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Identification of a novel TGF-β-miR-122-fibronectin 1/serum response factor signaling cascade and its implication in hepatic fibrogenesis.

Zeng C, Wang YL, Xie C, Sang Y, Li TJ, Zhang M, Wang R, Zhang Q, Zheng L, Zhuang SM - Oncotarget (2015)

Bottom Line: Notably, exposure to TGF-β led to significant downregulation of miR-122.Subsequent mechanism investigations revealed that miR-122 directly inhibited FN1 expression by binding to its 3'-untranslated region and indirectly reduced the transcription of α-SMA and COL1A1 by inhibiting the expression of serum response factor (SRF), a key transcription factor that mediated the activation of fibrogenic cells.Further in vivo studies disclosed that intravenous injection of miR-122-expressing lentivirus successfully increased miR-122 level and reduced the amount of collagen fibrils, FN1 and SRF in the livers of CCl4-treated mice.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Gene Engineering of The Ministry of Education, State Key Laboratory of Biocontrol, Collaborative Innovation Center for Cancer Medicine, School of Life Sciences, Sun Yat-sen University, Guangzhou, P.R. China.

ABSTRACT
Transforming growth factor-β (TGF-β) is a potent cytokine that promotes the development of fibrogenic cells, stimulates the expression of fibrosis-related genes, and consequently results in hepatic fibrogenesis. The involvement of miRNAs in this process remains largely unknown. We showed that miR-122 was substantially expressed in hepatic stellate cells (HSCs) and fibroblasts, the major sources of fibrogenic cells in liver tissues. Notably, exposure to TGF-β led to significant downregulation of miR-122. Furthermore, reintroduction of miR-122 suppressed TGF-β-induced expression of fibrosis-related genes, including alpha smooth muscle actin (α-SMA), fibronectin 1 (FN1) and α1 type I collagen (COL1A1), in HSCs and fibroblasts. Subsequent mechanism investigations revealed that miR-122 directly inhibited FN1 expression by binding to its 3'-untranslated region and indirectly reduced the transcription of α-SMA and COL1A1 by inhibiting the expression of serum response factor (SRF), a key transcription factor that mediated the activation of fibrogenic cells. Further in vivo studies disclosed that intravenous injection of miR-122-expressing lentivirus successfully increased miR-122 level and reduced the amount of collagen fibrils, FN1 and SRF in the livers of CCl4-treated mice. These findings disclose a novel TGF-β-miR-122-FN1/SRF signaling cascade and its implication in hepatic fibrogenesis, and suggest miR-122 as a promising molecular target for anti-fibrosis therapy.

No MeSH data available.


Related in: MedlinePlus

miR-122 inhibits the TGF-β-induced expression of fibrosis-related genes(A-D) Introduction of miR-122 repressed the TGF-β-stimulated expression of α-SMA, COL1A1 and FN1. LX2 cells (A, C) and NLFs (B, D) were transfected with negative control (NC) or miR-122 duplex for 24 hours, and then stimulated with 2 ng/ml TGF-β (+) or remained untreated (control, -) for 48 hours before qPCR analysis (A, B) or immunoblotting (C, D). For qPCR analysis, the levels of target genes were normalized to the expression of GAPDH. For immunoblotting, the intensity of each band was densitometrically quantified. The levels of target genes in each sample were normalized by that of GAPDH (internal control). (E) Ectopic expression of miR-122 inhibited the TGF-β-promoted contraction of collagen matrix containing LX2 cells. LX2 cells transfected with the indicated duplex were embedded in collagen matrix for 1 hour, and then incubated with 2 ng/ml TGF-β or remained untreated (control) for 24 hours before collagen lattice release. The contraction index denoted the change of collagen gel size at 24 hours after lattice release. * P < .05; ** P < .01; *** P < .001.
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Figure 2: miR-122 inhibits the TGF-β-induced expression of fibrosis-related genes(A-D) Introduction of miR-122 repressed the TGF-β-stimulated expression of α-SMA, COL1A1 and FN1. LX2 cells (A, C) and NLFs (B, D) were transfected with negative control (NC) or miR-122 duplex for 24 hours, and then stimulated with 2 ng/ml TGF-β (+) or remained untreated (control, -) for 48 hours before qPCR analysis (A, B) or immunoblotting (C, D). For qPCR analysis, the levels of target genes were normalized to the expression of GAPDH. For immunoblotting, the intensity of each band was densitometrically quantified. The levels of target genes in each sample were normalized by that of GAPDH (internal control). (E) Ectopic expression of miR-122 inhibited the TGF-β-promoted contraction of collagen matrix containing LX2 cells. LX2 cells transfected with the indicated duplex were embedded in collagen matrix for 1 hour, and then incubated with 2 ng/ml TGF-β or remained untreated (control) for 24 hours before collagen lattice release. The contraction index denoted the change of collagen gel size at 24 hours after lattice release. * P < .05; ** P < .01; *** P < .001.

