Exosomal circ-RBM23 sponging miR-139-5p to promote liver regeneration through RRM2/AKT/mTOR pathway

Background Exosomes are small nano-size membrane vesicles and are involved in intercellular interaction. Here, we examined if exosomes obtained from human placental stem cells promote liver regeneration after partial hepatectomy. Methods Exosomes generated from primary human placental stem cells were isolated and characterized. Cell co-culture model was used to clarify whether exosomes can induce hepatocytes proliferation in vitro . Partial hepatectomy mouse model was used to evaluate whether exosomes can promote hepatocytes proliferation in vivo . Results It is found that human placental-derived stem cells exosomes (hPDSCs-exo) can induce hepatocyte proliferation in vitro and in vivo . Mechanistically, exosomal circ-RBM23 served as a ceRNA for miR-139-5p, regulated RRM2 and accelerated proliferation through AKT/mTOR pathways. Ablation of exosomal circ-RBM23 suppressed the proliferative effect of exosomes. Conclusions The hPMSCs exosomal circ-RBM23 stimulated cell proliferation and liver regeneration after 70% partial hepatectomy by regulated RRM2. Our findings highlight a potential novel therapeutic avenue for liver regeneration after hepatectomy.


Introduction
Liver regeneration refers to a compensatory process that replaces functional liver mass lost to injury or disease (1,2). The mechanisms of this process are highly complex and have been researched for decades (3). Liver regeneration after partial hepatectomy (PH) is complex and well-orchestrated, implicating multiple factors including cytokines, exosomes, growth factors, and signaling pathways (2)(3)(4)(5).
Unravelling the mechanism and regulation of liver regeneration significantly helps liver resection patients.
Human placental mesenchymal stem cells (hPMSCs) exhibit self-renewing potential and differentiate into multiple cell type lineages (6). They secrete soluble factors with paracrine effects. The therapeutic potential of hPMSCs has been showed in tissue damage repair after ischemic diseases including stroke, and myocardial and cerebral infarctions (7)(8)(9)(10)(11). These studies suggest that hPMSCs-based therapies might be developed for future clinical applications.
However, exosomal circ-RNAs together with their function in liver regeneration are not completely understood. 5 5 In this study, we investigated the effects of human placental-derived stem cells exosomes (hPDSCs-exo) on murine/human hepatocytes (in vitro) and a 70% partial hepatectomy model (in vivo), as well as the underlying mechanisms.

Human placenta stem cell preparation
We previously described the methods for preparing hPMSCs (20). The collection and use of the samples were approved by the review board and ethics committee of Zhu Jiang Hospital (approval number: 2019-KY-015-02). All of the participants provided written informed consent prior to the sample collection.

RNase R treatment
RNase R (Epicentre Technologies) was utilized to degrade linear RNA. Total RNA (2μg) was incubated for 30 min at 37°C with zero units or 20 units of RNase R.
Subsequently, RNA was purified by phenol-chloroform extraction, retro-transcribed, and used for RT-qPCR via Actin as reference gene.

2.3.Exosomes isolation
For exosomes extraction from the conditioned medium of cell cultures, 5 × 10 7 cells were seeded in 25 mL media supplemented with 10% exo-depleted FBS in a 150cm 2 tissue culture plate for 48 h. Standard differential centrifugation was used to isolate exosomes from the conditioned medium. The conditioned medium was subjected to sequential centrifugation steps of 200 g for 10 min and 2000 g for 15 min to eliminate many cells and cellular debris. Then, the supernatant was centrifuged for 10,000 g for 30min and filtered using a 0.22 μm filter (Millipore) to eliminate cell debris membranes and EVs. A pellet was collected after 2 h of ultracentrifugation at 100,000 g using a P28Ti fixed angle rotor (Himac CP100xn, Japan) and resuspended in PBS. The resulting pellet was collected through ultracentrifugation for 70 min at 100,000 g and resuspended in PBS for downstream analyses.

2.4.Exosomes characterization
Exosomes were examined under transmission electron microscopy (H-600 HITACHI microscope, Japan) at 80 keV. Exosomes size and number were established and the data analyzed were using a Nanoparticle tracking analysis(NTA) ZetaView (Particle Metrix, GERMANY). Purity was examined by western blotting for exosome markers (CD9, CD63, CD81 and HSP70). The number of exosomes was examined by NTA.

