HOXA5-induced lncRNA DNM3OS promotes human embryo lung fibroblast fibrosis via recruiting EZH2 to epigenetically suppress TSC2 expression

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is an unrepairable disease that results in lung dysfunction and decreased quality of life. Prevention of pulmonary fibrosis is challenging, while its pathogenesis remains largely unknown. Herein, we investigated the effect and mechanism of long non-coding RNA (lncRNA) DNM3OS/Antisense RNA in the pathogenesis of pulmonary fibrosis.

Methods: EdU (5-ethynyl-2'-deoxyuridine) and wound healing assays were employed to evaluate the role of DNM3OS on cell proliferation and migration. Western blot detected the proteins expressions of alpha-smooth muscle actin (α-SMA), vimentin, and fibronectin. The interactions among genes were evaluated by RNA pull-down, luciferase reporter, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and chromatin Isolation by RNA purification (ChIRP) assays.

Results: DNM3OS was upregulated by transforming growth factor beta 1 (TGF-β1) in a dose- and time-dependent manner. DNM3OS knockdown repressed the growth and migration of lung fibroblast, and fibrotic gene expression (CoL1α1, CoL3α1, α-SMA, vimentin, and fibronectin), while suppression of TSC2 accelerated the above process. DNM3OS recruited EZH2 to the promoter region of TSC2, increased the occupancy of EZH2 and H3K27me3, and thereby suppressed the expression of TSC2. HOXA5 promoted the transcription of DNM3OS.

Conclusions: HOXA5-induced DNM3OS promoted the proliferation, migration, and expression of fibrosis-related genes in human embryo lung fibroblast via recruiting EZH2 to epigenetically suppress the expression of TSC2.

Keywords: EZH2; HOXA5; Idiopathic pulmonary fibrosis (IPF); TSC2; long noncoding RNA DNM3OS (lncRNA DNM3OS).

Figure 1

TGF-β1 upregulated lncRNA DNM3OS expression in lung fibroblast. (A) Lung fibroblast HELF and WI-38 were treated with indicated concentration of TGF-β1. LncRNA DNM3OS level was evaluated by qRT-PCR. (B) After treatment with TGF-β1 for indicated time, lncRNA DNM3OS level was assessed by qRT-PCR in HELF and WI-38. (C) HELF and WI-38 cells were subjected to nucleus and cytosol fractionation, and the levels of DNM3OS in the nucleus and cytosol were evaluated by qRT-PCR. *, P<0.05; **, P<0.01; ***, P<0.001. LncRNA, long noncoding RNA; HELF, human embryo lung fibroblast; TGF-β1, transforming growth factor beta 1; qRT-PCR, quantitative reverse transcription real-time polymerase chain reaction; GAPDH, glyceraldehyde phosphate dehydrogenase.

Figure 2

HOXA5 promoted the transcription of DNM3OS. (A) Potential HOXA5 binding site in the promoter of DNM3OS predicted by JASPAR database. (B) DNM3OS promoter luciferase reporter expressing HELF cells were transfected with pcDNA3.1 or HOXA5 plasmid, and luciferase activity was determined by dual-luciferase assay. (C) ChIP was performed to confirm the relationship between DNM3OS and HOXA5. The promoter of DNM3OS was amplified by PCR and the level was determined by agarose electrophoresis. (D) DNM3OS level in HELF cells transfected with pcDNA3.1 or HOXA5 plasmid was determined by qRT-PCR. *, P<0.05; **, P<0.01; ***, P<0.001. HELF, human embryo lung fibroblast; pcDNA3.1, the control plasmid; ChIP, chromatin immunoprecipitation; PCR, polymerase chain reaction; qRT-PCR, quantitative reverse transcription real-time polymerase chain reaction; IgG, immunoglobulin G; TSS, transcription start site; WT, wide type; MUT, mutant.

