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Cited by 16Extracellular vesicles in diseaseLiver physiology and pathologyMesenchymal stem cell research

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Engineered EVs from LncEEF1G - overexpressing MSCs promote fibrotic liver regeneration by upregulating HGF release from hepatic stellate cells

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Zhang, Jiebin, Qiu, Xiaotong, Lei, Yunguo, Chen, Haitian, Wu, Dongwei, Wang, Tingting, Sui, Xin, Xiao, Jiaqi, Jiang, Chenhao, Zhang, Huayao, Liu, Yasong, Liu, Xiaoquan, Zhang, Yingcai, Che, Xu, Lin, Ye, Yao, Jia, Pan, Zihao, Li, Rong, Zheng, Jun·2025·Experimental & Molecular Medicine
DOI 10.1038/s12276-025-01413-4PMC PMC11958733

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Abstract

Fibrosis is a disease that negatively affects liver regeneration, resulting in severe complications after liver surgery. However, there is still no clinically effective treatment for promoting fibrotic liver regeneration because the underlying hepatocellular mechanism remains poorly understood. Through microRNA microarrays combined with the application of AAV6, we found that high expression of miR-181a-5p in activated hepatic stellate cells (HSCs) suppressed the expression of hepatic growth factor (HGF) and partially contributed to impaired regeneration potential in mice with hepatic fibrosis that had undergone two-thirds partial hepatectomy. As nanotherapeutics, mesenchymal stem-cell-derived extracellular vesicles (MSC-EVs) have been verified as effective treatments for liver regeneration. Here we observe that MSC-EVs can also promote fibrotic liver regeneration via enriched lncEEF1G, which acts as a competing endogenous RNA to directly sponge miR-181a-5p, leading to the upregulated expression of HGF in HSCs. Finally, engineered MSC-EVs with high expression of lncEEF1G (lncEEF1G OE -EVs) were constructed, suggesting greater potential for this model. In summary, our findings indicate that lncEEF1G OE -EVs have a nanotherapeutic effect on promoting regeneration of fibrotic livers by modulating the miR-181a-5p/HGF pathway in HSCs, which highlights the potential of extracellular vesicle engineering technology for patients with hepatic fibrosis who have undergone hepatic surgery.

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Engineered mesenchymal stem cells that overexpress lncEEF1G can secrete extracellular vesicles that are rich in lncEEF1G (lncEEF1GOE-EVs). Upon injection of lncEEF1GOE-EVs into a fibrotic 70% partial hepatectomy mouse model, lncEEF1G competitively binds to miR-181a-5p in hepatic stellate cells, preventing the interaction between miR-181a-5p and the messenger RNA of hepatocyte growth factor. This consequently leads to an increase in the secretion of hepatocyte growth factor and the promotion of hepatocyte proliferation.
Engineered mesenchymal stem cells that overexpress lncEEF1G can secrete extracellular vesicles that are rich in lncEEF1G (lncEEF1GOE-EVs). Upon injection of lncEEF1GOE-EVs into a fibrotic 70% partial hepatectomy mouse model, lncEEF1G competitively binds to miR-181a-5p in hepatic stellate cells, preventing the interaction between miR-181a-5p and the messenger RNA of hepatocyte growth factor. This consequently leads to an increase in the secretion of hepatocyte growth factor and the promotion of hepatocyte proliferation.CC BY · PubMed Central
Fig. 1 Liver regeneration is weakened in fibrotic mice. a A schematic diagram of the preparation of a mouse hepatic fibrotic model through CCl4 treatment followed by two-thirds PHx. b Serum ALT, AST and LDH levels at the indicated time points after PHx (n = 4 independent biological mouse samples). c LW/BW ratios at the indicated time points after PHx (n = 4 independent biological mouse samples). d The representative images of H&E-stained samples at the indicated time points after PHx. Scale bar, 50 μm. e A quantification of the mitotic frequency determined by counting the numbers of mitotic nuclei and total nuclei in randomly selected fields (n = 3 independent experiments). f, g The representative images (f) and quantification (g) of Ki67 immunohistochemical staining at the indicated time points after PHx. Scale bar, 100 μm (n = 3 independent experiments). h, Western blotting (left) and relative quantification (right) of PCNA protein expression at the indicated time points after PHx in liver tissues (n = 3 independent biological mouse samples). i RT‒qPCR analysis of the levels of HGF and TGF-β in HSCs isolated from the PHx model (n = 4 independent cell experiments). j Western blotting (left) and relative quantification (right) showing the expression of HGF and TGF-β in HSCs isolated from the PHx model (n = 3 independent experiments). k–m In vitro experiments include: RT‒qPCR analysis of HGF in the LX-2 HSC line (n = 3 independent cell experiments) (k) western blotting (left) and relative quantification (right) showing the expression of HGF in the LX-2 HSC line (n = 3 independent cell experiments) (l) and ELISA results showing the HGF levels in LX-2 HSC line culture medium (n = 3 independent cell experiments) (m). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments or four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. n.s., no significance.
