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Cited by 109Circular RNAs in diseasesExtracellular vesicles in diseaseLiver physiology and pathology

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Mesenchymal stem cell-originated exosomal circDIDO1 suppresses hepatic stellate cell activation by miR-141-3p/PTEN/AKT pathway in human liver fibrosis

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Ma, Li, Wei, Junfeng, Zeng, Yanli, Liu, Junping, Xiao, Erhui, Kang, Yuehua, Kang, Yi·2022·Drug Delivery
DOI 10.1080/10717544.2022.2030428PMC PMC8812765
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Abstract

, thus enabling personalized treatment. This study aimed to investigate the role of exosome-based delivery of circDIDO1 in liver fibrosis. Levels of genes and proteins were examined by qRT-PCR and Western blot. Cell proliferation, apoptosis, and cell cycle were analyzed by using cell counting kit-8 (CCK-8) assay, EdU assay, and flow cytometry, respectively. The binding between circDIDO1 and miR-141-3p was confirmed by dual-luciferase reporter, RNA pull-down and RIP assays. Exosomes were isolated by ultracentrifugation, and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blot. CircDIDO1 overexpression or miR-141-3p inhibition suppressed the proliferation, reduced pro-fibrotic markers, and induced apoptosis as well as cell cycle arrest in hepatic stellate cells (HSCs) by blocking PTEN/AKT pathway. Mechanistically, circDIDO1 acted as an endogenous sponge for miR-141-3p, further rescue experiments showed that circDIDO1 suppressed HSC activation by targeting miR-141-3p. Extracellular circDIDO1 could be incorporated into exosomes isolated from mesenchymal stem cells (MSCs), and transmitted to HSCs to restrain HSC activation. Clinically, low levels of serum circDIDO1 in exosome were correlated with liver failure, and serum exosomal circDIDO1 had a well diagnostic value for liver fibrosis in liver failure patients. Transfer of circDIDO1 mediated by MSC-isolated exosomes suppressed HSC activation through the miR-141-3p/PTEN/AKT pathway, gaining a new insight into the prevention of liver fibrosis in liver failure patients.

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Crossref · 10.1080/10717544.2022.2030428

