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. 2019 Jun 5;14(6):e0214534.
doi: 10.1371/journal.pone.0214534. eCollection 2019.

TGF-β induces liver fibrosis via miRNA-181a-mediated down regulation of augmenter of liver regeneration in hepatic stellate cells

Parul Gupta  1 , Teja Naveen Sata  1 , Ajay K Yadav  1 , Amit Mishra  1 , Nisha Vats  2 , Md Musa Hossain  1 , M G Sanal  2 , Senthil Kumar Venugopal  1
Affiliations

TGF-β induces liver fibrosis via miRNA-181a-mediated down regulation of augmenter of liver regeneration in hepatic stellate cells

Parul Gupta et al. PLoS One. .

Abstract

Objective: To study the role of miRNA-181a and augmenter of liver regeneration in TGF-β-induced fibrosis in hepatic stellate cells.

Methods: LX2 cells were treated with 20 ng/ml TGF-β for 24 h. miRNA-181a, ALR plasmid and empty vectors were transfected using siPORT NeoFx reagent. Cells were harvested after 48 h or 72 h of transfection for protein or RNA analysis. Western blotting was performed for ALR, TGF-β receptor II (TGFβ-RII), collagen 1A1 (COLL1A1), alpha-smooth muscle cell actin (α-SMA), rac1, E-cadherin and β-actin. Quantitative RT-PCR was performed for ALR, GAPDH, miRNA-181a or 5S rRNA.

Results: TGF-β induced the expression of miRNA-181a, which in turn down-regulated ALR thereby induced the fibrosis markers, such as COLL1A1, α-SMA and rac1 in hepatic stellate cells. Over-expression of miRNA-181a down-regulated expression of ALR and up-regulated expression of fibrosis markers. On the other hand, ALR over-expression resulted in a decrease in miRNA-181a expression and fibrosis markers. Over-expression of ALR also inhibited the expression of TGFβ-RII and increased expression E-cadherin.

Conclusion: TGF-β induced miRNA-181a, which in turn induced fibrosis, at least in part, by inhibiting ALR. ALR inhibited TGF-β action by decreasing the expression of TGFβ-RII, thereby inhibiting miRNA-181a expression and fibrosis markers. ALR could serve as a potential molecule to inhibit liver fibrosis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Expression of miRNA-181a in liver tissues.
The liver tissues were obtained from the liver transplant participants. Total RNA was purified from the liver tissues of the donors (healthy liver samples) and recipients (cirrhotic patients). cDNA was synthesized and real-time RTPCR was performed to determine the expression of miRNA-181a (n = 5; *p<0.05, compared to healthy controls).
Fig 2
Fig 2. Effect of TGF-β on miRNA-181a, ALR and fibrogenic markers.
LX2 cells were incubated with TGF-β for 24 hours under serum-free conditions. A. Total RNA was purified and real-time RTPCR was performed for miRNA-181a expression (n = 3; *p<0.05, compared to control). Lane 1, control cells; Lane 2, TGF-β treated cells. B. The total cellular protein was isolated and Western blots were run for ALR protein. β-actin was used as an internal control. C. Western blots were performed for α-SMA, rac1, COLL1A1 and β-actin. A representative gel picture is shown (n = 3). Lane 1, control cells; and Lane 2, TGF-β treated cells. D. The Western blots from 3 experiments were quantitated using Image J software and presented as relative expression (*p<0.05, compared to control).
Fig 3
Fig 3. Role of miRNA-181a on the expression of ALR and fibrogenic markers.
A. LX2 cells were transfected with miRNA-181a mimics as described in materials and methods. The expression of ALR, α-SMA, rac1, COLL1A1 and β-actin were determined using Western blots. A representative picture from 3 experiments is shown. B. The bands from the Western blot experiments were quantitated using Image J software and the data are presented as relative expression (p<0.05, compared to control).
Fig 4
Fig 4. Effect of overexpression of ALR on miRNA-181a expression.
ALR was overexpressed in LX2 cells. As a transfection control empty vector was used. C, Control; EV, Empty Vector tranfected cells; and ALR, ALR expressing cells A. ALR protein expression was determined in the transfected cells by Western blots (n = 3). B. ALR mRNA expression was determined (n = 3; *p<0.05, compared to control). C. miRNA-181a expression was determined in ALR expressing cells (n = 3; *p<0.05, compared to control).
Fig 5
Fig 5. Effect of ALR on fibrogenic markers, TGF-β RII and E-Cadherin.
The total cellular protein was isolated from ALR overexpressing cells and Western blots were performed. C, Control; EV, Empty Vector tranfected cells; and ALR, ALR expressing cells. A. The representative picture shows the Western blot results of α-SMA, rac1, COLL1A1 and β-actin (n = 3). B. The Western blots from 3 experiments of the firbogenic markers were quantitated using Image J software and presented as relative expression (*p<0.05, compared to control). C. Western blots were performed to determined the expression of TGF-β RII and E-cadherin. D. The band intensities of TGF-β RII and E-cadherin was quantitated by Image J software and presented as relative expression (n = 3; *p<0.05, compared to control).
Fig 6
Fig 6. Proposed mechanism of TGF-β-induced miRNA-181a and fibrosis.
TGF-β induces fibrosis by upregulating miRNA-181a expression, which in turn inhibits ALR expression and fibrosis. Overexpression of ALR inhibited TGF-β-induced firbrogenic changes by inhibiting TGF-βRII expression.
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This work was supported by the Department of Science and Technology, Govt. of India, (SKV); University Grants Commission, Govt. of India (PG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.