Fig. 1 Liver regeneration is weakened in fibrotic mice. a A schematic diagram of the preparation of a mouse hepatic fibrotic model through CCl4 treatment followed by two-thirds PHx. b Serum ALT, AST and LDH levels at the indicated time points after PHx (n = 4 independent biological mouse samples). c LW/BW ratios at the indicated time points after PHx (n = 4 independent biological mouse samples). d The representative images of H&E-stained samples at the indicated time points after PHx. Scale bar, 50 μm. e A quantification of the mitotic frequency determined by counting the numbers of mitotic nuclei and total nuclei in randomly selected fields (n = 3 independent experiments). f, g The representative images (f) and quantification (g) of Ki67 immunohistochemical staining at the indicated time points after PHx. Scale bar, 100 μm (n = 3 independent experiments). h, Western blotting (left) and relative quantification (right) of PCNA protein expression at the indicated time points after PHx in liver tissues (n = 3 independent biological mouse samples). i RT‒qPCR analysis of the levels of HGF and TGF-β in HSCs isolated from the PHx model (n = 4 independent cell experiments). j Western blotting (left) and relative quantification (right) showing the expression of HGF and TGF-β in HSCs isolated from the PHx model (n = 3 independent experiments). k–m In vitro experiments include: RT‒qPCR analysis of HGF in the LX-2 HSC line (n = 3 independent cell experiments) (k) western blotting (left) and relative quantification (right) showing the expression of HGF in the LX-2 HSC line (n = 3 independent cell experiments) (l) and ELISA results showing the HGF levels in LX-2 HSC line culture medium (n = 3 independent cell experiments) (m). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments or four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. n.s., no significance.CC BY · PubMed Central
Fig. 2 The upregulation of miR-181a-5p in activated HSCs blocks HGF expression. a A heat map displaying the expression patterns of miRNAs in control and TGF-β-treated HSCs. The upregulated genes are shown in red, and the downregulated genes are shown in blue (n = 3 independent cell samples). b RT‒qPCR results showing the levels of differential miRNA expression in the HSC lines of each group (n = 3 independent cell samples). c RT‒qPCR results showing the levels of differential miRNA expression in the liver tissue of each group (n = 3 independent biological mice samples). d FISH results showing the localization of miR-181a-5p (red) in control and TGF-β-treated HSCs. Scale bar, 20 μm. e A schematic diagram of the construction of luciferase reporter plasmids with mutant and WT HGF sequences. f The luciferase activity of WT and mutant HGFs was detected by luciferase reporter assays after cotransfection with either the miR-181a-5p or control mimics (n = 3 independent experiments). g, h RT‒qPCR (g) and ELISA (h) results showing the levels of HGF in each HSC treatment group (n = 3 independent cell experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. n.s., no significance.
