Liver-derived EVs from HBV-ACLF patient inhibited liver regeneration
To determine whether liver-derived EVs were associated with impaired liver regeneration of HBV-ACLF, we collected discarded liver tissues from HBV-ACLF patients after liver transplantation. Healthy liver from was collected as control. Liver-derived EVs of HBV-ACLF and healthy control (ACLF_EVs and HC_EVs) were extracted from liver tissue with reference to the methods reported in previous research. We then analyzed the physicochemical features of the ACLF_EVs and HC_EVs. Under Transmission Electron Microscope (TEM), Both ACLF_EVs and HC_EVs showed clear cup sharp (FigS1A), and nanoparticle tracking analysis (NTA) showed that their diameter was about 130nm (FigS1B). Westernblot showed that both ACLF_EVs and HC_EVs were positive to classical EVs markers CD9, CD81 and TSG101(FigS1C). This indicated that there was no difference between ACLF_EVs and HC_EVs in terms of markers, morphology and diameter.
Jaemin Lee BS et al14. used 50% CCl4-induced acute liver injury (ALI) mouse model, a classical model for liver regeneration researches, to demonstrate that liver-derived EVs could promote liver regeneration. Therefore, in order to explore whether ACLF_EVs had functions that affected liver regeneration, we injected ACLF_EVs (Group ACLF_EVs) and HC_EVs (Group HC_EVs) into ALI mice through tail vein to observe the pathological changes of liver (Fig1A). 100ug EVs were injected 6 hours before intraperitoneal injection of CCl4, While PBS (Group PBS) was used as a blank control. The mice were sacrificed 48 hours later. We detected markers of liver injury in plasma. Compared with the group PBS, ALT, AST and LDH were up-regulated in group ACLF_EVs. In contrast, ALT, AST and LDH were significantly down-regulated in group HC_EVs (Fig1B). In addition, HE staining of liver tissue showed similar results (Fig1C). Intraperitoneal injection of CCl4 could induce severe liver necrosis, while in group ACLF_EVs, the area of liver necrosis was further enlarged. On the contrary, HC_EVs showed a certain degree of protective effect on the injury liver, reducing the area of liver necrosis. It proved that ACLF_EVs could aggravate the degree of liver injury in ALI mice. During ALI, hepatocytes rapidly entered the cell cycle and proliferated to repair injury liver. Therefore, PCNA and ki67 staining were used to detect the proliferation situation of hepatocytes. As shown in Fig1D, the number of PCNA-positive hepatocytes was significantly decreased in group ACLF_EVs, while it was increased in group PBS and group HC_EVs. Ki67 staining showed similar results. In conclusion, our results showed that ACLF_EVs were negatively correlated with liver injury repair by inhibiting liver regeneration.
Liver-derived EVs from HBV-ACLF patient suppressed proliferation of hepatocytes in vitro
As the main parenchymal cells of liver tissue, hepatocytes played an important role in the process of liver regeneration. Therefore, we attempted to determine the effect of ACLF_EVs on hepatocytes in vitro using AML12 cells, a normal mouse hepatocyte line. Studies had shown that EVs primarily relied on fusion with membranes of target cell to deliver substances, thus performed regulatory functions. Therefore, we first attempted to confirm whether ACLF_EVs could bind to AML12 cells. We used PKH26 dye (a fluorescent red linker compound) to label ACLF_EVs and HC_EVs, and used PKH67 dye (a fluorescent green linker compound) to label membranes of AML12 cell in vitro. Both ACLF_EVs and HC_EVs were co-cultured with AML12 cells at a concentration of 25ug/ml for 24h. Using laser confocal microscopy, we found that the cell membrane of AML12 cells excited both red and green fluorescent signals, indicating that EVs were successfully absorbed into the cells (Fig2A). Then, we co-cultured ACLF_EVs and HC_EVs with AML12 cells at different concentration gradients to explore their effects on the proliferation of AML12 cells. CCK8 assay showed that proliferation of AML12 cell was significantly downregulated with increasing concentrations of ACLF_EVs compared to treat with PBS (Fig2B). In contrast, within increasing concentrations of HC_EVs, proliferation ability of AML12 cell was significantly improved (Fig2B). In addition, similar results were observed with EDU staining assay of AML12 cells (Fig2C). ACLF_EVs treatment resulted in a significant decrease in EDU incorporation and a significant down-regulation of the green fluorescence ratio in AML12 cells, while the opposite result was obtained after HC_EVs treatment. In summary, in vitro studies revealed that liver-derived EVs had the ability to regulate hepatocyte proliferation. ACLF_EVs could deliver negative proliferative signals to inhibit hepatocyte proliferation, suggesting their importance role in the regeneration inhibition of HBV-ACLF.
