Hypoxia promotes the proliferation of mouse liver sinusoidal endothelial cells: miRNA-mRNA expression analysis

Abstract

During the initial stage of liver regeneration (LR), hepatocytes and liver sinusoidal endothelial cells (LSECs) initiate regeneration in a hypoxic environment. However, the role of LSECs in liver regeneration in hypoxic environments and their specific molecular mechanism is unknown. Therefore, this study aimed to explore the miRNA-mRNA network that regulates the proliferation of LSECs during hypoxia. In this study, first, we found that the proliferation ability of primary LSECs treated with hypoxia was enhanced compared with the control group, and then whole transcriptome sequencing was performed to screen 1837 differentially expressed (DE) genes and 17 DE miRNAs. Subsequently, the bioinformatics method was used to predict the target genes of miRNAs, and 309 pairs of interacting miRNA-mRNA pairs were obtained. Furthermore, the miRNA-gene action network was established using the negative interacting miRNA-mRNA pairs. The selected mRNAs were analyzed by Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and biological processes (BP) and signal pathways related to LSEC proliferation that were significantly enriched in GO-BP and KEGG were selected. Finally, 22 DE genes and 17 DE miRNAs were screened and the network was created. We also successfully verified the significant changes in the top six genes and miRNAs using qRT-PCR, and the results were consistent with the sequencing results. This study proposed that a specific miRNA-mRNA network is associated with hypoxia-induced proliferation of LSECs, which will assist in elucidating the potential mechanisms involved in hypoxia-promoting liver regeneration during LR.

Keywords: Liver sinusoidal endothelial cells; bioinformatics; hypoxia; liver regeneration; microrna.

Figure 1.

Changes in the proliferation of mouse liver sinusoidal endothelial cells (LSECs) cultured under hypoxic conditions. (a) Representative images of EdU staining assay of mouse LSECs cultured under hypoxia or normoxia for 24 h. (b) The average number of positive cells in Edu staining after LESCs were cultured in hypoxia or normoxia for 24 h. Data are shown as the mean ± SEM. n = 3 per group,***p < 0.001 vs control group (normoxia) by Student’s t-test

Figure 2.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis based on miRNA-targeted genes. (a), (b), and (c) illustrate significant GO-biological processes (BP) enrichment analysis of MF (molecular functions), CC (cellular components), and pathways, respectively, targeted by downregulated genes. Similarly, (d), (e), and (f) show the same content in upregulated genes

Figure 3.

The fine cluster map of miRNA and its predicted target genes were significantly altered in mouse liver sinusoidal endothelial cells (LSECs) cultured in hypoxia and normoxia (control group) for 24 h. (a) The expression profile of miRNA in mouse LSECs after 24 h of hypoxia and normoxia (control group). A1, A2, A3 served as control groups. (b) The target genes predicted by differentially expressed (DE) miRNA were enriched by GO-BP and KEGG, and the target intersecting genes related to LSECs and hepatocyte proliferation were intersected by the list of genes enriched in pathway and function. Combined with their initial expression values, the fine cluster map of mRNA was obtained. A total of 22 meaningful DE target genes were obtained, including 13 upregulated genes and 9 downregulated genes

Figure 4.

An interaction network of the miRNAs and their targets in liver sinusoidal endothelial cells (LSECs) cultured under hypoxia for 24 h

Figure 5.

Quantitative real-time PCR (qPCR) validation of the differentially expressed mRNAs (TOP 6) identified by transcriptomic analysis. (a),(b),(c),(d),(e),(f) show up-regulated mRNAs; (g),(h),(i),(j),(k),(l) show down-regulated mRNAs .Data are shown as the mean ± SEM,n = 3 per group,*p < 0.05, ***p < 0.001,****p < 0.0001 vs control group (normoxia), by Student’s t-test

Figure 6.

Quantitative real-time PCR (qPCR) validation of the differentially expressed miRNAs (TOP 6) identified by transcriptomic analysis.(a),(b),(c),(d) show down-regulated miRNAs;(e),(f) show up-regulated miRNAs. Data are shown as the mean ± SEM, n = 3 per group, ***p < 0.001,****p < 0.0001 vs control group (normoxia), by Student’s t-test