Fig. 2 The upregulation of miR-181a-5p in activated HSCs blocks HGF expression. a A heat map displaying the expression patterns of miRNAs in control and TGF-β-treated HSCs. The upregulated genes are shown in red, and the downregulated genes are shown in blue (n = 3 independent cell samples). b RT‒qPCR results showing the levels of differential miRNA expression in the HSC lines of each group (n = 3 independent cell samples). c RT‒qPCR results showing the levels of differential miRNA expression in the liver tissue of each group (n = 3 independent biological mice samples). d FISH results showing the localization of miR-181a-5p (red) in control and TGF-β-treated HSCs. Scale bar, 20 μm. e A schematic diagram of the construction of luciferase reporter plasmids with mutant and WT HGF sequences. f The luciferase activity of WT and mutant HGFs was detected by luciferase reporter assays after cotransfection with either the miR-181a-5p or control mimics (n = 3 independent experiments). g, h RT‒qPCR (g) and ELISA (h) results showing the levels of HGF in each HSC treatment group (n = 3 independent cell experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. n.s., no significance.CC BY · PubMed Central
Fig. 3 Blocking miR-181a-5p in HSCs strengthens liver regeneration in fibrotic PHx model mice. a A schematic diagram of the application of an AAV6-miR-181a-5p inhibitor to determine whether miR-181a-5p can modulate the expression of HGF in HSCs and affect the regeneration of the fibrotic liver. b Serum ALT, AST and LDH levels in different treatment groups (n = 4 independent biological mouse samples). c The ratio of LW/BW in different treatment groups (n = 4 independent biological mouse samples). d The representative images of H&E-stained samples from different treatment groups. Scale bar, 50 μm. e A quantification of the mitotic frequency determined by counting the numbers of mitotic nuclei and total nuclei in randomly selected fields (n = 4 independent biological mouse samples). f, g The representative images (f) and quantification (g) of Ki67 immunohistochemical staining in different treatment groups. Scale bar, 100 μm (n = 4 independent biological mouse samples). h Western blotting (left) and relative quantification (right) showing the expression of PCNA in different treatment groups (n = 3 independent experiments). i, j RT‒qPCR (i) and representative western blotting (j, left) results showing the expression of HGF in HSCs isolated from different treatment groups and a quantification of HGF protein levels (j, right) (n = 3 independent experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments or four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. n.s., no significance.
Fig. 3 Blocking miR-181a-5p in HSCs strengthens liver regeneration in fibrotic PHx model mice. a A schematic diagram of the application of an AAV6-miR-181a-5p inhibitor to determine whether miR-181a-5p can modulate the expression of HGF in HSCs and affect the regeneration of the fibrotic liver. b Serum ALT, AST and LDH levels in different treatment groups (n = 4 independent biological mouse samples). c The ratio of LW/BW in different treatment groups (n = 4 independent biological mouse samples). d The representative images of H&E-stained samples from different treatment groups. Scale bar, 50 μm. e A quantification of the mitotic frequency determined by counting the numbers of mitotic nuclei and total nuclei in randomly selected fields (n = 4 independent biological mouse samples). f, g The representative images (f) and quantification (g) of Ki67 immunohistochemical staining in different treatment groups. Scale bar, 100 μm (n = 4 independent biological mouse samples). h Western blotting (left) and relative quantification (right) showing the expression of PCNA in different treatment groups (n = 3 independent experiments). i, j RT‒qPCR (i) and representative western blotting (j, left) results showing the expression of HGF in HSCs isolated from different treatment groups and a quantification of HGF protein levels (j, right) (n = 3 independent experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments or four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. n.s., no significance.CC BY · PubMed Central
Fig. 4 Characterization of MSC-EVs. a A schematic diagram of the procedures for obtaining MSC-EVs from MSC-conditioned medium. b, c TEM (b) and NTA (c) were used to detect the morphology and size of MSC-EVs. Scale bar, 0.1 μm. d Representative western blotting results showing the expression of specific vesicle-related markers, namely, ALIX, CD63, CD81 and GRP94. e The representative fluorescence images of the uptake of MSC-EVs (fluorescent red) in HSCs were obtained via confocal microscopy. Scale bar, 20 μm.