2.5.High-throughput sequencing
High-throughput sequencing and OE Biotech (Shanghai, China) were used to determine the CircRNA expression profiles. Total RNA was extracted and determined, then a cDNA library constructed after removing ribosomal RNA and RNase R digestion. Illumina sequencer (HiSeqTM 2500) was utilized after passing quality inspection. Eventually, circRNAs were confirmed and statistically analyzed.

2.6.Bioinformatics analysis
Data Source: Gene expression profiles were obtained from GEO database (https://www.ncbi.nlm.nih.gov/geo/). Identification of DEGs and Heatmap and clustering analysis: Data were analyzed using R software (version 3.6.2) and its package, limma. |logFC|>1 and p=<0.05 were set as cut-off for identifying DEGs. 7 7 Heatmaps and clustering were generated using the R software and limma package.
Enrichment analysis of DEGs: The R software (version 3.6.2) was applied for functional and pathway enrichment analyses of the DEGs. The enrichment content included GO and KEGG pathways. Pathway enrichment analysis depended on references from KEGG pathways. False discovery rate (FDR) <0.05 was applied as cut-off.

2.9.RNA-binding protein immunoprecipitation (RIP) assay
Immunoprecipitation of the circ-RBM23 bound to Ago2 was done via a Magna RIP™ RNA-binding protein immunoprecipitation kit (Merck Millipore, Germany). 2×10 7 L02 cells were harvested in RIP lysis buffer and lysates stored at -80 ℃. 8 µg of anti-Ago2 (MA5-23515, Invitrogen, USA) or normal control IgG was incubated with magnetic beads for 2 h at RT and 100 µL of the RIP lysate mixed with 900 µL of RIP immunoprecipitation buffer. This was then added to bead-antibody complexes and incubated overnight at 4℃. The beads were then mixed with proteinase K buffer and incubated for 30 min at 55℃ and RNA extracted for PCR.

2.10.Dual-luciferase report assay
Dual-luciferase assay was conducted using a dual-luciferase reporter assay (Promega, Madison, WI, USA) as usual (22). L02 cells were seeded on 24-well plates and cultured to 50% confluence. Then, they were transfected with circ-RBM23-wt or circ-RBM23-mut plasmid and negative control renilla luciferase plasmid together with miR-139-5p for 48 h. Cells were lysed then luciferase and renilla substrates were added. This was followed by luciferase analysis after 24 h using a dual-luciferase reporter (Promega) based on the manufacturer's instructions.

2.11.Cell culture, exosomes labeling, cell transfection, flow cytometry, liver histological and serum analysis, western-blot, cell proliferation assays
Details are outlined in the supplemental materials.

2.12.Statistical analysis
Continuous variables are presented as mean±standard error of the mean (SEM).
Analysis of variance (ANOVA) and post hoc Bonferroni analysis was conducted for multiple comparisons using GraphPad Prism 8.0. P<0.05 was considered statistically 9 9 significant.

Identification of hPMSCs and hPDSCs-exo
The hPMSCs have the essential characteristics of MSCs and are positive for the surface cell marker CD38 and negative for CD34, CD45, and CD133 ( Fig 1A).
Transmission electron microscopy (TEM) revealed that hPDSCs-exo had a round, ball-like shape ( Fig 1B). NanoSight analysis (NTA) showed that hPDSCs-exo had diameters of 30-150 nm and the quantification of exosomes was showed in the right panels (Fig1C). Western blot analysis demonstrated that the collected hPDSCs-exo expressed precise exosomal surface markers including CD9, CD63, CD81, and HSP70 ( Fig 1D). These results suggest that hPDSCs-exo exhibited specific exosomes characteristics that are identical to those described previously (12,13).

