Upregulation of LncRNA Malat1 Induced Proliferation and Migration of Airway Smooth Muscle Cells via miR-150-eIF4E/Akt Signaling

Abstract

The increased proliferation and migration of airway smooth muscle cells (ASMCs) are critical processes in the formation of airway remodeling in asthma. Long non-coding RNAs (lncRNAs) have emerged as key mediators of diverse physiological and pathological processes, and are involved in the pathogenesis of various diseases, including asthma. LncRNA Malat1 has been widely reported to regulate the proliferation and migration of multiple cell types and be involved in the pathogenesis of various human diseases. However, it remains unknown whether Malat1 regulates ASMC proliferation and migration. Here, we explored the function of Malat1 in ASMC proliferation and migration in vitro stimulated by platelet-derived growth factor BB (PDGF-BB), and the underlying molecular mechanism involved. The results showed that Malat1 was significantly upregulated in ASMCs treated with PDGF-BB, and knockdown of Malat1 effectively inhibited ASMC proliferation and migration induced by PDGF-BB. Our data also showed that miR-150 was a target of Malat1 in ASMCs, and inhibited PDGF-BB-induced ASMC proliferation and migration, whereas the inhibition effect was effectively reversed by Malat1 overexpression. Additionally, translation initiation factor 4E (eIF4E), an important regulator of Akt signaling, was identified to be a target of miR-150, and both eIF4E knockdown and Akt inhibitor GSK690693 inhibited PDGF-BB-induced ASMC proliferation and migration. Collectively, these data indicate that Malat1, as a competing endogenous RNA (ceRNA) for miR-150, derepresses eIF4E expression and activates Akt signaling, thereby being involved in PDGF-BB-induced ASMC proliferation and migration. These findings suggest that Malat1 knockdown may present a new target to limit airway remodeling in asthma.

Keywords: Akt signaling; Malat1; asthma; eIF4E; miR-150.

Figure 1

Malat1 is upregulated in ASMCs stimulated with PDGF-BB, and Malat1 knockdown inhibits PDGF-BB-induced ASMC proliferation and migration. (A) Malat1 expression in ASMCs treated with PDGF-BB determined by RT-PCR. (B) Malat1 expression in ASMCs transfected with si-Malat1 or si-NC determined by RT-PCR. (C,D) The effect of Malat1 on PDGF-BB-induced ASMC proliferation and migration assessed by MTT and Transwell assays, respectively (scale bar: 100 μm). (E) The effect of Malat1 on PCNA and MMP-9 expression in PDGF-BB-treated ASMCs determined by western blot assay. *p < 0.05.

Figure 2

miR-150 is a target of Malat1. (A) Diagram of the putative binding sites of miR-20b, miR-145, miR-155, miR-25, miR-23b, miR-150, miR-142, miR-135a, and miR-21 in Malat1 sequences. (B) The effect of PDGF-BB stimulation on miR-20b, miR-145, miR-155, miR-25, miR-23b, miR-150, miR-142, miR-135a, and miR-21 expression in ASMCs determined by qRT-PCR. (C,D) The effect of Malat1 knockdown or overexpression on miR-20b, miR-155, miR-25, miR-150, and miR-135a expression in PDGF-BB-treated ASMCs determined by RT-PCR. (E) Luciferase reporter assays show that miR-150 mimic transfection significantly suppresses the luciferase activity of reporter vector containing Malat1-wt compared with the mimic-NC transfection, and has no influence on the luciferase activity of reporter vector containing Malat1-mut. *p < 0.05.

Figure 3

miR-150 inhibits PDGF-BB-induced ASMC proliferation and migration, and this inhibitory effect is markedly reversed by Malat1 overexpression. ASMCs are transfected with mimic-NC, miR-150 mimic, or miR-150 mimic + pcDNA-Malat1, and then used for proliferation and migration detection. (A) Cell proliferation assessed by MTT assay. (B) Cell migration detected by Transwell assay (scale bar: 100 μm). (C) PCNA and MMP-9 expression detected by western blot assay. *p < 0.05.

Figure 4

eIF4E is a direct target of Malat1/miR-150 signal axis. (A) Diagram of the miR-150 putative binding sites in eIF4E 3′-UTR. (B) Luciferase reporter assays show that miR-150 mimic transfection significantly reduces the luciferase activity of the reporter vector containing eIF4E-wt compared with mimic-NC transfection, whereas cotransfection with pcDNA-Malat1 and miR-150 mimic significantly relieves the inhibitory effect of miR-150 mimic transfection on luciferase activity. (C,D) eIF4E mRNA and protein expression in PDGF-BB-treated ASMCs transfected with mimic-NC, miR-150 mimic, miR-150 mimic + pcDNA-Malat1, si-NC, or si-Malat1 determined by RT-PCR and western blot, respectively. *p < 0.05.

Figure 5

eIF4E knockdown inhibits PDGF-BB-induced ASMC proliferation and migration. ASMCs are transfected with si-NC or si-eIF4E, and then are harvested for the following detections. (A,B) eIF4E mRNA and protein expression detected by RT-PCR and western blot, respectively. (C) Cell proliferation determined by MTT assay. (D) Cell migration detected by Transwell assay (scale bar: 100 μm). (E) PCNA and MMP-9 expression determined by western blot assay. *p < 0.05.

Figure 6

Malat1/miR-150/eIF4E signal axis regulates the Akt signaling in PDGF-BB-treated ASMCs. (A) Akt and p-Akt expression in PDGF-BB-treated ASMCs transfected with si-eIF4E, si-Malat1, miR-150 mimic, or their respective control. (B–D) The effects of GSK690693 on PDGF-BB-induced ASMC proliferation and migration determined by MTT, Transwell (scale bar: 100 μm), and western blot assays. *p < 0.05.