MiRNA in liver-derived EVs from HBV-ACLF patient dominated the inhibition of liver regeneration
MiRNAs were highly conserved among different species, and were abundant in EVs, which played a pivotal role in the regulation of recipient cell. Based on their important regulatory status, miRNAs in ACLF_EVs and HC_EVs were profiled using deep RNA sequencing to identify the miRNAs responsible for liver regeneration. Result showed that there were 106 differentially expressed miRNAs (DE-miRNAs) between ACLF_EVs and HC_EVs. Among them, 58 miRNAs were up-regulated and 48 miRNAs were down-regulated in ACLF_EVs compared with HC_EVs (Fig3A). Target genes of these DE-miRNAs were predicted by miRanda and RNAhybrid. Then, we performed GO and KEGG enrichment analysis on these target genes to investigate the function of miRNAs. GO enrichment analysis suggested that target genes of DE-miRNA were closely related to regeneration-related signaling modules such as cell-cell signaling, cell morphogenesis, regulation of multicellular organismal development (FigS2A). KEGG enrichment analysis revealed that target genes of DE-miRNA were mainly enriched in proliferation-related signaling pathways, mainly MAPK signaling pathway and Rap1 signaling pathway (Fig3A). Therefore, we suggested that DE-miRNAs were important substances in ACLF_EVs that regulating liver regeneration.
ACLF often occurred acutely in the setting of liver cirrhosis (LC). In this stage, persistent chronic injury impaired the ability of healthy hepatocytes to self-replicate, resulting in a severe decrease in the liver reserve function, which prevents compensatory liver regeneration in the face of acute injury. This implied a similar state of inhibition of liver regeneration in the diseased liver during the LC phase. Therefore, we extracted EVs from liver tissue of LC patients (LC_EVs) and co-cultured with AML12 cell in vitro. EDU staining assay revealed that, similar to ACLF_EVs, LC_EVs also had the ability to inhibit hepatocyte proliferation (FigS3A). The expression of miRNAs in LC_EVs were also profiled using deep RNA sequencing. It was found that there were 37 DE-miRNAs between LC_EVs and HC_EVs. Among them, 23 miRNAs were up-regulated and 14 miRNAs were down-regulated in LC_EVs compared with HC_EVs (Fig3B). GO enrichment analysis revealed that target genes of DE-miRNAs were closely related to regulation of cell differentiation, regulation of multicellular organismal development, cell development and other modules (FigS2B). KEGG enrichment analysis suggested that target genes of DE-miRNAs were mainly enriched in MAPK signaling pathway, WNT signaling pathway and HIPPO signaling pathway, which was closely associated with regeneration (Fig3B). Since miRNAs degraded mRNAs and thus inhibit protein translation and synthesis mainly through partial complementary binding to the 3' non-coding regions (3'UTRs) of target mRNA, here, we concentrated on DE-miRNAs upregulated in ACLF_EVs and LC_EVs. Thus, we searched for DE-miRNAs that were upregulated in both ACLF_EVs and LC_EVs. A total of 15 miRNAs were finally screened (Fig3C). Among them, three miRNAs (miR-146b-3p, miR218-5p, miR-200c-3p) were significantly up-regulating (p<0.05 and Log2FD>2). Expression of these three miRNAs in ACLF_EVs and HC_EVs was demonstrated by qPCR. Compared with HC_EVs, all of these three miRNAs increased significantly in ACLF_EVs (p<0.001) (Fig3D). In addition, we compared the sequence of these miRNAs between human and mouse (Fig3E). We found that the sequence of has-miR-146b-3p and mmu-miR-146b-3p did not match exactly, suggesting that the miR-146b-3p were not conserved between different species, while miR-218-5p and miR-200c-3p were highly conserved. Thus, we suggested that miR-218-5p, the most significantly DE-miRNA, might be the most important factor in ACLF_EVs that inhibited liver regeneration.
