Long noncoding RNA ANRIL up-regulates CCND1 via sponging miR-98-5p to promote TGF-β1-induced human airway smooth muscle cell proliferation, migration, and extracellular matrix deposition

Abstract

Excessive proliferation and migration of airway smooth muscle cell (ASMC) contribute to asthma pathogenesis. Long noncoding RNAs (lncRNAs) are reported to take part in asthma pathogenesis. This study is targeted at deciphering the role of the lncRNA antisense noncoding RNA in the INK4 locus (ANRIL) in ASMC proliferation, migration and extracellular matrix (ECM) deposition. qRT-PCR was performed to determine ANRIL, miR-98-5p, and cyclin D1 (CCND1) mRNA expression levels in transforming growth factor-β1 (TGF-β1)-treated ASMCs. CCK-8 and Transwell assays were employed to examine ASMC proliferation and migration, respectively. Dual-luciferase reporter gene assay and RNA immunoprecipitation assay were carried out for analyzing the targeted relationship of miR-98-5p with ANRIL or CCND1 mRNA 3'-UTR. The levels of CCND1 and ECM proteins (such as fibronectin, COL3A1, and COL1A2) in ASMCs were detected through Western blot. In this work, we found that ANRIL and CCND1 were up-regulated in TGF-β1-treated ASMCs, whereas miR-98-5p was down-regulated. ANRIL overexpression facilitated the proliferation, ECM deposition and migration of TGF-β1-induced ASMCs, while knocking down ANRIL had the opposite effect. Furthermore, ANRIL targeted miR-98-5p directly, and CCND1 was miR-98-5p's downstream target. ANRIL indirectly increased CCND1 expression in ASMCs via competitively binding to miR-98-5p. MiR-98-5p inhibition or CCND1 overexpression counteracted the inhibiting effect that ANRIL knockdown had on TGF-β1-stimulated ASMC proliferation, migration and ECM deposition. In conclusion, ANRIL indirectly up-regulates CCND1 expression by targeting miR-98-5p to promote ASMC proliferation, migration and ECM deposition, thus facilitating the pathogenesis of asthma.

Keywords: ANRIL; CCND1; MiR-98-5p; airway smooth muscle cell; asthma.

FIGURE 1

Effects of TGF‐β1 on the expressions of ANRIL, miR‐98‐5p and CCND1 in ASMCs. (A–C) qRT‐PCR was used to detect the expression levels of ANRIL (A), miR‐98‐5p (B), and CCND1 mRNA (C) in ASMCs treated with or without 10 ng/ml TGF‐β1. (D) The CCND1 level in ASMCs treated with or without 10 ng/ml TGF‐β1 was determined by Western blot. ***p < 0.001

FIGURE 2

Effects of ANRIL on TGF‐β1‐stimulated ASMC proliferation, migration and ECM deposition. (A,B) ANRIL expression level in ASMCs with ANRIL knockdown or overexpression was determined through qRT‐PCR. (C) ASMCs were transfected with si‐ANRIL‐2 or ANRIL overexpression plasmids, and then treated with 10 ng/ml TGF‐β1 for 24 h. Then ASMC proliferation was examined via the CCK‐8 method. (D) Transwell assay was conducted to detect TGF‐β1‐treated ASMC migration after ANRIL knockdown or overexpression. Scale bar, 250 μm. (E) Western blot analysis was conducted to determine the levels of fibronectin, COL1A2 and COL3A1 in TGF‐β1‐induced ASMCs after ANRIL knockdown or overexpression. (F) Immunofluorescence staining was carried out to detect COL1A2 and COL3A1 expressions in TGF‐β1‐induced ASMCs after ANRIL overexpression or knockdown. Scale bar, 100 μm. **p < 0.01, ***p < 0

FIGURE 3

ANRIL directly targets and binds to miR‐98‐5p. (A) qRT‐PCR was carried out to examine the relative expression levels of ANRIL, U6 mRNA and GAPDH mRNA in ASMC nucleus and cytoplasm. (B) The binding sequence of miR‐98‐5p with ANRIL was predicted by bioinformatics analysis. (C) The targeted relationship of miR‐98‐5p with ANRIL was verified through dual‐luciferase reporter gene assay. (D) RIP assay was conducted to detect the enrichment of ANRIL and miR‐98‐5p in the immunoprecipitate of the anti‐Ago2 and anti‐IgG groups. (E) qRT‐PCR was utilized for detecting miR‐98‐5p expression in ASMCs with ANRIL overexpression or knockdown. **p < 0.01 and ***p < 0.001

FIGURE 4

CCND1 is miR‐98‐5p's downstream target in ASMCs. (A) The StarBase online tool was employed to analyze and predict the binding sequences of miR‐98‐5p to CCND1 3'‐UTR. (B) Dual‐luciferase reporter gene assay was carried out to verify the targeted relationship of miR‐98‐5p with CCND1 3'‐UTR. (C) qRT‐PCR was performed for detecting miR‐98‐5p expression in ASMCs transfected with miR‐98‐5p mimics or inhibitors. (D,E) CCND1 mRNA and protein expressions in ASMCs transfected with miR‐98‐5p mimics or inhibitors were detected via qRT‐PCR (D) and Western blot (E). ***p < 0.001

FIGURE 5

MiR‐98‐5p inhibition or CCND1 overexpression reverses the inhibiting impact of ANRIL knockdown on TGF‐β1‐induced ASMC proliferation, migration and ECM deposition. ASMCs were co‐transfected with si‐ANRIL‐2 or/and miR‐98‐5p inhibitors or CCND1 overexpression plasmids, and subsequently treated with 10 ng/ml TGF‐β1 for 24 h. Untreated ASMCs served as the control. (A,B) CCND1 mRNA and protein expressions in the transfected ASMCs were examined via qRT‐PCR (A) and Western blot (B). (C,D) CCK‐8 method (C) and Transwell assay (D) were employed to detect ASMC proliferation and migration. Scale bar, 250 μm. (E) Western blotting was utilized to detect fibronectin, COL1A2 and COL3A1 expression levels in ASMCs. (F) COL1A2 and COL3A1 expression levels in ASMCs were determined by immunofluorescence staining. Scale bar, 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001