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Figures

Figure 1. Characterization of circDIDO1 in LX2 cells. (A) The Schematic illustration showing the production of circDIDO1 from its host gene, and the validation by Sanger sequencing. (B) The existence of circDIDO1 was validated using divergent and convergent primers from cDNA or genomic DNA (gDNA) of LX2 cells. (C) Random hexamer or Oligo(dT)18 primers were used in the reverse transcription experiments, and the products were examined in by qRT-PCR. (D) The relative RNA levels of circDIDO1 and GAPDH were determined by qRT-PCR after actinomycin D treatment at the indicated times. (E) The expression of circDIDO1 and GAPDH mRNA was examined in LX2 cells treated with or without RNase R. (F) Subcellular fractionation assay indicating the distribution of circDIDO1 in the cytoplasmic and nuclear fractions of LX2 cells. *p < .05, **p < .01, and ***p < .001.
Figure 1. Characterization of circDIDO1 in LX2 cells. (A) The Schematic illustration showing the production of circDIDO1 from its host gene, and the validation by Sanger sequencing. (B) The existence of circDIDO1 was validated using divergent and convergent primers from cDNA or genomic DNA (gDNA) of LX2 cells. (C) Random hexamer or Oligo(dT)18 primers were used in the reverse transcription experiments, and the products were examined in by qRT-PCR. (D) The relative RNA levels of circDIDO1 and GAPDH were determined by qRT-PCR after actinomycin D treatment at the indicated times. (E) The expression of circDIDO1 and GAPDH mRNA was examined in LX2 cells treated with or without RNase R. (F) Subcellular fractionation assay indicating the distribution of circDIDO1 in the cytoplasmic and nuclear fractions of LX2 cells. *p < .05, **p < .01, and ***p < .001.CC BY-NC · PubMed Central
Figure 2. CircDIDO1 suppresses the activation of HSCs by PTEN/AKT pathway. (A–G) LX2 cells were transfected with circDIDO1 or vector. (A) qRT-PCR analysis for the expression level of circDIDO1 in LX2 cells after transfection. (B, C) The proliferation ability analysis of LX2 cells using CCK-8 and EdU assays. (D, E) Flow cytometry for cell cycle and cell apoptosis in LX2 cells. (F, G) Western blot analysis for the protein levels of α-SMA, collagen I, PTEN, AKT, and p-AKT in LX2 cells. *p < .05, **p < .01, and ***p < .001.
Figure 2. CircDIDO1 suppresses the activation of HSCs by PTEN/AKT pathway. (A–G) LX2 cells were transfected with circDIDO1 or vector. (A) qRT-PCR analysis for the expression level of circDIDO1 in LX2 cells after transfection. (B, C) The proliferation ability analysis of LX2 cells using CCK-8 and EdU assays. (D, E) Flow cytometry for cell cycle and cell apoptosis in LX2 cells. (F, G) Western blot analysis for the protein levels of α-SMA, collagen I, PTEN, AKT, and p-AKT in LX2 cells. *p < .05, **p < .01, and ***p < .001.CC BY-NC · PubMed Central
Figure 3. MiR-141-3p is a target of circDIDO1. (A) qRT-PCR analysis for the expression of miR-141-3p, miR-128-3p, miR-188-5p, miR-942-5p, and miR-217 in LX2 cells transfected with circDIDO1 or vector. (B) RIP experiment was performed with Ago2 antibody and negative control IgG, followed by qRT-PCR to detect the expression of miR-141-3p and circDIDO1. (C) RNA pull-down assay was performed using the miR-141-3p-specific probe and control probe, and the enrichment of circDIDO1 was detected by qRT-PCR. (D) The starBase database showing the binding site of miR-141-3p on circDIDO1, and the mutated sequence of circDIDO1 in the binding site was established. (E) qRT-PCR analysis for miR-141-3p expression level in LX2 cells after miR-141-3p mimic or NC mimic transfection. (F) Dual-luciferase reporter assay for the luciferase activity of wild and mutated circDIDO1 reporter after miR-141-3p overexpression in LX2 cells. *p < .05, **p < .01, ***p < .001.
Figure 3. MiR-141-3p is a target of circDIDO1. (A) qRT-PCR analysis for the expression of miR-141-3p, miR-128-3p, miR-188-5p, miR-942-5p, and miR-217 in LX2 cells transfected with circDIDO1 or vector. (B) RIP experiment was performed with Ago2 antibody and negative control IgG, followed by qRT-PCR to detect the expression of miR-141-3p and circDIDO1. (C) RNA pull-down assay was performed using the miR-141-3p-specific probe and control probe, and the enrichment of circDIDO1 was detected by qRT-PCR. (D) The starBase database showing the binding site of miR-141-3p on circDIDO1, and the mutated sequence of circDIDO1 in the binding site was established. (E) qRT-PCR analysis for miR-141-3p expression level in LX2 cells after miR-141-3p mimic or NC mimic transfection. (F) Dual-luciferase reporter assay for the luciferase activity of wild and mutated circDIDO1 reporter after miR-141-3p overexpression in LX2 cells. *p < .05, **p < .01, ***p < .001.CC BY-NC · PubMed Central
Figure 4. MiR-141-3p knockdown suppresses the activation of HSCs by PTEN/AKT pathway. (A–G) LX2 cells were transfected with miR-141-3p inhibitor or NC inhibitor. (A) qRT-PCR analysis for the expression level of miR-141-3p in LX2 cells after transfection. (B, C) The proliferation ability analysis of LX2 cells using CCK-8 and EdU assays. (D, E) Flow cytometry for cell cycle and cell apoptosis in LX2 cells. (F, G) Western blot analysis for the protein levels of α-SMA, collagen I, PTEN, AKT, and p-AKT in LX2 cells. *p < .05, **p < .01, and ***p < .001.
