TGF-β2-induced NEAT1 regulates lens epithelial cell proliferation, migration and EMT by the miR-26a-5p/FANCE axis

Abstract

Aim: To explore the regulatory mechanism of nuclear paraspeckle assembly transcript 1 (NEAT1) in the pathogenesis of posterior capsule opacification (PCO).

Methods: Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was executed to analyze NEAT1 and microRNA (miR)-26a-5p expression in transforming growth factor-beta 2 (TGF-β2)-disposed lens epithelial cells (LECs). The proliferation, cell cycle progression, apoptosis, and migration of TGF-β2-disposed LECs were evaluated. The relationship between NEAT1 or fanconi anemia (FA) complementation group E (FANCE) and miR-26a-5p was verified by dual-luciferase reporter assay.

Results: TGF-β2 induced NEAT1 expression in LECs. NEAT1 inhibition accelerated apoptosis, cell cycle arrest, decreased proliferation, epithelial-mesenchymal transition (EMT), and migration of TGF-β2-disposed LECs. NEAT1 sponged miR-26a-5p to further regulate FANCE expression. Rescue experiments presented that miR-26a-5p downregulation overturned NEAT1 silencing-mediated impacts on TGF-β2-disposed LEC biological behaviors. Additionally, FANCE overexpression reversed miR-26a-5p mimic-mediated impacts on TGF-β2-disposed LEC biological behaviors.

Conclusion: TGF-β2-induced NEAT1 facilitates LEC proliferation, migration, and EMT by upregulating FANCE via sequestering miR-26a-5p.

Keywords: fanconi anemia complementation group E; miRNA-26a-5p; nuclear paraspeckle assembly transcript 1; posterior capsule opacification; transforming growth factor-beta 2.

Figure 1. TGF-β2 stimulation elevated NEAT1 levels in SRA01/04 cells

RT-qPCR exhibiting NEAT1 levels in SRA01/04 cells with TGF-β2 treatment. ^aP<0.05.

Figure 2. NEAT1 silencing weakened PCO progression

A: The knockdown efficiencies of si-NEAT1#1 and si-NEAT1#2 on NEAT in SRA01/04 cells were verified by RT-qPCR. B-G: After transfection with si-NEAT1#2 or si-NC, SRA01/04 cells were treated with 5 ng/mL of TGF-β2. B-F: The proliferation, apoptosis, cycle progression, and migration of the above-mentioned cells were assessed. G: WB exhibiting α-SMA, E-cadherin, and vimentin levels in the above-mentioned cells. ^aP<0.05.

Figure 3. NEAT1 was a miR-26a-5p sponge

A: The complementary sequences between NEAT1 and miR-26a-5p. B: The luciferase activities of NEAT1-MUT and NEAT1-WT reporters in cells with miR-26a-5p mimic or miR-NC. C, D: RT-qPCR presenting the miR-26a-5p level in SRA01/04 cells after TGF-β2 treatment. E: RT-qPCR showing the miR-26a-5p level in SRA01/04 cells with different treatments. ^aP<0.05.

Figure 4. MiR-26a-5p inhibitor reversed the impacts of NEAT1 downregulation on PCO progression

SRA01/04 cells were transfected with si-NC, si-NEAT1#2, si-NEAT1#2+anti-miR-NC, or si-NEAT1#2+anti-miR-26a-5p and then treated with TGF-β2 (5 ng/mL). A-G: Analysis of the proliferation, apoptosis, cell cycle progression, and migration of the above-mentioned cells. H: WB exhibiting α-SMA, E-cadherin, and vimentin protein levels in the above-mentioned cells. ^aP<0.05.

Figure 5. MiR-26a-5p directly targeted FANCE

A: The sites of miR-26a-5p complementary to FANCE. B: Dual-luciferase reporter assay exhibiting the targeting relationship between FANCE and miR-26a-5p mimic. C, D: WB detection of FANCE protein levels in SRA01/04 cells with TGF-β2 treatment. E: The overexpression efficiency of miR-26a-5p mimic was verified by RT-qPCR. F: After TGF-β2 treatment, the level of FANCE protein in SRA01/04 cells transfected with miR-NC, miR-26a-5p, miR-26a-5p+pcDNA, or miR-26a-5p+FANCE was assessed by WB. ^aP<0.05.

Figure 6. FANCE overexpression overturned miR-26a-5p mimic-mediated impacts on PCO progression

After transfection, SRA01/04 cells were treated with TGF-β2 (5 ng/mL). A-G: The proliferation, apoptosis, cell cycle progression, and migration of the above-mentioned cells were detected. H: WB exhibiting α-SMA, E-cadherin, and vimentin protein levels in the above-mentioned cells. ^aP<0.05.

Figure 7. NEAT1 sponged miR-26a-5p to regulate FANCE expression

WB presenting the impact of miR-26a-5p silencing on FANCE protein levels in NEAT1-inhibting SRA01/04 cells under TGF-β2 treatment. ^aP<0.05.