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. 2023 Mar 31;11(6):259.
doi: 10.21037/atm-23-835.

Promotion of liver fibrosis by Y-box binding protein 1 via the attenuation of transforming growth factor-beta 3 transcription

Qiong Liao #  1 , Yuwei Dong #  2 , Binghang Li  2 , Jie Qin  3 , Yangwei Cao  3 , Wenjuan Tu  3 , Lungen Lu  4
Affiliations

Promotion of liver fibrosis by Y-box binding protein 1 via the attenuation of transforming growth factor-beta 3 transcription

Qiong Liao et al. Ann Transl Med. .

Abstract

Background: Spurred by the seriousness of liver fibrosis, we evaluated the correlation between Y-box binding protein 1 (YB-1) and transforming growth factor-beta 3 (TGF-β3) expression levels in the signaling pathways of the disease.

Methods: Based on a mouse model of carbon tetrachloride-induced liver fibrosis, YB-1 overexpression lentivirus was used to explore the effect of YB-1 on liver fibrosis in vivo. In addition, a hepatic stellate cell (HSC) activation model in the HSC line LX-2 was developed using TGF-β1. Western blot assays were used to investigate the effects of YB-1 overexpression and knockdown on liver fibrosis. Finally, chromatin immunoprecipitation and luciferase reporter assays were used to elucidate the relationship between YB-1 and its downstream signaling pathways.

Results: YB-1 was overexpressed in fibrotic liver tissue, which enhanced both fibrosis and the relative protein expressions of the TGF-β pathway. Moreover, YB-1 overexpression promoted HSC activation in response to TGF-β1 stimulation, but its knockdown inhibited liver fibrosis in vitro. Both in vitro and in vivo experiments indicated the expression of TGF-β3 in the YB-1 overexpression group to be suppressed, and liver fibrosis was more obvious in the YB-1-overexpression group than in the YB-1-inhibition group. YB-1 attenuated TGF-β3 transcription by binding to its promoter, which is involved in the effect of YB-1 on liver fibrosis.

Conclusions: YB-1 overexpression in HSCs promoted liver fibrosis by attenuating TGF-β3 transcription.

Keywords: Hepatic stellate cell (HSC); Y-box binding protein 1 (YB-1); liver fibrosis; transforming growth factor-beta 3 (TGF-β3).