Fig. 4 Characterization of MSC-EVs. a A schematic diagram of the procedures for obtaining MSC-EVs from MSC-conditioned medium. b, c TEM (b) and NTA (c) were used to detect the morphology and size of MSC-EVs. Scale bar, 0.1 μm. d Representative western blotting results showing the expression of specific vesicle-related markers, namely, ALIX, CD63, CD81 and GRP94. e The representative fluorescence images of the uptake of MSC-EVs (fluorescent red) in HSCs were obtained via confocal microscopy. Scale bar, 20 μm.CC BY · PubMed Central
Fig. 5 The biodistribution of MSC-EVs in fibrotic PHx model mice. a The representative in vivo imaging system (IVIS) images of the fibrotic PHx mouse model injected with DiR supernatant-treated control (left) or DiR-labeled MSC-EVs (right) at 3, 6 and 48 h. b Representative IVIS images of organs (from top to bottom: heart, lung, liver, spleen and kidneys) collected at 3, 6 and 48 h. c A quantification of the fluorescence intensity in livers at 3, 6 and 48 h (n = 4 independent biological mouse samples). d The representative fluorescent sections of different organs (heart, lung, liver, spleen and kidneys) used to investigate the biodistribution of DiR-labeled MSC-EVs (red). Scale bar, 50 μm. The statistical data are presented as mean ± s.d., and the error bars represent the means of four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig. 5 The biodistribution of MSC-EVs in fibrotic PHx model mice. a The representative in vivo imaging system (IVIS) images of the fibrotic PHx mouse model injected with DiR supernatant-treated control (left) or DiR-labeled MSC-EVs (right) at 3, 6 and 48 h. b Representative IVIS images of organs (from top to bottom: heart, lung, liver, spleen and kidneys) collected at 3, 6 and 48 h. c A quantification of the fluorescence intensity in livers at 3, 6 and 48 h (n = 4 independent biological mouse samples). d The representative fluorescent sections of different organs (heart, lung, liver, spleen and kidneys) used to investigate the biodistribution of DiR-labeled MSC-EVs (red). Scale bar, 50 μm. The statistical data are presented as mean ± s.d., and the error bars represent the means of four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001.CC BY · PubMed Central
Fig. 6 MSC-EVs promote fibrotic liver regeneration via the upregulation of HGF expression in HSCs. a A schematic diagram of the application of MSC-EVs to determine whether MSC-EVs can stimulate the regeneration of the fibrotic liver. b The serum ALT, AST and LDH levels in different treatment groups (n = 4 independent biological mouse samples). c The LW/BW ratio in different treatment groups (n = 4 independent biological mouse samples). d The representative images of H&E-stained samples from different treatment groups. Scale bar, 50 μm). e A quantification of the mitotic frequency determined by counting the numbers of mitotic nuclei and total nuclei in randomly selected fields (n = 4 independent biological mouse samples). f, g The representative images (f) and quantification (g) of Ki67 immunohistochemical staining in different treatment groups. Scale bar, 100 μm (n = 4 independent biological mouse samples). h Western blotting (left) and relative quantification (right) showing the expression of PCNA in different treatment groups (n = 3 independent experiments). i, j RT‒qPCR (i) and representative western blotting (j, left) results showing the expression of HGF in HSCs isolated from different treatment groups and a quantification of HGF protein levels (j, right) (n = 3 independent experiments). In vitro experiments including LX-2 HSCs treated with TGF-β were given PBS or MSC-EVs. k, l RT‒qPCR (k) and ELISA (l) were used to measure the HGF level in the LX-2 HSC line (n = 3 independent cell experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments or four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig. 6 MSC-EVs promote fibrotic liver regeneration via the upregulation of HGF expression in HSCs. a A schematic diagram of the application of MSC-EVs to determine whether MSC-EVs can stimulate the regeneration of the fibrotic liver. b The serum ALT, AST and LDH levels in different treatment groups (n = 4 independent biological mouse samples). c The LW/BW ratio in different treatment groups (n = 4 independent biological mouse samples). d The representative images of H&E-stained samples from different treatment groups. Scale bar, 50 μm). e A quantification of the mitotic frequency determined by counting the numbers of mitotic nuclei and total nuclei in randomly selected fields (n = 4 independent