hPDSCs-exo promoted liver regeneration in vivo and hepatocyte proliferation in vitro
To determine whether hPDSCs-exo induced hepatocyte regeneration in vivo, we administered hPDSCs-exo by intraperitoneal injection before 70% partial hepatectomy (PH) in mice. Relative to PH-only mice, exosomes treatment significantly increased liver mitosis 2 days after PH as revealed by H&E staining (Fig   2A-B). Additionally, PCNA IHC staining revealed that exosomes promoted cell proliferation in mice after PH (Fig 2C-D). Furthermore, we showed that relative to exosomes treated mice, untreated mice exhibited markedly higher levels of ALT and AST, suggested greater liver injury (Fig 2E). To determine whether hPDSCs-exo affected hepatocyte proliferation, we treated cultured AML-12 and L02 cells with hPDSCs-exo which labeled with PKH26 (a cell membrane marker). Confocal analysis found that exosomes were directly taken up by the cells (Fig 2F). Moreover, CCK8 assays showed elevated cell proliferation after hPDSCs-exo treatment for 24 h ( Fig 2G). These results indicating that hPDSCs-exo plays a proliferative and protective role in PH mice.

The identification and characteristics of circ-RBM23 in hPDSCs-exo
We profiled circular RNA expression in hPDSCs-exo using high-throughput sequencing. We first selected 14 possible circular RNAs based on their expression ( Fig 3A) and found that circ-RBM23, circ-SWAP70, circ-MAPK9, circ-FKBP8, and circ-EPHB4 were highly expressed in hPDSCs-exo. RT-qPCR revealed that circ-RBM23 had the highest expression ( Fig 3B, primer of circ-RNAs were showed in supplementary Table 1), hence it was therefore selected for subsequent analysis.
Then we found that relative to linear RBM23, circ-RBM23 was resistant to RNase R digestion ( Fig 3C). Primer of linear RBM23 and circ-RBM23 were showed in supplementary Table 2. Furthermore, Sanger sequencing and circPrimer2.0 were used to confirm the head-to-tail splicing, circular features and characterized circ-RBM23 expression in hPMSCs ( Fig 3D). Next, we knocked down circ-RBM23 using siRNA in L02 cells and confirmed the silencing result using RT-qPCR (p<0.01, Fig   3E). CCK8 assays also showed that downregulating circ-RBM23 blocked the proliferation of L02 cells (Fig 3F). 11

Circ-RBM23 was served as a sponge for miR-139-5p
Previous studies showed that circular RNA mainly functions as miRNA sponges.

Circ-RBM23 regulated miR-139-5p/RRM2 in cell proliferation
In order to identify the important genes in liver regeneration, we used R software as usual to analyze dataset GSE97429 from GEO for genes involved in liver regeneration after PH (23). Gene expression profiles from 12 samples were retrieved from GEO database. The samples were then divided into the SHAM and 70% PH groups (Fig 4A). Volcano plot analyses were used to visualize differentially expressed genes (Fig 4B). Functional enrichment analysis was conducted to analyze differentially expressed genes (DEGs) functions. Gene Ontology (GO) analysis showed that the DEGs were implicated in "DNA replication" and "nuclear division" (Fig 4C). KEGG pathway analysis displayed a significant involvement in cell cycle signaling and DNA replication pathways (Fig 4D-E). Among the DEGs, RRM2 (Ribonucleotide Reductase Regulatory Subunit M2) elicited our attention. RRM2 provides DNA synthesis precursors and accelerates biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides. RRM2 is the primary target for miR-139-5p. Therefore, we hypothesized that circ-RBM23 might regulate RRM2 via the miR-139-5p.
Next, we intraperitoneally injected mice with si-circ-RBM23-exo 24 h prior to 70% PH. WB analysis showed similar results, the expression of RRM2, p-AKT, p-mTOR and eIF4G were downregulated in si-circ-RBM23-exo treated mice (Fig 6C). H&E staining showed that the mitotic count was significantly lower in si-circ-RBM23-exo mice than hPDSCs-exo treated mice detected in the hepatocellular area. In the other hand, PCNA IHC staining showed a similar trend in si-circ-RBM23-exo treated mice compared to the hPDSCs-exo treatment mice. Interestingly, liver mitosis and PCNA IHC staining did not differ in si-circ-RBM23-exo treated mice compared with the controls (Fig 6D).