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-23-835/coif). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
YB-1 was upregulated but TGF-β3 was downregulated in liver fibrosis. (A) Masson staining and Sirius red staining of liver sections from the control and CCl4 groups. (B) Quantitative statistics of Masson and Sirius red staining. ***, P<0.001 control vs. CCl4. (C) Western blots of YB-1 and TGF-β3 in the control and CCl4 groups. (D) Quantitative statistics of Western blotting. **, P<0.01 Ctrl vs. CCl4. Ctrl, control group.
Figure 2
Figure 2
YB-1 promoted liver fibrosis in an in vivo mouse model of CCl4-induced liver fibrosis. (A) Masson and Sirius red staining of liver sections from the control, CCl4*, and CCl4 + YB-1-OE groups. (B) Immunofluorescence of α-SMA in the control, CCl4* and CCl4 + YB-1-OE groups. (C) Quantitative statistics of immunofluorescence. ***, P<0.01 control vs. CCl4; ##, P<0.01 CCl4* vs. CCl4 + YB-1 OE. (D) Western blots of YB-1, collagen I, TGF-β3, TGFBR, p-SMAD2/SAMD2, and p-SMAD3/SAMD3 in the control, CCl4*, and CCl4 + YB-1-OE groups. (E) Quantitative statistics of Western blotting. *, P<0.05 control vs. CCl4; **, P<0.01 Ctrl vs. CCl4*; #, P<0.05 CCl4 vs. CCl4 + YB-1-OE; ##, P<0.01 CCl4* vs. CCl4 + YB-1 OE; ###, P<0.001 CCl4* vs. CCl4 + YB-1 OE. Ctrl, control group; OE, overexpression.
Figure 3
Figure 3
YB-1 promoted liver fibrosis in a model of TGF-β1–induced HSC. (A) Western blot of α-SMA, collagen I, collagen III, YKL-40, and Timp1 in YB-1 KD or YB-1 OE HSCs following TGF-β1 stimulation. (B) Quantitative statistics of Western blotting. ***, P<0.001 NC* vs. NC; #, P<0.05 TGF-β1 + YB-1 KD/OE vs. NC*; ##, P<0.01 TGF-β1 + YB-1 KD/OE vs. NC*; ###, P<0.001 TGF-β1 + YB-1 KD/OE vs. NC*. (C) Immunofluorescence images of α-SMA in YB-1 KD or YB-1 OE HSCs following TGF-β1 stimulation (blue: nucleus; green: α-SMA). (D) Quantitative statistics of immunohistochemical assays. **, P<0.01 NC* vs. NC; ##, P<0.01 TGF-β1 + YB-1 KD/OE vs. NC*. NC, normal control; NC*, NC + TGF-β1. HSC, hepatic stellate cell; KD, knockdown; OE, overexpression.
Figure 4
Figure 4
YB-1 promoted liver fibrosis in the LX-2 cell line by attenuating TGF-β3 transcription. (A) Enrichment of each region of the TGF-β3 promoter in the YB-1 DNA fragments from chromatin immunoprecipitation. ***, P<0.001 YB-1 OE vs. NC. (B) Luciferase reporter assay showing the interactions between YB-1 and the TGF-β3 promoter region. ***, P<0.001 YB-1 KD vs. NC. (C) ELISA of TGF-β3 from YB-1 OE or YB-1 KD hepatic stellate cells following TGF-β1 stimulation. ***, P<0.001 NC + TGF-β1 vs. NC, ##, P<0.01 TGF-β1 + YB-1 KD/OE vs. NC + TGF-β1. KD, knockdown; OE, overexpression; NC, normal control; NC*, NC + TGF-β1; ELISA, enzyme-linked immunosorbent assay.
Figure 5
Figure 5
HSCs activated by YB-1 further activated surrounding cells through TGF-β3. (A) Western blots of α-SMA, collagen I, collagen III, YKL-40, and Timp1 expression levels in HSCs treated with the culture supernatant of YB-1 OE or YB-1 KD HSCs following TGF-β1 stimulation. (B) Quantitative statistics of Western blotting. *, P<0.05 NC + TGF-β1 vs. NC; **, P<0.01 NC + TGF-β1 vs. NC; ***, P<0.001 NC + TGF-β1 vs. NC; #, P<0.05 TGF-β1 + YB-1 KD/OE vs. NC + TGF-β1; ##, P<0.01 TGF-β1 + YB-1 KD/OE vs. NC + TGF-β1; ###, P<0.001 TGF-β1 + YB-1 KD/OE vs. NC + TGF-β1. (C) Immunofluorescence of α-SMA in HSCs exposed to the culture supernatant of YB-1 OE or YB-1 KD HSCs following TGF-β1 stimulation (blue: nucleus; green: α-SMA). (D) Quantitative statistics of immunohistochemical assays. ***, P<0.001 NC + TGF-β1 vs. NC; ###, P<0.001 TGF-β1 + YB-1 KD/OE vs. NC + TGF-β1. HSC, hepatic stellate cell; KD, knockdown; OE, overexpressing; NC, normal control; NC*, NC + TGF-β1.
Figure 6
Figure 6
TGF-β signaling in liver fibrosis. (A) Western blots of TGFBR, p-SMAD2/SAMD2, and p-SMAD3/SAMD3 in the control and CCl4 groups. (B) Quantitative statistics of Western blotting. *, P<0.05 vs. Ctrl; **, P<0.01 vs. Ctrl; ***, P<0.001 Ctrl vs. CCl4. KD, knockdown; Ctrl, control group.
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