miR-338-3p blocks TGFβ-induced myofibroblast differentiation through the induction of PTEN

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease. The pathogenesis of IPF is not completely understood. However, numerous genes are associated with the development and progression of pulmonary fibrosis, indicating there is a significant genetic component to the pathogenesis of IPF. Epigenetic influences on the development of human disease, including pulmonary fibrosis, remain to be fully elucidated. In this paper, we identify miR-338-3p as a microRNA severely downregulated in the lungs of patients with pulmonary fibrosis and in experimental models of pulmonary fibrosis. Treatment of primary human lung fibroblasts with miR-338-3p inhibits myofibroblast differentiation and matrix protein production. Published and proposed targets of miR-338-3p such as TGFβ receptor 1, MEK/ERK 1/2, Cdk4, and Cyclin D are also not responsible for the regulation of pulmonary fibroblast behavior by miR-338-3p. miR-338-3p inhibits myofibroblast differentiation by preventing TGFβ-mediated downregulation of phosphatase and tensin homolog (PTEN), a known antifibrotic mediator.

Keywords: fibroblast; lung; miRNA; pulmonary fibrosis.

Figure 1.

miR-338-3p is downregulated in pulmonary fibrosis. A: human lung tissue from nonfibrotic donors (n = 3 subjects) and donors with IPF (n = 10 subjects; patient characteristics listed in Table 1) was analyzed for miR338-3p expression. B: mice (n = 7 mice/group) were treated with vehicle or 1.5 U/kg bleomycin by oropharyngeal aspiration and lung tissue was harvested after 21 days. C: lung tissue from mice exposed to a single dose of 5 Gy total body and 10 Gy thoracic radiation (n = 7–9 mice/group) were harvested 26 wk after exposure and tissue was analyzed for miR-338-3p. All data was analyzed by t test (**P < 0.01, ***P < 0.005).

Figure 2.

miR-338-3p blocks myofibroblast differentiation. Primary human lung fibroblasts from three different donors were treated with 200 nM nontargeting control miRNA or miR-338-3p and/or 1 ng/mL of TGFβ. Each donor is graphed on a separate axis with one representative Western blot shown. Raw blots are provided in the supplement. A–C: protein was harvested after 72 h to examine αSMA protein expression. D: representative western blot for αSMA protein expression. E–G: protein was harvested after 72 h to assess calponin expression. H: representative Western blot for calponin protein expression. Data analysis was performed with three technical replicates (*P < 0.05, **P < 0.01, ***P < 0.005, by ANOVA).

Figure 3.

miR-338-3p blocks TGFβ-induced matrix protein production. Primary human lung fibroblasts from three different donors were treated with 200 nM nontargeting control microRNA or miR-338-3p ±1 ng/mL TGFβ. A–C: protein was harvested at 72 h to assess fibronectin expression. D: representative Western blot. E–G: protein was harvested at 72 h to assess collagen 1 expression. H: representative Western blot. Secreted matrix proteins were evaluated from cells isolated from two donors. I and J: supernatants were collected at 72 h posttreatment to assess secreted fibronectin. F–H and K and L: supernatants were collected at 72 h posttreatment to assess secreted collagen. All experiments performed in technical triplicate (P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 by ANOVA).

Figure 3.

miR-338-3p blocks TGFβ-induced matrix protein production. Primary human lung fibroblasts from three different donors were treated with 200 nM nontargeting control microRNA or miR-338-3p ±1 ng/mL TGFβ. A–C: protein was harvested at 72 h to assess fibronectin expression. D: representative Western blot. E–G: protein was harvested at 72 h to assess collagen 1 expression. H: representative Western blot. Secreted matrix proteins were evaluated from cells isolated from two donors. I and J: supernatants were collected at 72 h posttreatment to assess secreted fibronectin. F–H and K and L: supernatants were collected at 72 h posttreatment to assess secreted collagen. All experiments performed in technical triplicate (P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 by ANOVA).

Figure 4.

miR-338-3p blocks TGFβ-induced matrix proliferation. Primary human lung fibroblasts from three different donors were treated with 200 nM nontargeting control microRNA or miR-338-3p ±1 ng/mL TGFβ. A: 72 h after treatment cells were counted utilizing trypan blue, only live cells were counted, n = 4/group. B: ATP production was assessed by cleavage of a luminescent substrate 72 h after treatment, n = 8/group. Cell death was assessed via LDH release prior to treatment (C) and 72 h after treatment (D), n = 5/group (***P < 0.005, ****P < 0.0001 by ANOVA).

Figure 5.

miR-338-3p does not alter glycolytic enzyme expression. Primary human lung fibroblasts were treated with 200 nM nontargeting control miRNA or miR-338-3p ±1 ng/mL TGFβ. Protein was harvested at 72 h to assess expression of LDHA (A), PKM2 (B), PFKFB3 (C), and PFKFB4 (D). n = 3/group (***P < 0.005 by ANOVA). miRNA, microRNA.

Figure 6.

miR-338-3p does not inhibit TGFβ-induced signaling pathways. Primary human lung fibroblasts were treated with 200 nM nontargeting control microRNA or miR-338-3p ±1 ng/mL TGFβ. Protein was harvested 24 h after treatment to assess expression of TGFβR1 (A) and phosphorylation of Smad2 (B), MEK (C), and ERK (D). RNA was harvested 48 h posttreatment and evaluated for expression of SPARC (E), KLF9 (F), and ADAMTS6 (G). All experiments were performed in triplicate (*P < 0.05, **P < 0.01 by ANOVA).

Figure 7.

miR-338-3p does not inhibit target cell cycle proteins. Primary human lung fibroblasts were treated with 200 nM nontargeting control microRNA or miR-338-3p ±1 ng/mL TGFβ. Protein was harvested 72 h after treatment to assess expression of Cdk4 (A) Cdk6 (B), Cyclin D (C), and Cyclin E (D). All experiments were performed in triplicate, statistical analysis by ANOVA (*P < 0.05, **P < 0.01 by ANOVA).

Figure 8.

miR-338-3p prevents TGFβ-mediated downregulation of PTEN and targets the PREX2-PTEN axis to inhibit myofibroblast differentiation. A: human lung tissue (Table 1) was examined for PTEN gene expression. PTEN knockdown was performed, and protein expression of PTEN (B) and αSMA (C) was assessed 72 h after transfection. Fibroblasts were transfected with 200 nM nontargeting miRNA or miR-338-3p with or without 1 ng/mL TGFβ to assess PTEN protein expression (D) and observe the effect of TGFβ on PTEN expression (E). In addition, phosphorylation status of PTEN (F) examined. Primary human lung fibroblasts were cotransfected with 100 nM of a nontargeting control siRNA (siCon) or siRNA targeting PTEN (siPTEN) and 100 nM of a nontargeting control miRNA (con miR) or miR-338-3p and 1 ng/mL TGFβ where indicated. 72 h after transfection, PTEN expression was assessed to determine knockdown efficiency (G–H) and αSMA protein expression was examined (I). Cells transfected with a nontargeting miRNA or miR-338-3p were examined for the expression of β-catenin (J) and PREX2 (K). We propose miR-338-3p is an antifibrotic regulator, which is capable of inducing PTEN expression through the inhibition of PREX2, a negative regulator of PTEN, which then inhibits myofibroblast differentiation (L and M). All experiments were performed in triplicate. Data were analyzed by t test and ANOVA (*P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001). miRNA, microRNA.