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LncRNA GAS5 Promotes Apoptosis As a Competing Endogenous RNA for miR-21 via Thrombospondin 1 in Ischemic AKI
34 Pages Posted: 18 Jun 2019
More...Abstract
Background: Mounting evidence has indicated that long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) played an important role in renal ischemia/reperfusion (I/R) injury. However, the involvement of lncRNA growth arrest specific transcripts 5 (GAS5) in acute kidney injury (AKI) remained largely unexplored. This study aimed to determine the functions of GAS5 and possible molecular mechanisms during the renal I/R process.
Methods: Bioinformatic analysis revealed possible interaction involving GAS5, miR-21 and thrombospondin 1(TSP-1). Dural Luciferase reporter assays were employed to identify the regulatory relationship between GAS5 and miR-21 as well as between miR-21 and TSP-1. In this study, renal I/R and delayed IPC models were established in vivo. Besides, hypoxia/reoxygenation for different time (H6R0.5 and H24R3) was induced in vitro. Then we performed real-time PCR, western blot, flow cytometry, TUNEL assay to explore possible downstream regulatory molecules. LNA modified anti-miR-21 and anti-scramble were delivered intraperitoneally 1h before procedure or transfected into HK-2 cells to knock down the expression of miR-21. Also, small interfering RNA and plasmids were used to knock down and overexpress the level of GAS5 in HK-2 cells, respectively.
Findings: GAS5, noticeably upregulated by renal I/R injury, was further suppressed by delayed IPC while knockdown of miR-21 in vivo before IPC could significantly increased the expression of GAS5. Concurrently, TSP-1 was negatively regulated by miR-21 in vivo and vitro. Additionally, Reciprocal repression of GAS5 and miR-21 was identified. Knockdown of miR-21 in H6R0.5 treated HK-2 cells promoted apoptosis. Co-transfection of miR-21 mimic and pcDNA-GAS5 or pcDNA-Vector were performed, results of which showed that co-transfection of inhibition of miR-21 on TSP-1 could be rescued by overexpression of GAS5.
Interpretation: GAS5 facilitated the apoptosis by competitively sponging miR-21, which negatively regulated TSP-1 in renal I/R injury. This novel regulatory axis could act as a therapeutic target for AKI in the future.
Funding Statement: National Natural Science Foundation of China grants 81770734 (to Dr. Xu) and 81430015 (to Dr. Ding); Science and Technology Commission of Shanghai (14DZ2260200) and Construction Project of Shanghai Renal Disease Clinical Medical Center (2017ZZ01015).
Declaration of Interests: The authors declare no conflict of interest.
Ethics Approval Statement: All protocols were approved by Institutional Animal Care Use Committee of Fudan University.
Keywords: lncRNA GAS5; miR-21; ischemia/reperfusion; TSP-1; apoptosis
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