Abstract
ObjectiveEpithelial-mesenchymal transition (EMT) is an important factor leading to peritoneal fibrosis (PF) in end-stage renal disease (ESRD) patients. The current research aimed to evaluate the effect of long non-coding RNA growth arrest-specific 5 (lncRNA GAS5) in human peritoneal mesothelial cells (HPMCs) EMT and explore the potential molecular mechanisms.Materials and MethodsHPMCs were cultured under control conditions or with high glucose (HG). The cells were then treated with lncRNA GAS5, lncRNA GAS5 siRNA, with or without miR-21 inhibitor and PTEN transfection. Expression of lncRNA GAS5, miR-21, α-SMA, Vimentin, E-cadherin, phosphatase and tensin homolog deleted on chromosome ten (PTEN), Wnt3a, and β-catenin were measured by real time PCR and Western blotting. Bioinformatics analyses were used to test the specific binding sites between the 3′ UTR of the PTEN gene, miR-21, and lncRNA GAS5. Rescue experiments were performed to confirm the lncRNA GAS5/miR-21/PTEN axis in HPMC EMT.ResultsWe found that HG-induced EMT decreased lncRNA GAS5 and that overexpression of lncRNA GAS5 can attenuate EMT in HPMCs. In addition, lncRNA GAS5 regulated HG-induced EMT through miR-21/PTEN. Cotransfection of miR-21 inhibitors remarkably increased PTEN expression and attenuated EMT in lncRNA GAS5 knockdown HPMCs. Moreover, rescue experiments showed that overexpression of PTEN attenuated the EMT effects of lncRNA GAS5 siRNA in HPMCs. We also confirmed that the Wnt/β-catenin pathway was stimulated in lncRNA GAS5/miR-21/PTEN-mediated EMT.ConclusionOur research showed that lncRNA GAS5 competitively combined with miR-21 to regulate PTEN expression and influence EMT of HPMCs via the Wnt/β-catenin signaling pathway. This study provides novel evidence that lncRNA GAS5 may be a potential therapeutic target for HPMC EMT.
Highlights
Peritoneal dialysis (PD) is one of the important alternative therapies for end-stage renal disease (ESRD), and it has been more widely used recently
Following our previously published methods (Yang et al, 2018), human peritoneal mesothelial cells (HPMCs) were cultured with 5.5 mmol/L glucose, 2.5% HG (126 mmol/L) and mannitol as the high osmotic pressure group (5.5 mM glucose + 120.5 mmol/L mannitol)for 24 h, cells were collected for subsequent experiments. long non-coding RNAs (lncRNAs) GAS5 overexpression plasmids and lncRNA GAS5 siRNA were purchased from GenePharma (Shanghai, China)
In order to test whether lncRNA GAS5 could modulate HG-induced epithelial-mesenchymal transition (EMT), HPMCs were transfected with pcDNA3.1-GAS5 and GAS5 siRNA
Summary
Peritoneal dialysis (PD) is one of the important alternative therapies for end-stage renal disease (ESRD), and it has been more widely used recently. Researchers have found that epithelial-mesenchymal transition (EMT), the initial reversible step in the peritoneal fibrosis (PF) process, is an important factor leading to PF in patients with PD. One study found that 232 lncRNAs were differentially expressed in the PF mouse model used, indicating that lncRNAs are involved in the regulation of PF (Liu et al, 2015) Among these lncRNAs, lncRNA GAS5 has been shown to regulate organ fibrosis in liver (Gong et al, 2018), heart Our previous research demonstrated that miR21 targeting of phosphatase and tensin homolog deleted on chromosome ten (PTEN) played an important role in the HGinduced EMT of human peritoneal mesothelial cells (HPMCs) (Yang et al, 2018)
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