Proangiogenic role of circRNA‐007371 in liver fibrosis

Abstract Circular RNAs (circRNAs) are crucially involved in cancers as competing endogenous RNA (ceRNA) or microRNA (miRNA) sponges. However, the function and mechanism of circRNAs in liver fibrosis remain unknown and are the focus of this study. Murine fibrotic models were induced by thioacetamide (TAA) or carbon tetrachloride (CCl4). Increased angiogenesis is accompanied by liver fibrosis in TAA‐ and CCl4‐induced murine fibrotic livers. circRNA microarray and argonaute 2 (AGO2)‐RNA immunoprecipitation (RIP) sequencing (AGO2‐RIP sequencing) were performed in murine livers to screen for functional circRNAs. Compared to control livers, 86 differentially expressed circRNAs were obtained in TAA‐induced murine fibrotic livers using circRNA microarray. In addition, 551 circRNAs were explored by AGO2‐RIP sequencing of murine fibrotic livers. The circRNA‐007371 was then selected and verified for back‐spliced junction, resistance to RNase R, and loop formation. In vitro, murine hemangioendothelioma endothelial (EOMA) cells were transfected with circRNA‐007371 overexpressing plasmid or empty plasmid. circRNA‐007371 overexpression promoted tube formation, migration, and cell proliferation of EOMA cells. RNA sequencing and miRNA sequencing were then performed to explore the mechanism of the proangiogenic effects of circRNA‐007371. circRNA‐007371 promotes liver fibrosis via miRNA sponges or ceRNA mechanisms. Stag1, the parent gene of circRNA‐007371, may play a significant role in proangiogenic progression. In conclusion, circRNA‐007371 enhances angiogenesis via a miRNA sponge mechanism in liver fibrosis. The antiangiogenic effect of circRNA‐007371 inhibition may provide a new strategy for treating patients with liver cirrhosis.

Angiogenesis is defined as new blood vessel formation from the preexisting vasculature. 13 Pathological angiogenesis leads to dysfunctional liver sinusoidal endothelial cells (LSECs) in the transport and exchange of oxygen and nutrients, enhances inflammation aggravation, and raises vascular resistance, eventually leading to the progression of liver cirrhosis. 13,14 Traditionally, the vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF) pathways are master regulators in pathological angiogenesis, 13,15,16 and angiocrine factors in LSECs contribute to pathological angiogenesis. 17,18 However, it remains unclear whether other regulators also contribute to pathological angiogenesis.
As endogenous noncoding and covalently closed RNAs, circular RNAs (circRNAs) are produced by pre-messenger RNA (mRNA) through back-splicing. 19 circRNAs play crucial roles in liver diseases as microRNA (miRNA) sponges or competing endogenous RNAs (ceRNAs). 19,20 By binding to the parent mRNA, miRNAs combine with the argonaute (AGO) protein to form an RNA-induced silencing complex (RISC), which leads to mRNA degradation, translation blockade, and parent gene expression inhibition. 21,22 However, as miRNA sponges or ceRNAs, cir-cRNAs compete with the target sites of miRNA-RISC, which further relieves its inhibitory effects on parent genes. 21 circRNAs can regulate HSC activation 23,24 and quiescence 25,26 through miRNA sponges in liver fibrosis. Previous studies have emphasized the crucial effects of cir-cRNAs in angiogenesis in HCC. 27,28 Nevertheless, it remains unclear that the function and potential mechanism of circRNAs in the angiogenesis of liver fibrosis. circRNAs can compete with miRNAs to abrogate the inhibitory effects of miRNA on parent genes. Moreover, miRNAs participate in angiogenesis in the context of HCC 29,30 and liver fibrosis. 31 Whether circRNAs can regulate angiogenesis in liver fibrosis as miRNA sponges or ceRNAs remains unexplored.
This study aimed to detect the proangiogenic function and potential mechanism of circRNAs in liver fibrosis. Increased angiogenesis is accomplished with liver fibrosis in murine fibrotic models. As screened by circRNA microarray and AGO2 RNA immunoprecipitation (RIP) sequencing (AGO2-RIP sequencing) in thioacetamide (TAA)-induced murine fibrotic livers, circRNA-007371 was chosen as the target cir-cRNA. circRNA-007371 is characterized by back-splicing of exon 2 and exon 8 of stromal antigen 1 (Stag1). In vitro, circRNA-007371 overexpression promoted cell migration, vessel formation, and cell proliferation in murine hemangioendothelioma endothelial (EOMA) cells. Then, the genes, miRNAs, and signalling pathways affected by circRNA-007371 were explored in circRNA-007371 overexpressing EOMA cells using mRNA sequencing and miRNA sequencing.
Together, the angiogenic role of circRNA-007371 might promote liver fibrosis via miRNA sponges or ceRNA mechanisms.