biological mouse samples). f, g The representative images (f) and quantification (g) of Ki67 immunohistochemical staining in different treatment groups. Scale bar, 100 μm (n = 4 independent biological mouse samples). h Western blotting (left) and relative quantification (right) showing the expression of PCNA in different treatment groups (n = 3 independent experiments). i, j RT‒qPCR (i) and representative western blotting (j, left) results showing the expression of HGF in HSCs isolated from different treatment groups and a quantification of HGF protein levels (j, right) (n = 3 independent experiments). In vitro experiments including LX-2 HSCs treated with TGF-β were given PBS or MSC-EVs. k, l RT‒qPCR (k) and ELISA (l) were used to measure the HGF level in the LX-2 HSC line (n = 3 independent cell experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments or four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001.CC BY · PubMed Central
Fig. 7 LncEEF1G derived from MSC-EVs upregulates HGF expression in HSCs by acting as a sponge to target miR-181a-5p. a A Venn diagram showing lncRNAs from three independent samples of MSC-EVs obtained through whole-transcriptome sequencing analysis. b Bioinformatic analysis revealing that lncEEF1G acts as a miRNA sponge for miR-181a-5p in HSCs. c RT‒qPCR analysis of the miRNAs (let-7a-5p, miR-20b-5p, miR-100-3p, miR-124-3p, miR-149-5p, miR-217-3p, miR-326, miR-378b, miR-484 and miR-181a-5p) that potentially interact with lncEEF1G (n = 3 independent cell experiments). d A RIP assay was conducted to confirm the interaction between lncEEF1G and miR-181a-5p (n = 3 independent cell experiments). e The RNAalifold online database was used to predict the site at which lncEEF1G binds to miR-181a-5p for further mutation. f The luciferase activity of WT and mutant lncEEF1G was detected by luciferase reporter assays after cotransfection with either miR-181a-5p mimics or control mimics (n = 3 independent cell experiments). g A biotin-labeled miR-181a-5p probe was used to confirm that lncEEF1G could be captured by miR-181a-5p (n = 3 independent cell experiments). h FISH results showing the localization of lncEEF1G (green) and miR-181a-5p (red) in HSCs. Scale bar, 20 μm. For HSCs, PBS, TGF-β, TGF-β + MSC-EVs, TGF-β + MSC-EVssilncEEF1G or TGF-β + MSC-EVssilncEEF1G + miR-181a-5p were added to each group. This finding further verified that lncEEF1G in MSC-EVs regulates HGF levels by interacting with miR-181a-5p. i, j RT‒qPCR (i) and representative western blotting (j, upper) results showing the expression of HGF in HSCs in different treatment groups and a quantification (j, down) of HGF protein levels (n = 3 independent cell experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. n.s., no significance.
Fig. 7 LncEEF1G derived from MSC-EVs upregulates HGF expression in HSCs by acting as a sponge to target miR-181a-5p. a A Venn diagram showing lncRNAs from three independent samples of MSC-EVs obtained through whole-transcriptome sequencing analysis. b Bioinformatic analysis revealing that lncEEF1G acts as a miRNA sponge for miR-181a-5p in HSCs. c RT‒qPCR analysis of the miRNAs (let-7a-5p, miR-20b-5p, miR-100-3p, miR-124-3p, miR-149-5p, miR-217-3p, miR-326, miR-378b, miR-484 and miR-181a-5p) that potentially interact with lncEEF1G (n = 3 independent cell experiments). d A RIP assay was conducted to confirm the interaction between lncEEF1G and miR-181a-5p (n = 3 independent cell experiments). e The RNAalifold online database was used to predict the site at which lncEEF1G binds to miR-181a-5p for further mutation. f The luciferase activity of WT and mutant lncEEF1G was detected by luciferase reporter assays after cotransfection with either miR-181a-5p mimics or control mimics (n = 3 independent cell experiments). g A biotin-labeled miR-181a-5p probe was used to confirm that lncEEF1G could be captured by miR-181a-5p (n = 3 independent cell experiments). h FISH results showing the localization of lncEEF1G (green) and miR-181a-5p (red) in HSCs. Scale bar, 20 μm. For HSCs, PBS, TGF-β, TGF-β + MSC-EVs, TGF-β + MSC-EVssilncEEF1G or TGF-β + MSC-EVssilncEEF1G + miR-181a-5p were added to each group. This finding further verified that lncEEF1G in MSC-EVs regulates HGF levels by interacting with miR-181a-5p. i, j RT‒qPCR (i) and representative western blotting (j, upper) results showing the expression of HGF in HSCs in different treatment groups and a quantification (j, down) of HGF protein levels (n = 3 independent cell experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. n.s., no significance.CC BY · PubMed Central
