Yonsei Med J.  2019 Jun;60(6):561-569. 10.3349/ymj.2019.60.6.561.

miR-140-3p Knockdown Suppresses Cell Proliferation and Fibrogenesis in Hepatic Stellate Cells via PTEN-Mediated AKT/mTOR Signaling

• Wu SM
• Li TH
• Yun H
• Ai HW
• Zhang KH

• KMID: 2446962
• DOI: http://doi.org/10.3349/ymj.2019.60.6.561

Abstract

PURPOSE
Liver fibrosis is a major cause of morbidity and mortality and the outcome of various chronic liver diseases. Activation of hepatic stellate cells (HSCs) is the key event in liver fibrosis. Studies have confirmed that miR-140-3p plays a potential regulatory effect on HSC activation. However, whether miR-140-3p mediates the liver fibrosis remains unknown.
MATERIALS AND METHODS
Expression of miR-140-3p was detected by real-time quantitative PCR (qPCR). Cell proliferation was measured by MTT, while cell apoptosis rate was determined via flow cytometry. Western blot assay was used to detect the expression of cleaved PARP. The fibrogenic effect was evaluated by expression of α-smooth muscle actin and desmin. Functional experiments were performed in transforming growth factor β1 (TGF-β1)-induced HSC-T6 cells with transfection of anti-miR-140-3p and/or siPTEN. Target binding between miR-140-3p and PTEN was predicted by the TargetScan database and identified using luciferase reporter assay and RNA immunoprecipitation.
RESULTS
TGF-β1 induced the activation of HSC-T6 cells, and miR-140-3p expression varied according to HSC-T6 cell activation status. Knockdown of miR-140-3p reduced cell proliferation and the expressions of α-SMA and desmin, as well as increased apoptosis, in TGF-β1-induced HSC-T6 cells, which could be blocked by PTEN silencing. Additionally, inactivation of the AKT/mTOR signaling pathway stimulated by miR-140-3p knockdown was abolished when silencing PTEN expression. PTEN was negatively regulated by miR-140-3p via direct binding in HSC-T6 cells.
CONCLUSION
miR-140-3p is an important mediator in HSC-T6 cell activation, and miR-140-3p knockdown suppresses cell proliferation and fibrogenesis in TGF-β1-induced HSC-T6 cells, indicating that miR-140-3p may be a potential novel molecular target for liver fibrosis.

Keyword

miR-140-3p; PTEN; liver fibrosis; TGF-β1; hepatic stellate cells (HSCs)

MeSH Terms

Actins
Apoptosis
Blotting, Western
Cell Proliferation*
Desmin
Flow Cytometry
Hepatic Stellate Cells*
Immunoprecipitation
Liver Cirrhosis
Liver Diseases
Luciferases
Mortality
Polymerase Chain Reaction
RNA
Transfection
Transforming Growth Factors
Actins
Desmin
Luciferases
RNA
Transforming Growth Factors

Figure

• 

  Fig. 1 miR-140-3p expression according to HSC-T6 cell activation status. Expression of miR-140-5p was measured in HSC-T6 cells by qPCR. (A) Cells were incubated with 5% FBS for 48 h. The control was FBS-free cells. (B) Cells were treated with 10 ng/mL of PDGF-BB (PDGF) for 48 h. The control was no treatment cells. (C) Cells were exposed to TGF-β1 for 48 h. (D) Levels of the marker of hepatic stellate cell activation, α-SMA in TGF-β1 treated cells were detected using western blot assay. The control was no treatment cells. All experiments were performed in triplicate, and *p<0.05, †p<0.01. PDGF, platelet derived growth factor; α-SMA, α-smooth muscle actin; TGF-β1, transforming growth factor β1.

• 

  Fig. 2 miR-140-3p knockdown reduces cell fibrosis in HSC-T6 cells. miR-140-3p knockdown was obtained after transfection of anti-miR-140-3p into TGF-β1-induced HSC-T6 cells. (A) Relative expression levels of miR-140-3p. (B) Cell proliferation was measured with cell proliferation assay. (C) Cell apoptosis was measured on flow cytometry, and apoptosis rate was calculated. (D) Expressions of PCNA, caspase 3, and cleaved caspase 3 (cle-caspase 3) were detected using Western blot assay, and the gray intensity was analyzed. (E) Cell migration was determined using transwell assay (0.5% crystal violet, ×100). (F and G) Expressions of α-SMA, desmin, COL1A1, and fibronectin were detected using Western blot assay, and the gray intensity was analyzed. All experiments were compared with cells transfected of anti-NC and performed in triplicate, *p<0.05. TGF-β1, transforming growth factor β1; α-SMA, α-smooth muscle actin; anti-miR-140-3p, miR-140-3p inhibitor; PCNA, proliferating cell nuclear antigen; COL1A1, collagen type 1A1.

• 

  Fig. 3 PTEN is negatively regulated by miR-140-3p target binding in HSC-T6 cells. (A) Potential target sites (red) on PTEN 3′UTR were predicted on TargetScan software and mutated. (B) Luciferase reporter assay was conducted to identify the binding sites. Relative luciferase activity was significantly reduced in cells transfected with PTEN wild type (wt) 3′UTR and miR-140-3p mimic (miR-140-3p). (C) RIP were performed. Dramatically high expression of PTEN were obtained from Ago2, separately. (D) Effect of miR-140-3p on expression of PTEN in cells. Relative expression levels of PTEN were evaluated using Western blotting, and the gray intensity was analyzed. All experiments were performed in triplicate, *p<0.05. RIP, RNA immunoprecipitation; PTEN, phosphatase and tensin homolog deleted on chromosome.

• 

  Fig. 4 PTEN silencing blocks the effect of miR-140-3p knockdown in activated HSC-T6 cells. Silencing expression of PTEN was obtained after transfection with specific siRNA against PTEN (siPTEN) into TGF-β1-induced HSC-T6 cells. Effects of siPTEN on anti-miR-140-3p action were measured. (A) Expression levels of PTEN in cells transfected with anti-miR-140-3p and siPTEN or not. (B and C) Cell proliferation and apoptosis was analyzed in cells transfected with anti-miR-140-3p and siPTEN or not. (D) Expressions of PCNA, caspase 3, and cle-caspase 3 were analyzed in cells transfected with anti-miR-140-3p and siPTEN or not, and the gray intensity was calculated. (E) Cell migration was determined using transwell assay after transfection with anti-miR-140-3p and siPTEN or not. (F and G) Expressions of α-SMA, desmin, COL1A1, and fibronectin were analyzed in cells transfected with anti-miR-140-3p and siPTEN or not, and the gray intensity was calculated. All experiments were performed in triplicate, *p<0.05. TGF-β1, transforming growth factor β1; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PCNA, proliferating cell nuclear antigen; α-SMA, α-smooth muscle actin; COL1A1, collagen type 1A1.

• 

  Fig. 5 miR-140-3p knockdown inactivates the AKT/mTOR signaling pathway, which is abolished by PTEN silencing. Effects of siPTEN on anti-miR-140-3p-inactivated signaling pathway were measured. Expressions of p-AKT and p-mTOR and PTEN were detected using Western blot assay, and the gray intensity was calculated. All experiments were performed in triplicate. *p<0.05. PTEN, phosphatase and tensin homolog deleted on chromosome.

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