| Murine models for liver fibrosis
The animal procedures were approved and conducted by the Animal Use and Care Committee of West China Hospital, Sichuan University.
All animals were freely fed food and water under 12-h light/dark cycles.
Wild-type male C57BL/6J mice 8-12 weeks old were obtained from GemPharmate Co. Ltd. The mice were randomly assigned to four groups with eight animals in each group, including the saline, TAA, olive oil (OO), and carbon tetrachloride (CCl 4 ) groups. For the murine fibrotic models, mice received an intraperitoneal (ip) injection of TAA (200 mg/kg, Sigma #163678) twice a week for 8 weeks or CCl 4 (4 μL/g with 1:3 diluted in OO, RHAWN #R033168) twice a week for 6 weeks. For the control groups, mice were ip injected with normal saline or OO. The mice were sacrificed with an overdose of sodium pentobarbital 48 h after the last injection.

| circRNA microarray and data analysis
Five liver tissues from the TAA group and five liver tissues from the saline group were sent to the circRNA microarray. The circRNA microarray procedure and data analysis were performed by KangCheng Biotech. The raw data of the circRNA microarray were deposited in the NCBI Gene Expression Omnibus (GEO; GSE218574).

| Anti-AGO2-RIP sequencing and data analysis
Anti-AGO2-RIP followed by RNA sequencing (AGO2-RIP sequencing) was used to explore the expression of RNAs that bind to AGO2. Liver tissues from the TAA group were utilized for AGO2-RIP sequencing. AGO2-RIP sequencing was performed by Shanghai Cloud-Seq Biotech. The raw data of AGO2-RIP sequencing were deposited in the NCBI GEO (GSE218577).

| mRNA sequencing and miRNA sequencing
The control and circRNA-007371 overexpressing EOMA cells were subjected to mRNA and miRNA sequencing. The RNA sequencing process and data analysis were performed by Novogene. The raw mRNA sequencing (GSE218578) and miRNA sequencing (GSE218579) data have been deposited in the NCBI GEO.

| Statistical analysis
All data are shown as the mean ± standard deviation and were analysed using GraphPad software (version 9; GraphPad Software Inc.).
Then, t-tests and one-way ANOVA followed by the Bonferroni post hoc test were utilized for statistical analysis. A significant difference was defined as p < 0.05.

| Accomplishment of angiogenesis with liver fibrosis in mice
Angiogenesis is defined as new blood vessel formation from the preexisting vasculature. 32 As the VEGF-mediated angiogenesis signalling pathway is predicted to be involved in nonalcoholic steatohepatitisand hepatitis B virus-related liver cirrhosis, 33,34 we first determined angiogenesis in murine fibrotic models induced by TAA and 3.2 | Expression profiling and potential functions of circRNAs in murine fibrotic livers circRNAs play crucial roles in the pathogenesis of angiogenesis and liver diseases. 19 To explore hepatic circRNA expression profiling in