MiR-218-5p exerted an inhibitory effect in liver regeneration mediated by liver-derived EVs from HBV-ACLF patient
To investigate whether miR-218-5p had the function of regulating hepatocyte proliferation, we transfected AML12 cells in vitro with miR-218-5p-mimic to observe its function on liver regeneration. CCK8 assay revealed that the proliferation of AML12 cells transfected with miR-218-5p-mimic (group miR_mimic) was significantly inhibited compared to cells transfected with miR-218-5p-vehicle (group miR-veh) (Fig4A). Similarly, EDU staining assay revealed a significant reduction in green fluorescence in group miR_mimic (Fig4B). These results confirmed that miR-218-5p had the function of inhibiting the proliferation of hepatocytes. In order to further determine the function of miR-218-5p, we injected miR-218-5p-agomir (a powerful miRNA mimic) into ALI mice by tail vein injection to investigate its effect on liver regeneration. We performed immunohistochemical staining of mouse liver for PCNA and Ki67 and found that miR-218-5p had the ability to inhibit liver regeneration in mice (Fig4C). Next, we desired to investigate whether miR-218-5p was important for ACLF_EVs inhibition of hepatocyte proliferation. We co-cultured ACLF_EVs with AML12 cells, while using miR-218-5p-inhibitor (an inhibitory nucleic acid fragment of miR-218-5p) to transfect AML12 cells, in order to observe whether competitive inhibition of miR-218-5p, which is highly expressed within ACLF_EVs, could antagonized the inhibitory effect of ACLF_EVs on hepatocyte proliferation. CCK8 assay revealed that ACLF_EVs significantly inhibited the proliferation of AML12 cells, while miR-218-5p-inhibitor transfection reversed the proliferation inhibitory ability of ACLF_EVs on AML12 cells to some extent, which was not achieved after miR-veh transfection (Fig4D). Meanwhile, similar results were observed for EDU staining assay (Fig4E). In addition, we also injected ACLF_EVs and miR-218-5p-antagomir (a powerful miRNA inhibitor) together into ALF mice. Immunohistochemical staining for PCNA and Ki67 suggested that competitive inhibition of miR-218-5p, which was highly expressed within ACLF_EVs, effectively reversed the inhibitory effect of ACLF_EVs on liver regeneration (Fig4F). Therefore, we suggested that miR-218-5p was a key factor in ACLF_EVs that exerted regenerative inhibitory ability.
MiR‐218‐5p inactivated the ERK1/2 signaling pathway by targeting FGFR2
Next, we attempted to identify how miR-218-5p regulated hepatocyte proliferation. Usually, miRNA regulated cell function by repressing target mRNA. Therefore, it was crucial to identify the target mRNA of miR-218-5p that inhibited hepatocyte proliferation. We used targetscan tool to predict the target genes of hsa-miR-218-5p, and a total of 1102 potential target genes were detected. In addition, we performed single-cell RNA sequencing of liver tissues from HBV-ACLF patients and healthy humans to observe the actual changes in the expression of mRNA. We sorted out subpopulation of hepatocytes from the data of single-cell RNA sequencing of liver tissues and compared the differential changes of mRNAs between HBV-ACLF and healthy human. We screened a total of 469 differentially expressed mRNAs (DE-mRNAs, p<0.05 and Log2FC >0.5/Log2FC<-0.5), of which 198 DE-mRNAs were down-regulated and 271 DE-mRNAs were up-regulated. Since the function of miRNAs was mainly to repress target mRNAs, we focused on the down-regulated part of DE-mRNAs. In addition, in order to predict the function of miRNAs more accurately, the species conserved nature of miRNAs must be taken into account when screening target mRNAs. Therefore, we predicted the target mRNAs of mmu-miR-218-5p, and a total of 963 potential target mRNAs were predicted. Next, we performed a cross-screening of the three mRNA datasets. A total of three DE-mRNAs were screened, which were significantly down-regulated in hepatocytes of ACLF patients and were potential target mRNAs for hsa/mmu-miR-218-5p (Fig5A). Among them, FGFR2 showed the most significant differential changes and was closely associated with hepatocyte proliferation and liver regeneration (Fig5B)23. Therefore, we suggested that FGFR2 might be an important potential target mRNA for miR-218-5p. We used RNAhybrid software to predict the potential relationship between miR-218-5p and FGFR2, and found that miR-218-5p had a complementary pairing sequence at the 3'UTR of FGFR2 (Fig5C), and luciferase reporter assay confirmed the interaction between miR-218-5p and