Figure 4. MiR-141-3p knockdown suppresses the activation of HSCs by PTEN/AKT pathway. (A–G) LX2 cells were transfected with miR-141-3p inhibitor or NC inhibitor. (A) qRT-PCR analysis for the expression level of miR-141-3p in LX2 cells after transfection. (B, C) The proliferation ability analysis of LX2 cells using CCK-8 and EdU assays. (D, E) Flow cytometry for cell cycle and cell apoptosis in LX2 cells. (F, G) Western blot analysis for the protein levels of α-SMA, collagen I, PTEN, AKT, and p-AKT in LX2 cells. *p < .05, **p < .01, and ***p < .001.CC BY-NC · PubMed Central
Figure 5. CircDIDO1 suppresses the activation of HSCs by miR-141-3p/PTEN/AKT pathway. (A–F) LX2 cells were co-transfected with circDIDO1 and miR-141-3p mimic. (A, B) The proliferation ability analysis of LX2 cells using CCK-8 and EdU assays. (C, D) Flow cytometry for cell cycle and cell apoptosis in LX2 cells. (E, F) Western blot analysis for the protein levels of α-SMA, collagen I, PTEN, AKT, and p-AKT in LX2 cells. *p < .05, **p < .01, and ***p < .001.
Figure 5. CircDIDO1 suppresses the activation of HSCs by miR-141-3p/PTEN/AKT pathway. (A–F) LX2 cells were co-transfected with circDIDO1 and miR-141-3p mimic. (A, B) The proliferation ability analysis of LX2 cells using CCK-8 and EdU assays. (C, D) Flow cytometry for cell cycle and cell apoptosis in LX2 cells. (E, F) Western blot analysis for the protein levels of α-SMA, collagen I, PTEN, AKT, and p-AKT in LX2 cells. *p < .05, **p < .01, and ***p < .001.CC BY-NC · PubMed Central
Figure 6. Identification of exosomes isolated from circDIDO1 transfected MSCs. (A) qRT-PCR analysis of circDIDO1 expression level in MSCs transfected circDIDO1 or vector. (B) TEM images of exosomes isolated from circDIDO1 or vector transfected-MSCs. Scale bar, 200 nm. (C) Exosomes isolated from circDIDO1 or vector transfected-MSCs underwent an NTA to determine exosomal size distribution. (D) Western blot analysis for exosomes markers CD63, TSG101, and CD81. (E) qRT-PCR analysis of circDIDO1 expression level in exosomes isolated from circDIDO1 or vector transfected-MSCs. **p < .01, ***p < .001.
Figure 6. Identification of exosomes isolated from circDIDO1 transfected MSCs. (A) qRT-PCR analysis of circDIDO1 expression level in MSCs transfected circDIDO1 or vector. (B) TEM images of exosomes isolated from circDIDO1 or vector transfected-MSCs. Scale bar, 200 nm. (C) Exosomes isolated from circDIDO1 or vector transfected-MSCs underwent an NTA to determine exosomal size distribution. (D) Western blot analysis for exosomes markers CD63, TSG101, and CD81. (E) qRT-PCR analysis of circDIDO1 expression level in exosomes isolated from circDIDO1 or vector transfected-MSCs. **p < .01, ***p < .001.CC BY-NC · PubMed Central
Figure 7. MSC-exosome-mediated transfer of circDIDO1 suppresses the activation of HSCs by PTEN/AKT pathway. (A–H) LX2 cells were co-cultured with PBS, MSC-circDIDO1 Exo or MSC-vector Exo. (A) qRT-PCR analysis of circDIDO1 and miR-141-3p expression level in LX2 cells after incubation. (C, D) The proliferation ability analysis of LX2 cells using CCK-8 and EdU assays. (E, F) Flow cytometry for cell cycle and cell apoptosis in LX2 cells. (G, H) Western blot analysis for the protein levels of α-SMA, collagen I, PTEN, AKT, and p-AKT in LX2 cells. *p < .05, **p < .01, and ***p < .001.
Figure 7. MSC-exosome-mediated transfer of circDIDO1 suppresses the activation of HSCs by PTEN/AKT pathway. (A–H) LX2 cells were co-cultured with PBS, MSC-circDIDO1 Exo or MSC-vector Exo. (A) qRT-PCR analysis of circDIDO1 and miR-141-3p expression level in LX2 cells after incubation. (C, D) The proliferation ability analysis of LX2 cells using CCK-8 and EdU assays. (E, F) Flow cytometry for cell cycle and cell apoptosis in LX2 cells. (G, H) Western blot analysis for the protein levels of α-SMA, collagen I, PTEN, AKT, and p-AKT in LX2 cells. *p < .05, **p < .01, and ***p < .001.CC BY-NC · PubMed Central
Figure 8. Serum exosomal circDIDO1 level is down-regulated in liver failure patients. (A) TEM images of exosomes isolated from the serum of healthy individuals and liver failure patients. Scale bar, 200 nm. (B) Western blot analysis for exosomes markers CD63, TSG101, and CD81. (C) Exosomes isolated from the serum of healthy individuals and liver failure patients underwent an NTA to determine exosomal size distribution. (D) qRT-PCR analysis of exosomal circDIDO1 expression level in the serum of healthy individuals and liver failure patients. (E) ROC curve analysis of the diagnostic value of exosomal circDIDO1 in liver failure patients. (F–H) Exosomal circDIDO1 expression level was not significantly affected by long exposure (F), PH values (G), or repeat freezing and thawing (H). ***p < .001.
Figure 8. Serum exosomal circDIDO1 level is down-regulated in liver failure patients. (A) TEM images of exosomes isolated from the serum of healthy individuals and liver failure patients. Scale bar, 200 nm. (B) Western blot analysis for exosomes markers CD63, TSG101, and CD81. (C) Exosomes isolated from the serum of healthy individuals and liver failure patients underwent an NTA to determine exosomal size distribution. (D) qRT-PCR analysis of exosomal circDIDO1 expression level in the serum of healthy individuals and liver failure patients. (E) ROC curve analysis of the diagnostic value of exosomal circDIDO1 in liver failure patients. (F–H) Exosomal circDIDO1 expression level was not significantly affected by long exposure (F), PH values (G), or repeat freezing and thawing (H). ***p < .001.CC BY-NC · PubMed Central