| Screening of functional circRNAs in fibrotic murine livers
By binding with AGO2, circRNAs can enhance the expression of target genes as ceRNAs or miRNA sponges. 21 To determine which cir-cRNAs can regulate gene expression as ceRNAs, AGO2-RIP sequencing was utilized in murine fibrotic livers induced by TAA. In total, 551 circRNAs were explored by AGO2-RIP sequencing of murine fibrotic livers. The 551 circRNAs obtained from AGO2-RIP sequencing were merged with 86 DEcircRNAs, and 23 shared circRNAs were displayed ( Figure 3A). As shown by the heatmap, these 23 circRNAs were DEcircRNAs in the murine fibrotic liver compared to the control liver with ceRNA potential (Figure 3B,C).
As ceRNAs can act as miRNA sponges, TargetScan and miRandabased miRNA target prediction software was applied to predict the miRNAs bound to these 23 circRNAs. In total, 55 miRNAs were found.
Furthermore, differentially expressed miRNAs (DEmiRNAs) in the fibrotic murine liver (GSE66278) were retrieved 35 and merged with the 55 predicted miRNAs ( Figure 3D). Two miRNAs, miR-200a-5p and miR-34a-5p, were eventually identified ( Figure 3D). By sequence analysis, miR-200a-5p can bind with circRNA-007371, and miR-34a-5p can bind with circRNA-018381 ( Figure 3E). As circRNA-007371 has  D). (E-G) Total RNA was extracted from fibrotic murine livers and treated with or without RNase R. Agarose gel electrophoresis was utilized to detect the RNA (E) and polymerase chain reaction (PCR) products (F). Gapdh was used as a control. qPCR was utilized to quantify the level of circRNA-007371 in the fibrotic and control livers (G), and β-actin was used as a control. n = 8/group. (H) PCR-base agarose gel electrophoresis was applied to evaluate the level of circRNA-007371 amplified by cDNA and gDNA with divergent and convergent primers. Gapdh was used as a control. (I) The back-spliced junction of circRNA-007371 was analysed by Sanger sequencing for PCR products obtained with divergent primers. NS, not significant; **p < 0.01; ***p < 0.001. more miRNA binding sites ( Figure 3E), circRNA-007371 was selected for further investigation. In summary, circRNA-007371 may participate in the development of liver fibrosis as a miRNA sponge.