the 3′-UTR of FGFR2 (Fig5D). We used immunofluorescence assay to observe the expression level of FGFR2 in liver tissues of ACLF patients (Fig5E). Compared with NC, the expression level of FGFR2 in hepatocyte was significantly downregulated in HBV-ACLF patients. In addition, we analyzed the expression level of FGFR2 in AML12 cells after transfecting with miR-mimic and found that FGFR2 level was significantly downregulated (Fig5F). Meanwhile, the downregulation of FGFR2 was reversed by transfection of miR-inh into AML12 cell co-cultured with ACLF_EVs (Fig5G). KEGG pathway Database suggested that ERK1/2 signaling pathway was the main downstream signaling pathway of FGFR2 and was closely related to liver regeneration24. Meanwhile, KEGG enrichment analysis of miRNAs in ACLF_EVs also suggested the importance of MAPK signaling pathway. Therefore, we subsequently detected the changes in ERK1/2 pathway. The results suggested that ERK1/2 activation was significantly downregulated in AML12 cells transfected with miR-mimic, and expression of its downstream cyclinD1 was also repressed (Fig5F). Furthermore, activation of ERK1/2 and cyclinD1 levels were also significantly decreased in AML12 cells after ACLF_EVs treatment, which was improved by transfection with miR-inh (Fig5G). Thus, our results suggested that miR-218-5p, which was highly expressed within ACLF_EVs, functioned as an inhibitor of liver regeneration by downregulating the expression of FGFR2 in hepatocytes to suppress the activation of ERK1/2 pathway.
Inhibition of miR-218-5p effectively enhanced liver regeneration
Our results above revealed the importance of miR-218-5p, so we sought to investigate whether ACLF mice had similar alterations as ACLF patients. We constructed an ACLF mouse model according to the method reported in previous literature and examined the expression levels of miR-218-5p in EVs of liver tissue from ACLF mice. The results showed that the expression of miR-218-5p was significantly increased (Fig6A). Immunofluorescence assay of liver tissue also demonstrated a significant downregulation of FGFR2 expression level in hepatocytes of ACLF mice (Fig6B).
Based on the inhibitory function of miR-218-5p on liver regeneration, we sought to see whether inhibition of miR-218-5p in vivo could exert a therapeutic effect. MiR-218-5p-antagomir, a specially chemically modified miR-218-5p antagonist, inhibited function of miR-218-5p by strongly competing with mature miR-218-5p in vivo to prevent complementary binding of miRNA with its target gene mRNA. Administered through the tail vein, it could be transported to the whole body through the blood circulation. However, miRNAs were susceptible to degradation during blood transportation and could not target specific tissues and cells, which greatly affected its therapeutic effects. Yang et al. found that LNP could target hepatocytes in fibrosis mice through interaction with apolipoprotein E and was subsequently internalized into hepatocytes through endocytosis25. Kentaro Gokita et al. demonstrated the ability of LNP to deliver miRNA26. Therefore, in order to enhance its stability in vivo and its targeting ability to hepatocytes, we used LNP as a delivery system for miR-218-5p-antagomir. We administered LNP-miR-218-5p-antagomir (Group LNP-ant), LNP-miR-vehicle (Group LNP-veh), miR-218-5p-antagomir (Group miR-ant), miR-vehicle (Group miR-veh) and PBS (Group PBS) to treat ACLF mice (Fig6C). Mice were sacrificed 48 hours after acute CCl4 injection. To investigate liver regeneration in ACLF mice, we performed PCNA and Ki67 staining on mouse liver tissues (Fig6D). Results revealed that PNCA and Ki67 expression levels in liver tissues of group miR-ant and LNP-ant were significantly improved. This suggested that inhibition of miR-218-5p could enhance the regenerative ability of the liver. Then, to verify the potential mechanism of restoration of liver regenerative capacity in ACLF mice, we performed immunofluorescence staining on mouse liver tissues. We found that in Group miR-ant and LNP-ant, FGFR2 expression levels in liver tissues were significantly increased and co-localized mainly with hepatocytes (Fig7A). In addition, we examined the activation of ERK1/2 pathway in liver tissue. The result showed that the levels of p-ERK1/2 and its downstream protein cyclinD1 were significantly up-regulated in group miR-ant and LNP-ant, suggesting that inhibition of miR-218-5p in mice could improve liver regeneration by increasing the activation of ERK1/2 signaling pathway (Fig7B).