Mentions: Further investigations were conducted using LX2 and NLFs. Consistent with primary HSCs, TGF-β stimulation induced downregulation of miR-122 in LX2 cells (Figure 1D). As expected, TGF-β treatment resulted in increased mRNA levels of fibrosis-related genes, like α-SMA, COL1A1 and FN1, in LX2 and NLFs (Figure 2A and 2B; Supplementary Figure 2A and 2B). Interestingly, introduction of miR-122 attenuated TGF-β-induced elevation in α-SMA and COL1A1 mRNA levels (Figure 2A and 2B), but did not affect TGF-β-promoted increase of FN1 mRNA (Supplementary Figure 2A and 2B). However, ectopic expression of miR-122 abrogated TGF-β-induced upregulation of FN1 protein level in LX2 and NLFs (Figure 2C and 2D). These findings were also reproducible in SFs (Supplementary Figure 3A-D).


Identification of a novel TGF-β-miR-122-fibronectin 1/serum response factor signaling cascade and its implication in hepatic fibrogenesis.

Zeng C, Wang YL, Xie C, Sang Y, Li TJ, Zhang M, Wang R, Zhang Q, Zheng L, Zhuang SM - Oncotarget (2015)

miR-122 inhibits the TGF-β-induced expression of fibrosis-related genes(A-D) Introduction of miR-122 repressed the TGF-β-stimulated expression of α-SMA, COL1A1 and FN1. LX2 cells (A, C) and NLFs (B, D) were transfected with negative control (NC) or miR-122 duplex for 24 hours, and then stimulated with 2 ng/ml TGF-β (+) or remained untreated (control, -) for 48 hours before qPCR analysis (A, B) or immunoblotting (C, D). For qPCR analysis, the levels of target genes were normalized to the expression of GAPDH. For immunoblotting, the intensity of each band was densitometrically quantified. The levels of target genes in each sample were normalized by that of GAPDH (internal control). (E) Ectopic expression of miR-122 inhibited the TGF-β-promoted contraction of collagen matrix containing LX2 cells. LX2 cells transfected with the indicated duplex were embedded in collagen matrix for 1 hour, and then incubated with 2 ng/ml TGF-β or remained untreated (control) for 24 hours before collagen lattice release. The contraction index denoted the change of collagen gel size at 24 hours after lattice release. * P < .05; ** P < .01; *** P < .001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: miR-122 inhibits the TGF-β-induced expression of fibrosis-related genes(A-D) Introduction of miR-122 repressed the TGF-β-stimulated expression of α-SMA, COL1A1 and FN1. LX2 cells (A, C) and NLFs (B, D) were transfected with negative control (NC) or miR-122 duplex for 24 hours, and then stimulated with 2 ng/ml TGF-β (+) or remained untreated (control, -) for 48 hours before qPCR analysis (A, B) or immunoblotting (C, D). For qPCR analysis, the levels of target genes were normalized to the expression of GAPDH. For immunoblotting, the intensity of each band was densitometrically quantified. The levels of target genes in each sample were normalized by that of GAPDH (internal control). (E) Ectopic expression of miR-122 inhibited the TGF-β-promoted contraction of collagen matrix containing LX2 cells. LX2 cells transfected with the indicated duplex were embedded in collagen matrix for 1 hour, and then incubated with 2 ng/ml TGF-β or remained untreated (control) for 24 hours before collagen lattice release. The contraction index denoted the change of collagen gel size at 24 hours after lattice release. * P < .05; ** P < .01; *** P < .001.
Mentions: Further investigations were conducted using LX2 and NLFs. Consistent with primary HSCs, TGF-β stimulation induced downregulation of miR-122 in LX2 cells (Figure 1D). As expected, TGF-β treatment resulted in increased mRNA levels of fibrosis-related genes, like α-SMA, COL1A1 and FN1, in LX2 and NLFs (Figure 2A and 2B; Supplementary Figure 2A and 2B). Interestingly, introduction of miR-122 attenuated TGF-β-induced elevation in α-SMA and COL1A1 mRNA levels (Figure 2A and 2B), but did not affect TGF-β-promoted increase of FN1 mRNA (Supplementary Figure 2A and 2B). However, ectopic expression of miR-122 abrogated TGF-β-induced upregulation of FN1 protein level in LX2 and NLFs (Figure 2C and 2D). These findings were also reproducible in SFs (Supplementary Figure 3A-D).

