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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

See all articles by Xuemei Geng

Xuemei Geng

Fudan University - Division of Nephrology

Xialian Xu

Fudan University - Division of Nephrology

Nana Song

Fudan University - Division of Nephrology

Shuan Zhao

Fudan University - Division of Nephrology

Jiarui Xu

Fudan University - Division of Nephrology

Yong Liu

Medical College of Wisconsin

Haoxuan Li

Fudan University - Division of Nephrology

Xin Chen

Fudan University - Division of Nephrology

Mingyu Liang

Medical College of Wisconsin

Xiaoqiang Ding

Fudan University - Division of Nephrology; Shanghai Medical Center of Kidney Disease; Fudan University - Kidney and Dialysis Institute of Shanghai; Kidney and Blood Purification Laboratory; Hemodialysis Quality Control Center; Shanghai Laboratory of Kidney Disease and Dialysis

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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

Suggested Citation

Geng, Xuemei and Xu, Xialian and Song, Nana and Zhao, Shuan and Xu, Jiarui and Liu, