Limits...
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

Silencing of SRF attenuates the effect of TGF-β in LX2 and NLFs(A) Transfection of miR-122 repressed the expression of SRF. (B) Inhibition of miR-122 elevated the level of SRF. (C and D) Knockdown of SRF attenuated the TGF-β-induced increase of α-SMA and COL1A1 mRNA levels. (E) Inhibition of SRF impaired the TGF-β-induced expression of α-SMA protein. LX2 and NLFs transfected with the indicated duplex were stimulated with 2 ng/ml TGF-β (+) or remained untreated (−, control) for 48 hours before immunoblotting (A, B and E) or qPCR analysis (C and D). For (A-E), GAPDH was used as an internal control. For immunoblotting, the intensity of each band was densitometrically quantified. The levels of target genes in each sample were normalized by that of GAPDH. * P < .05; ** P < .01; *** P < .001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494934&req=5

Figure 4: Silencing of SRF attenuates the effect of TGF-β in LX2 and NLFs(A) Transfection of miR-122 repressed the expression of SRF. (B) Inhibition of miR-122 elevated the level of SRF. (C and D) Knockdown of SRF attenuated the TGF-β-induced increase of α-SMA and COL1A1 mRNA levels. (E) Inhibition of SRF impaired the TGF-β-induced expression of α-SMA protein. LX2 and NLFs transfected with the indicated duplex were stimulated with 2 ng/ml TGF-β (+) or remained untreated (−, control) for 48 hours before immunoblotting (A, B and E) or qPCR analysis (C and D). For (A-E), GAPDH was used as an internal control. For immunoblotting, the intensity of each band was densitometrically quantified. The levels of target genes in each sample were normalized by that of GAPDH. * P < .05; ** P < .01; *** P < .001.

Mentions: We next explored the mechanisms responsible for miR-122-induced downregulation of α-SMA and COL1A1 expression. Serum response factor (SRF) is an identified target for miR-122 in HCC cells [6], and SRF interacts with myocardin-related transcription factor to drive transcription of α-SMA and COL1A1 [2, 13, 14]. As expected, we found that introduction of miR-122 repressed SRF expression (Figure 4A), whereas inhibition of endogenous miR-122 elevated SRF level (Figure 4B) in both LX2 and NLFs. Furthermore, similar to miR-122 overexpression, knockdown of SRF (Supplementary Figure 5) significantly attenuated TGF-β-induced elevation of α-SMA and COL1A1 levels (Figure 4C-E).


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)

Silencing of SRF attenuates the effect of TGF-β in LX2 and NLFs(A) Transfection of miR-122 repressed the expression of SRF. (B) Inhibition of miR-122 elevated the level of SRF. (C and D) Knockdown of SRF attenuated the TGF-β-induced increase of α-SMA and COL1A1 mRNA levels. (E) Inhibition of SRF impaired the TGF-β-induced expression of α-SMA protein. LX2 and NLFs transfected with the indicated duplex were stimulated with 2 ng/ml TGF-β (+) or remained untreated (−, control) for 48 hours before immunoblotting (A, B and E) or qPCR analysis (C and D). For (A-E), GAPDH was used as an internal control. For immunoblotting, the intensity of each band was densitometrically quantified. The levels of target genes in each sample were normalized by that of GAPDH. * P < .05; ** P < .01; *** P < .001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494934&req=5

Figure 4: Silencing of SRF attenuates the effect of TGF-β in LX2 and NLFs(A) Transfection of miR-122 repressed the expression of SRF. (B) Inhibition of miR-122 elevated the level of SRF. (C and D) Knockdown of SRF attenuated the TGF-β-induced increase of α-SMA and COL1A1 mRNA levels. (E) Inhibition of SRF impaired the TGF-β-induced expression of α-SMA protein. LX2 and NLFs transfected with the indicated duplex were stimulated with 2 ng/ml TGF-β (+) or remained untreated (−, control) for 48 hours before immunoblotting (A, B and E) or qPCR analysis (C and D). For (A-E), GAPDH was used as an internal control. For immunoblotting, the intensity of each band was densitometrically quantified. The levels of target genes in each sample were normalized by that of GAPDH. * P < .05; ** P < .01; *** P < .001.
Mentions: We next explored the mechanisms responsible for miR-122-induced downregulation of α-SMA and COL1A1 expression. Serum response factor (SRF) is an identified target for miR-122 in HCC cells [6], and SRF interacts with myocardin-related transcription factor to drive transcription of α-SMA and COL1A1 [2, 13, 14]. As expected, we found that introduction of miR-122 repressed SRF expression (Figure 4A), whereas inhibition of endogenous miR-122 elevated SRF level (Figure 4B) in both LX2 and NLFs. Furthermore, similar to miR-122 overexpression, knockdown of SRF (Supplementary Figure 5) significantly attenuated TGF-β-induced elevation of α-SMA and COL1A1 levels (Figure 4C-E).

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