Discussion
This study described a novel and significant function of hPMSCs secreted exosomes. Our findings revealed that hPDSCs-exo directly fused with hepatocytes, promoted their proliferation in vitro and in vivo. Mechanistically, hPMSCs exosomal circ-RBM23 regulated hepatocyte proliferation by sponging miR-139-5p via RRM2/AKT/mTOR signaling. These results improved our current understanding of hPDSCs-exo and highlight their therapeutic potential against liver disease.
CircRNA is a special type of ncRNA with a stable structure and conserved sequence in different biological tissues and cells (15,16). Studies indicate that circRNA have critical roles in various diseases, including colorectal cancer, pulmonary fibrosis, and diabetes . We found that circ-RBM23 was a potential key circRNA in hPDSCs-exo, and regulated hepatocyte cell proliferation. In 2011,  et al. suggested a regulatory mechanism of competing endogenous RNAs (ceRNA) (19). Our luciferase reporter assay indicated an interaction between miR-139-5p and circ-RBM23 in L02 cells. RIP assay showed that circ-RBM23 interacted with miR-139-5p. These findings suggested that circ-RBM23 could sponges miR-139-5p to inhibit its function in mouse after PH.
RRM2 comprises two 44-kDa proteins only expressed during late G1/early S phase of cell cycle (29). RRM2 overexpression is linked to cancer cell invasiveness, metastasis, tumorigenesis, and poor patient outcome (29,30). Our analysis revealed that RRM2 regulates PH development and promotes liver proliferation.
Several studies have recently showed that MSC exosomes have hepatoprotective effect (31-33). Du et al. reported that hiPSC-MSCs-Exo administration immediately after reperfusion harbored hepatoprotective effect (34). MSCs-exo increased hepatocyte proliferation after liver injury via carbon tetrachloride (35,36). Similarly, we observed a proliferative effect of hPDSCs-exo in PH mice. More importantly, our results demonstrated that hPMSCs exosomal circ-RBM23 induced hepatocyte proliferation and activated AKT/mTOR/eIF4G pathway by sponging miR-139-5p and modulated RRM2 in PH mice. In contrast, downregulated exosomal circ-RBM23 had no effect in hepatocyte proliferation relative to control mice. These findings suggested that hPDSCs-exo promoted their proliferation, which was in line with previous studies.
In summary, for the first time, we describe the proliferative and protective impacts of hPDSCs-exo on hepatocytes post PH. Furthermore, we find that exosomal circ-RBM23 promotes hepatocyte proliferation via miR-139-5p/RRM2/AKT/mTOR signaling (Fig 7). These results suggest a new mechanism of liver regeneration, which provide a potential therapeutic approach against liver disease and in transplantation.

Conclusion
Liver regeneration after partial hepatectomy is complex and well-orchestrated, hPDSCs-exo are membrane nano vesicles (30-100 nm) secreted by hPMSCs. This study shown that the hPMSCs exosomal circ-RBM23 serve as efficient miR-139-5p sponges and regulate liver regeneration of mouse after 70% partial hepatectomy by regulated RRM2/mTOR pathway. This highlighted the importance of ceRNA mechanism in pathogenesis and therapeutic development for liver regeneration in the future.

Ethics approval and consent to participate
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Availability of data and materials
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Competing interests
The authors declare that they have no competing interests.

Author contributions
All authors contributed to the study conception and design.     23 23 counted. c) PCNA staining 24h and 48h after surgery. d) The PCNA-positive cells were counted. e) Serum ALT and AST levels were determined. f) Immunofluorescent images of PKH26 (red) together with DAPI for nuclei (blue). g) Cell viabilities were measured by CCK8 in AML12 and L02 cells treated with hPDSCs-exo at the concentrations of 0%, 10%, 20%, 30% and 40% for 24h. Error bars represent the mean±standard error of the mean (SEM). *P<0.05, ***P<0.001.    Western blots were quantified. c) Liver tissues were lysed from circ-RBM23-siRNA pretreated hPDSCs-exo or vehicle treated PH mice, RRM2 and mTOR pathway protein levels were determined by western blots. Western blots were quantified. d) Liver tissues were collected and followed by H&E and PCNA staining of the liver postoperative from hPDSCs-exo, circ-RBM23-siRNA pretreated hPDSCs-exo or vehicle treated PH mice. The number of mitotic hepatocytes and PCNA-positive cells were counted. Error bars represent the SEM. *P<0.05, ***P<0.001.m