Bottom Line: Notably, exposure to TGF-β led to significant downregulation of miR-122.Subsequent mechanism investigations revealed that miR-122 directly inhibited FN1 expression by binding to its 3'-untranslated region and indirectly reduced the transcription of α-SMA and COL1A1 by inhibiting the expression of serum response factor (SRF), a key transcription factor that mediated the activation of fibrogenic cells.Further in vivo studies disclosed that intravenous injection of miR-122-expressing lentivirus successfully increased miR-122 level and reduced the amount of collagen fibrils, FN1 and SRF in the livers of CCl4-treated mice.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Gene Engineering of The Ministry of Education, State Key Laboratory of Biocontrol, Collaborative Innovation Center for Cancer Medicine, School of Life Sciences, Sun Yat-sen University, Guangzhou, P.R. China.

ABSTRACT
Transforming growth factor-β (TGF-β) is a potent cytokine that promotes the development of fibrogenic cells, stimulates the expression of fibrosis-related genes, and consequently results in hepatic fibrogenesis. The involvement of miRNAs in this process remains largely unknown. We showed that miR-122 was substantially expressed in hepatic stellate cells (HSCs) and fibroblasts, the major sources of fibrogenic cells in liver tissues. Notably, exposure to TGF-β led to significant downregulation of miR-122. Furthermore, reintroduction of miR-122 suppressed TGF-β-induced expression of fibrosis-related genes, including alpha smooth muscle actin (α-SMA), fibronectin 1 (FN1) and α1 type I collagen (COL1A1), in HSCs and fibroblasts. Subsequent mechanism investigations revealed that miR-122 directly inhibited FN1 expression by binding to its 3'-untranslated region and indirectly reduced the transcription of α-SMA and COL1A1 by inhibiting the expression of serum response factor (SRF), a key transcription factor that mediated the activation of fibrogenic cells. Further in vivo studies disclosed that intravenous injection of miR-122-expressing lentivirus successfully increased miR-122 level and reduced the amount of collagen fibrils, FN1 and SRF in the livers of CCl4-treated mice. These findings disclose a novel TGF-β-miR-122-FN1/SRF signaling cascade and its implication in hepatic fibrogenesis, and suggest miR-122 as a promising molecular target for anti-fibrosis therapy.

No MeSH data available.


Related in: MedlinePlus