Fig. 8 LncEEF1G derived from MSC-EVs strengthens liver regeneration in fibrotic PHx model mice. a A schematic diagram of the application of MSC-EVssilncEEF1G to determine whether the ability of MSC-EVs to stimulate fibrotic liver regeneration depends on the direct interaction of lncEEF1G with miR-181a-5p to modulate HGF expression in HSCs. b Serum ALT, AST and LDH levels in different treatment groups (n = 4 independent biological mouse samples). c The LW/BW ratio in different treatment groups (n = 4 independent biological mouse samples). d The representative images of H&E-stained samples from different treatment groups. Scale bar, 50 μm). e A quantification of the mitotic frequency determined by counting the numbers of mitotic nuclei and total nuclei in randomly selected fields (n = 4 independent biological mouse samples). f, g The representative images (f) and quantification (g) of Ki67 immunohistochemical staining in different treatment groups. Scale bar, 100 μm (n = 4 independent biological mouse samples). h Western blotting (left) and relative quantification (right) showing the expression of PCNA in different treatment groups (n = 3 independent experiments). i, j RT‒qPCR (i) and ELISA (j) results showing the HGF levels of primary HSCs from each group (RT‒qPCR, n = 4 independent cell experiments; ELISA, n = 3 independent cell experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments or four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig. 8 LncEEF1G derived from MSC-EVs strengthens liver regeneration in fibrotic PHx model mice. a A schematic diagram of the application of MSC-EVssilncEEF1G to determine whether the ability of MSC-EVs to stimulate fibrotic liver regeneration depends on the direct interaction of lncEEF1G with miR-181a-5p to modulate HGF expression in HSCs. b Serum ALT, AST and LDH levels in different treatment groups (n = 4 independent biological mouse samples). c The LW/BW ratio in different treatment groups (n = 4 independent biological mouse samples). d The representative images of H&E-stained samples from different treatment groups. Scale bar, 50 μm). e A quantification of the mitotic frequency determined by counting the numbers of mitotic nuclei and total nuclei in randomly selected fields (n = 4 independent biological mouse samples). f, g The representative images (f) and quantification (g) of Ki67 immunohistochemical staining in different treatment groups. Scale bar, 100 μm (n = 4 independent biological mouse samples). h Western blotting (left) and relative quantification (right) showing the expression of PCNA in different treatment groups (n = 3 independent experiments). i, j RT‒qPCR (i) and ELISA (j) results showing the HGF levels of primary HSCs from each group (RT‒qPCR, n = 4 independent cell experiments; ELISA, n = 3 independent cell experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments or four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001.CC BY · PubMed Central
Fig. 9 Engineered MSC-EVs with high lncEEF1G expression further upregulate HGF expression in HSCs to stimulate fibrotic liver regeneration. a TEM and NTA were used to detect the morphology and size of the MSC-EVs and lncEEF1GOE-EVs. Scale bar, 0.1 μm. b Representative western blots of specific vesicle-related markers (ALIX, CD63, CD81 and GRP94). c Representative fluorescence images of the uptake of lncEEF1GOE-EVs (fluorescent red) in HSCs were obtained via confocal microscopy. Scale bar, 20 μm). d RT‒qPCR showing the expression of lncEEF1G in MSC-EVs and lncEEF1GOE-EVs. e A schematic diagram of the application of lncEEF1GOE-EVs to detect whether lncEEF1GOE-EVs stimulate fibrotic liver regeneration. f Serum ALT, AST and LDH levels in different treatment groups (n = 4 independent biological mouse samples). g The LW/BW ratio in different treatment groups (n = 4 independent biological mouse samples). h The representative images of H&E-stained samples from different treatment groups. Scale bar, 50 μm). i A quantification of the mitotic frequency determined by counting the numbers of mitotic nuclei and total nuclei in randomly selected fields (n = 4 independent biological mouse samples). j, k The representative images (j) and quantification (k) of Ki67 immunohistochemical staining in different treatment groups. Scale bar, 100 μm (n = 4 independent biological mouse samples). l Western blotting (left) and relative quantification (right) showing the expression of PCNA in liver samples subjected to different treatments (n = 3 independent experiments). m, n RT‒qPCR (m) and