| Characterization of circRNA-007371 as circRNA in murine livers
circRNAs are endogenous noncoding and covalently closed RNAs produced by pre-mRNA through back-splicing. 19 By sequence comparison, we found that circRNA-007371 is a 976 bp circRNA derived from the parent gene Stag1 and located at chromosome 9 (NC_000075.7; Figure S1). Presumably, circRNA-007371 may be back-spliced from exons 2 and 8 of Stag1 ( Figure 4A). Quaking RNA binding protein (QKI) is an RNA binding protein that can promote the formation of cir-cRNAs. 36 Compared to controls, the protein level of QKI was increased in TAA-and CCl 4 -induced murine fibrotic livers ( Figure 4B-D). circRNA is characterized by high stability to RNase R digestion. RNase R can degrade linear RNA, but circRNAs resist RNase R digestion ( Figure 4E).
To this end, murine liver total RNA was first treated with RNase R and then reverse transcribed into cloned, reverse-transcribed mRNA (cDNA), and PCR followed by agarose gel electrophoresis was performed. The linear RNA Gapdh was degraded by RNase R, whereas circRNA-007371 was resistant to RNase R treatment ( Figure 4F).
Under normal conditions, compared to that in the control livers, the level of circRNA-007371 in the murine fibrotic livers was significantly increased ( Figure 4G). Similar results were obtained when the RNAs were pretreated with RNase R ( Figure 4G). These results indicate that circRNA-007371 was upregulated in fibrotic livers and was resistant to RNase R.
3.6 | The underlying mechanism of circRNA-007331 as a miRNA sponge As miRNA sponges or ceRNAs, circRNAs can abrogate the target gene inhibition effects of miRNAs. 19,21,22 Given that upregulated circRNA-007371 has multiple miRNA binding sites, we examined whether upregulated circRNA-007371 regulates angiogenesis by sponging miRNA. RNA sequencing was utilized for EOMA endothelial cells transfected with empty plasmid or circRNA-007371 overexpressing plasmid in vitro. Compared with control cells, 47 upregulated differentially expressed genes (DEGs) and 3 downregulated DEGs were found after circRNA-007371 overexpression ( Figure 6A,B). Of note, Stag1, the parent gene of circRNA-007371, was the most upregulated DEG ( Figure 6A,B). In addition, the KEGG pathway and GO analysis of DEGs are displayed ( Figure 6C,D). In the KEGG pathway analysis, circRNA-007371 promoted the activation of angiogenesis-related signalling pathways, including the phagosome, HIF1, glycolysis, focal adhesion, and cell cycle signalling pathways ( Figure 6C). In addition, GO analysis indicated that circRNA-007371 enhanced the organization of organelles and chromosomes, the cell cycle, and cell division ( Figure 6D).
To explore the miRNA sponge or ceRNA mechanism of circRNA- In liver fibrosis, most research on angiogenesis focuses on the VEGF-HIF signalling pathway in LSECs or the crosstalk of LSECs with HSCs. 13,15,40 When hypoxia occurs in a tissue, HIF1α is upregulated, 41 which induces the increased expression of VEGFA and angiopoietin 2 to promote angiogenesis. 37 Consistently, increased angiogenesis and VEGFA/VEGFR2 axis were confirmed in TAA-and CCl 4 -induced murine fibrotic livers. Recently, DNA nanoparticles have been used to F I G U R E 8 Proposed mechanism. Increased angiogenesis is accomplished with liver fibrosis. circRNA-007371 enhances angiogenesis in liver fibrosis via increasing STAG1 in a miRNA sponge mechanism. regulate angiogenesis in some diseases. 42,43 However, whether other regulators can promote angiogenesis in the context of liver fibrosis remains unclear. circRNAs are a subtype of noncoding RNA with a covalently closed continuous loop. 19 It has been demonstrated that cir-cRNAs can induce angiogenesis in HCC. 27,28 However, the function and mechanism of circRNAs in angiogenesis in liver fibrosis have not been well characterized. By screening with circRNA microarray and AGO2-RIP sequencing, circRNA-007371 was selected as the target cir-cRNA in this study. circRNA-007371 overexpression promotes angiogenesis in EOMA cells. It seems that circRNA-007371 might involve in the pathogenesis of liver cirrhosis by enhancing angiogenesis.
The covalent loop of circRNA is formed by back-splicing from parent linear pre-mRNA. 19 circRNA-007371 is a 976 bp novel circRNA by back-splicing from exon 2 and exon 8 of Stag1. Similar to other well-studied circRNAs with the advantages of stability, circRNA-007371 is resistant to RNase R. However, as a novel circRNA, it is a great challenge to explore the functions of circRNA-007371 in liver cirrhosis. circRNAs, as miRNA sponges or ceRNAs, can compete with the target sites of miRNA-RISC, further relieving the parent gene suppression of miRNAs to regulate the gene expression process. 21,22 The miRNA sponge role of circRNAs has been illustrated in liver fibrosis by controlling HSC activation 24 and quiescence. 25,26 Whether circRNA-007371 also promotes angiogenesis as a miRNA sponge needs to be determined. In this study, circRNA-007371 was enriched in AGO2-RIP sequencing of the murine fibrotic livers, which means that circRNA-007371 combined with miRNA-RISC to serve as a miRNA sponge. Consistently, circRNA-007371 overexpression induced the upregulation of Stag1 and the disturbance of several miRNAs in EOMA endothelial cells. STAG1 contributes to genome organization, sister chromatid cohesion, and gene expression. 38 In this study, STAG1 was accomplished with circRNA-007371 overexpression, angiogenesis, and cell proliferation. Angiogenesis is regulated by several signalling pathways, including the MAPK, Notch, and HIF1α signalling pathways. 13 Collectively, the current study displayed that the circRNA-007371-STAG1-HIF1α axis may regulate angiogenesis during liver fibrosis.