
A total of 10,389 circRNAs were identified by microarray screening, and 69 differentially expressed circRNAs were detected in the fibrotic liver tissues with >2-fold difference in expression level relative to normal liver tissues (P < 0.05); 14 circRNAs were up-regulated and 55 were down-regulated. Five differentially expressed circRNAs in fibrotic liver and JS1 cells were verified by RT-qPCR, while all five showed similar trends with the microarray results in the liver, only 3 circRNAs in the JS1 activation model were consistent with the microarray results while one showed no significant change and one circRNA was not detected. Bioinformatics analysis predicted that the "mmu_circ_34116/miR-22-3P/BMP7" signal axis might be involved in the activation of HSC. Transfection experiment confirmed that the expression of α-SMA is significantly elevated as a result of inhibitory expression of mmu_circ_34116.
The circRNAs expression profile of liver tissue had changed in fibrosis mouse model, and some of these circRNAs may be associated with HSC activation. For instance, mmu_circ_34116 would inhibit HSC activation.
通过监测肝纤维化模型中肝 circRNA 表达的变化，筛选与肝星状细胞（HSC）激活相关的环状 RNA（circRNA）。
通过组织病理学检查验证经典的 CCl 诱导的肝纤维化小鼠模型。用 TGF-β1 激活 JS1 细胞，在体外模拟 HSC 激活。采用 circRNA 微阵列检测纤维化肝组织和 JS1 细胞中差异表达的 circRNA，并用 RT-qPCR 验证部分 circRNA。然后通过生物信息学分析预测上述 circRNA 的靶基因，并总结为“circRNA-miRNA-mRNA”网络图。用 Lipo2000 将构建的质粒 mmu_circ_34116 siRNA 转染到 JS1 细胞中，然后检测 α-SMA 的表达变化。
通过微阵列筛选共鉴定出 10389 个 circRNA，与正常肝组织相比，纤维化肝组织中有 69 个 circRNA 的表达水平差异超过 2 倍（P<0.05）；其中 14 个上调，55 个下调。用 RT-qPCR 验证了纤维化肝和 JS1 细胞中 5 个差异表达的 circRNA，而这 5 个在肝组织中的结果与微阵列结果相似，JS1 激活模型中只有 3 个 circRNA 与微阵列结果一致，1 个无明显变化，1 个未检测到。生物信息学分析预测，“mmu_circ_34116/miR-22-3P/BMP7”信号轴可能参与 HSC 的激活。转染实验证实，抑制 mmu_circ_34116 的表达可显著提高 α-SMA 的表达。
纤维化小鼠模型肝组织 circRNA 表达谱发生改变，其中一些 circRNA 可能与 HSC 激活有关。例如，mmu_circ_34116 可抑制 HSC 激活。