Figure 3

Loss of DNM3OS attenuated growth and the expression of fibrosis-related genes. (A) The level of DNM3OS in HELF and WI-38 cells expression sh-NC and sh-DNM3OS were evaluated by qRT-PCR. (B-E) WT and shRNA expressing lung fibroblasts were challenged with vehicle control or TGF-β1. The proliferation (B) and migration activity (C) of HELF and WI-38 cells were examined by EdU and wound healing assay, respectively. For EdU assay, the EdU probe was conjugated with a fluorescent dye, and the nucleus was stained by Hoechst 33342 and was examined under a fluorescent microscope. CoL1α1 and CoL3α1 expression (D) in HELF and WI-38 cell was assessed by qRT-PCR. The levels of α-SMA, vimentin, and fibronectin (E) in HELF and WI-38 cell were evaluated by western blot. *, P<0.05; **, P<0.01; ***, P<0.001. HELF, human embryo lung fibroblast; qRT-PCR, quantitative reverse transcription real-time polymerase chain reaction; WT, wide type; TGF-β1, transforming growth factor beta 1; shRNA, short hairpin RNA; sh-NC, short hairpin RNA negative control; EdU, 5-ethynyl-2'-deoxyuridine; α-SMA, alpha-smooth muscle actin; GAPDH, glyceraldehyde phosphate dehydrogenase.

Figure 4

LncRNA DNM3OS suppressed the expression of TSC2 through recruiting EZH2. (A) RIP was performed in HELF and WI-38 cells. DNM3OS level in control IgG or EZH2 antibody-enriched fraction was determined by PCR. (B) RNA pull-down assay was performed in HELF and WI-38 cells with DNM3OS and antisense RNA. EZH2 level was evaluated by Western blot. (C,D) The mRNA (C) and protein (D) levels of TSC2 in sh-NC or sh-DNM3OS HELF and WI-38 cells were examined by qRT-PCR and Western blot. (E) ChIP was performed in sh-NC and sh-DNM3OS HELF cells. (F,G) The mRNA (F) and protein (G) levels of TSC2 in HELF and WI-38 cells transfected with sh-NC or sh-EZH2 were determined by qRT-PCR and western blot analysis. *, P<0.05; **, P<0.01. LncRNA, long noncoding RNA; RIP, RNA immunoprecipitation; HELF, human embryo lung fibroblast; sh-NC, short hairpin RNA negative control; ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative reverse transcription real-time polymerase chain reaction; IgG, immunoglobulin G; GAPDH, glyceraldehyde phosphate dehydrogenase.

Figure 5

LncRNA DNM3OS suppressed TSC2 to promote the expression of fibrosis-related genes of in lung fibroblast. (A) The level of TSC2 in HELF and WI-38 cells with sh-NC or sh-TSC2 was determined by qRT-PCR. (B-E) sh-DNM3OS or sh-DNM3OS plus sh-TSC2 were expressed in HELF and WI-38 cells. WT and shRNA expressing lung fibroblasts were treated with control or TGF-β1. The proliferation (B) and migration activity (C) of HELF and WI-38 cells were examined by EdU and wound healing assay, respectively. For EdU assay, the EdU probe was conjugated with a fluorescent dye, and the nucleus was stained by Hoechst 33342 and was examined under a fluorescent microscope. CoL1α1 and CoL3α1 levels (D) in HELF and WI-38 cell were evaluated by qRT-PCR. The expressions of α-SMA, vimentin, and fibronectin (E) in HELF and WI-38 cell were evaluated by western blot assay. *, P<0.05; **, P<0.01; ***, P<0.001. LncRNA, long noncoding RNA; HELF, human embryo lung fibroblast; TGF-β1, transforming growth factor beta 1; sh-NC, short hairpin RNA negative control; WT, wide type; qRT-PCR, quantitative reverse transcription real-time polymerase chain reaction; EdU, 5-ethynyl-2'-deoxyuridine; α-SMA, alpha-smooth muscle actin; GAPDH, glyceraldehyde phosphate dehydrogenase.