Differential expression of circular RNAs in hepatic tissue in a model of liver fibrosis and functional analysis of their target genes

Abstract

Aim To measure the expression profile of circular RNA (circRNA) in hepatic tissues in a liver fibrosis model and to explore their function using molecular biology and bioinformatic techniques. Methods The classic CCl4 mouse liver fibrosis model was established alongside a normal control group. The circRNA expression profile of hepatic tissue from the two groups was compared using a high‐throughput circRNA microarray. The differentially expressed circRNAs were identified, and real‐time quantitative polymerase chain reaction (RT‐qPCR) was used to verify a subset of the differentially expressed circRNAs (target genes). At the same time, the mouse oxidative stress injury, macrophage inflammation, and hepatic stellate cell activation models were established, and the expression of target circRNA in the above cells was measured by RT‐qPCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to predict the biological functions of target genes. Finally, one of the circRNAs was selected and its cellular function was verified using siRNA. Results A total of 10 389 circRNAs were analyzed by microarray. Compared with the normal group, there were 69 circRNAs that were differentially expressed in the liver fibrosis model group (>2‐fold differential expression, P < 0.05), of which 14 were upregulated and 55 were downregulated. Five circRNAs and their differential expression were verified by RT‐qPCR, and the findings were consistent with the microarray results. Of these, three circRNAs were differentially expressed (P < 0.05) in the JS1 model, one circRNA was differentially expressed (P < 0.05) in the AML12 model, and four circRNAs were differentially expressed (P < 0.05) in the RAW264.7 model. The GO analysis showed that the differentially expressed circRNAs might be involved in cell autophagy, composition of extracellular matrix components, synthesis and metabolism of retinoic acid, retinol dehydrogenase activity, ubiquitin‐like protein ligase activity, histone methylation, and other biological functions. The KEGG analysis showed that the target genes of the differentially expressed circRNAs might be involved in transforming growth factor‐β1/smads, Hippo, Rap1, vascular endothelial growth factor, and other signaling pathways. Lipofection experiments showed that the expression of α‐smooth muscle actin (α‐SMA) in JS1 cells increased significantly after the expression of mmu_circ_34116 was decreased. Conclusion The circRNA expression profile in liver fibrosis tissue shows significant changes. Partially differentially expressed circRNA could be involved in hepatic fibrosis related to hepatic oxidative stress injury, macrophage inflammation, and stellate cell activation. For instance, mmu_circ_34116 can significantly inhibit the activation of hepatic stellate cells.

Hsa_circ_0070963 inhibits liver fibrosis via regulation of miR-223-3p and LEMD3

“…As a result, circRNAs are considered to be suitable as biomarkers or prognosis factors in various diseases. The expression pattern of circRNAs in liver fibrosis has been previously reported [14,15]. We found that hsa_circ_0070963 expression was abnormally downregulated in irradiated HSCs.…”

Role of ncRNAs in modulation of liver fibrosis by extracellular vesicles

“…In a model of radiation-induced HSC activation, they found 179 upregulated and 630 downregulated circRNAs compared with quiescent HSCs. In a CCl 4 -induced murine model of hepatic fibrosis, Zhou and colleagues have shown the differential expression of 69 circRNAs, of which 14 were upregulated and 55 downregulated in injured livers in respect to healthy controls [86]. In both experimental settings, bioinformatic analyses indicated that the dysregulated circRNAs may target miRNAs and proteins involved in the activation of HSCs, thus contributing to the development of liver fibrosis [85,86].…”

“…In a CCl 4 -induced murine model of hepatic fibrosis, Zhou and colleagues have shown the differential expression of 69 circRNAs, of which 14 were upregulated and 55 downregulated in injured livers in respect to healthy controls [86]. In both experimental settings, bioinformatic analyses indicated that the dysregulated circRNAs may target miRNAs and proteins involved in the activation of HSCs, thus contributing to the development of liver fibrosis [85,86]. Among the anti-fibrogenic circRNAs, hsa_circ_0007874 (also known as circ-MTO1) has proved to inhibit HSC activation and liver fibrogenesis by regulating miR-17-5p and Smad7 [87].…”

Harnessing noncoding RNA‐based macrophage polarization: Emerging therapeutic opportunities for fibrosis

“…These differentially expressed circRNAs are involved in the cell composition, biological processes, molecular function, and several cell signal pathways. 34 A novel ligand of CCR2, PC3-secreted microprotein/microseminoprotein (PSMP/MSMP), which can give rise to inflammatory macrophage infiltration and pro-inflammatory cytokines production in HF patients. 35 The antibody of PSMP is a potential therapeutic agent for the treatment of liver fibrosis.…”