representative western blotting (n, left) results showing the expression of HGF in HSCs isolated from different treatment groups and a quantification (n, right) of HGF protein levels (n = 3 independent experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments or four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig. 9 Engineered MSC-EVs with high lncEEF1G expression further upregulate HGF expression in HSCs to stimulate fibrotic liver regeneration. a TEM and NTA were used to detect the morphology and size of the MSC-EVs and lncEEF1GOE-EVs. Scale bar, 0.1 μm. b Representative western blots of specific vesicle-related markers (ALIX, CD63, CD81 and GRP94). c Representative fluorescence images of the uptake of lncEEF1GOE-EVs (fluorescent red) in HSCs were obtained via confocal microscopy. Scale bar, 20 μm). d RT‒qPCR showing the expression of lncEEF1G in MSC-EVs and lncEEF1GOE-EVs. e A schematic diagram of the application of lncEEF1GOE-EVs to detect whether lncEEF1GOE-EVs stimulate fibrotic liver regeneration. f Serum ALT, AST and LDH levels in different treatment groups (n = 4 independent biological mouse samples). g The LW/BW ratio in different treatment groups (n = 4 independent biological mouse samples). h The representative images of H&E-stained samples from different treatment groups. Scale bar, 50 μm). i A quantification of the mitotic frequency determined by counting the numbers of mitotic nuclei and total nuclei in randomly selected fields (n = 4 independent biological mouse samples). j, k The representative images (j) and quantification (k) of Ki67 immunohistochemical staining in different treatment groups. Scale bar, 100 μm (n = 4 independent biological mouse samples). l Western blotting (left) and relative quantification (right) showing the expression of PCNA in liver samples subjected to different treatments (n = 3 independent experiments). m, n RT‒qPCR (m) and representative western blotting (n, left) results showing the expression of HGF in HSCs isolated from different treatment groups and a quantification (n, right) of HGF protein levels (n = 3 independent experiments). The statistical data are presented as mean ± s.d., and the error bars represent the means of three independent experiments or four independent biological mouse samples. Statistical significance was determined by a Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001.CC BY · PubMed Central

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Sorted by citations

Metadata

Authors
Zhang, J., Qiu, X., Lei, Y., Chen, H., Wu, D., Wang, T., Sui, X., Xiao, J., Jiang, C., Zhang, H., Liu, Y., Liu, X., Zhang, Y., Che, X., Lin, Y., Yao, J., Pan, Z., Li, R., & Zheng, J.
Affiliations
  • Third Affiliated Hospital of Sun Yat-sen University (CN)
  • Chinese Academy of Medical Sciences & Peking Union Medical College (CN)
  • Guangdong Academy of Medical Sciences (CN)
Corresponding author
  • Yao, Jia
  • Pan, Zihao
  • Li, Rong
  • Zheng, Jun
ORCID
Venue
Experimental & Molecular Medicine
Publisher
Springer Science and Business Media LLC
Published
2025-03-03
Volume
57
Issue
3
Pages
584-600
ISSN
  • 1226-3613 (ISSN-L)
  • 2092-6413
DOI
10.1038/s12276-025-01413-4
PMC
PMC11958733
OpenAlex
W4408082218
Cited by
16
References
1
Citations by year
2026: 12 · 2025: 4
FWCI
4.81
Type
journal article
Language
en
Open access
Licence
Locations
  • Experimental & Molecular Medicine (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (81900597, 2021A1515011156, 202102020237, 82300747, 81970567, 81802897)
  • Sun Yat-sen University
  • China Postdoctoral Science Foundation (2022M713621, 2022M713617)
  • Natural Science Foundation of Guangdong Province (202102020237, 2021A1515011156, 2021A1515010571, 2023A1515030052, 2021A1515012136, 81802897, 2023A1515010346)
  • National Key Research and Development Program of China (2022YFA1104903)
  • Basic and Applied Basic Research Foundation of Guangdong Province (2021A1515111058, 202102020237)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
Keywords
liver fibrosis, liver regeneration, mesenchymal stem cell-derived extracellular vesicles, miR-181a-5p, hepatocyte growth factor, hepatic stellate cells, competing endogenous RNA, microRNA sponging, AAV6, partial hepatectomy, nanotherapeutics
MeSH terms
Extracellular Vesicles, Animals, Disease Models, Animal, Humans, Liver Cirrhosis, Liver Regeneration, Male, Mice, Inbred C57BL, Up-Regulation, Hepatocyte Growth Factor, MicroRNAs, Mice, Hepatic Stellate Cells, Mesenchymal Stem Cells, RNA, Long Noncoding

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