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Metadata

Authors
Ma, L., Wei, J., Zeng, Y., Liu, J., Xiao, E., Kang, Y., & Kang, Y.
Affiliations
Zhengzhou University (CN)
Corresponding author
Kang, Yi
ORCID
Venue
Drug Delivery
Publisher
Informa UK Limited
Published
2022-01-31
Volume
29
Issue
1
Pages
440-453
ISSN
  • 1071-7544 (ISSN-L)
  • 1521-0464
DOI
10.1080/10717544.2022.2030428
PMC
PMC8812765
OpenAlex
W4210563368
Cited by
109
References
39
Citations by year
2026: 14 · 2025: 22 · 2024: 26 · 2023: 30 · 2022: 17
FWCI
7.76
Type
journal article
Language
en
Open access
Licence
Locations
  • Drug Delivery (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Circular RNAs in diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
Keywords
liver fibrosis, hepatic stellate cell activation, mesenchymal stem cell-derived exosomes, miR-141-3p, PTEN/AKT pathway, microRNA sponge, exosome therapy, liver failure, serum biomarkers, cell cycle arrest
MeSH terms
RNA, Circular, Cell Line, DNA-Binding Proteins, Humans, Liver Cirrhosis, Signal Transduction, Apoptosis, MicroRNAs, Cell Proliferation, Proto-Oncogene Proteins c-akt, PTEN Phosphohydrolase, Hepatic Stellate Cells, Exosomes, Mesenchymal Stem Cells

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