环状 RNA 的综合网络分析揭示了人诱导多能干细胞衍生内胚层肝脏特化的调控机制。
Integrative network analysis of circular RNAs reveals regulatory mechanisms for hepatic specification of human iPSC-derived endoderm.
机构信息
Organ Transplant Center, The First Affiliated Hospital, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation), Guangzhou, Guangdong, China.
Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China.
出版信息
Stem Cell Res Ther. 2022 Sep 8;13(1):468. doi: 10.1186/s13287-022-03160-z.
BACKGROUND
Human-induced pluripotent stem cell (hiPSC)-derived functional hepatic endoderm (HE) is supposed to be an alternative option for replacement therapy for end-stage liver disease. However, the high heterogeneity of HE cell populations is still challenging. Hepatic specification of definitive endoderm (DE) is an essential stage for HE induction in vitro. Recent studies have suggested that circular RNAs (circRNAs) determine the fate of stem cells by acting as competing endogenous RNAs (ceRNAs). To date, the relationships between endogenous circRNAs and hepatic specification remain elusive.
METHODS
The identities of DE and HE derived from hiPSCs were determined by qPCR, cell immunofluorescence, and ELISA. Differentially expressed circRNAs (DEcircRNAs) were analysed using the Arraystar Human circRNA Array. qPCR was performed to validate the candidate DEcircRNAs. Intersecting differentially expressed genes (DEGs) of the GSE128060 and GSE66282 data sets and the DEcircRNA-predicted mRNAs were imported into Cytoscape for ceRNA networks. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were involved in the enrichment analysis. Hepatic markers and Wnt/β-catenin were detected in hsa_circ_004658-overexpressing cells by western blotting. Dual-luciferase reporter assays were used to evaluate the direct binding among hsa_circ_004658, miRNA-1200 and CDX2. DE cells were transfected with miR-1200 mimics, adenovirus containing CDX2, and Wnt/β-catenin was detected by western blotting.
RESULTS
hiPSC-derived DE and HE were obtained at 4 and 9 days after differentiation, as determined by hepatic markers. During hepatic specification, 626 upregulated and 208 downregulated DEcircRNAs were identified. Nine candidate DEcircRNAs were validated by qPCR. In the ceRNA networks, 111 circRNA-miRNA-mRNA pairs were involved, including 90 pairs associated with hsa_circ_004658. In addition, 53 DEGs were identified among the intersecting mRNAs of the GSE128060 and GSE66282 data sets and the hsa_circ_004658-targeted mRNAs. KEGG and GO analyses showed that the DEGs associated with hsa_circ_004658 were mainly enriched in the WNT signalling pathway. Furthermore, hsa_circ_004658 was preliminarily verified to promote hepatic specification, as determined by hepatic markers (AFP, ALB, HNF4A, and CK19) (p < 0.05). This promotive effect may be related to the inhibition of the Wnt/β-catenin signalling pathway (detected by β-catenin, p-β-catenin, and TCF4) when hsa_circ_004658 was overexpressed (p < 0.05). Dual-luciferase reporter assays showed that there were binding sites for miR-1200 in the hsa_circ_004658 sequence, and confirmed the candidate DEG (CDX2) as a miR-1200 target. The level of miR-1200 decreased and the level of CDX2 protein expression increased when hsa_circ_004658 was overexpressed (p < 0.05). In addition, the results showed that CDX2 may suppress the Wnt/β-catenin signalling during hepatic specification (p < 0.05).
CONCLUSIONS
This study analysed the profiles of circRNAs during hepatic specification. We identified the hsa_circ_004658/miR-1200/CDX2 axis and preliminarily verified its effect on the Wnt/β-catenin signalling pathway during hepatic specification. These results provide novel insight into the molecular mechanisms involved in hepatic specification and could improve liver development in the future.
背景
诱导多能干细胞（hiPSC）衍生的功能性肝内胚层（HE）被认为是终末期肝病替代治疗的一种选择。然而，HE 细胞群体的高度异质性仍然具有挑战性。DE 向 HE 的诱导是体外诱导 HE 的一个重要阶段。最近的研究表明，环状 RNA（circRNA）通过充当竞争性内源性 RNA（ceRNA）来决定干细胞的命运。迄今为止，内源性 circRNA 与肝特异性之间的关系仍不清楚。
方法
通过 qPCR、细胞免疫荧光和 ELISA 确定 hiPSC 来源的 DE 和 HE 的特性。使用 Arraystar Human circRNA Array 分析差异表达的 circRNAs（DEcircRNAs）。qPCR 用于验证候选 DEcircRNAs。将 GSE128060 和 GSE66282 数据集的差异表达基因（DEGs）和 DEcircRNA 预测的 mRNA 导入 Cytoscape 进行 ceRNA 网络分析。基因本体论（GO）和京都基因与基因组百科全书（KEGG）参与了富集分析。通过 Western blot 检测 hsa_circ_004658 过表达细胞中的肝标志物和 Wnt/β-catenin。使用双荧光素酶报告基因检测评估 hsa_circ_004658、miR-1200 和 CDX2 之间的直接结合。用 miR-1200 模拟物转染 DE 细胞，用含有 CDX2 的腺病毒转染，通过 Western blot 检测 Wnt/β-catenin。
结果
通过肝标志物在分化后 4 天和 9 天获得 hiPSC 来源的 DE 和 HE。在肝特异性形成过程中，鉴定出 626 个上调和 208 个下调的 DEcircRNA。通过 qPCR 验证了 9 个候选 DEcircRNA。在 ceRNA 网络中，涉及 111 个 circRNA-miRNA-mRNA 对，包括与 hsa_circ_004658 相关的 90 对。此外，在 GSE128060 和 GSE66282 数据集的 intersecting mRNAs 和 hsa_circ_004658 靶向 mRNAs 中鉴定出 53 个 DEG。KEGG 和 GO 分析表明，与 hsa_circ_004658 相关的 DEGs 主要富集在 WNT 信号通路中。此外，hsa_circ_004658 初步验证可通过肝标志物（AFP、ALB、HNF4A 和 CK19）（p<0.05）促进肝特异性。这种促进作用可能与 hsa_circ_004658 过表达时抑制 Wnt/β-catenin 信号通路（通过β-catenin、p-β-catenin 和 TCF4 检测）有关（p<0.05）。双荧光素酶报告基因检测显示 hsa_circ_004658 序列中有 miR-1200 的结合位点，并证实候选 DEG（CDX2）是 miR-1200 的靶标。当 hsa_circ_004658 过表达时，miR-1200 的水平降低，CDX2 蛋白表达水平升高（p<0.05）。此外，结果表明 CDX2 可能在肝特异性形成过程中抑制 Wnt/β-catenin 信号（p<0.05）。
结论
本研究分析了肝特异性形成过程中环状 RNA 的谱。我们鉴定了 hsa_circ_004658/miR-1200/CDX2 轴，并初步验证了其在肝特异性形成过程中对 Wnt/β-catenin 信号通路的影响。这些结果为肝特异性形成的分子机制提供了新的见解，并可能为未来的肝脏发育提供帮助。
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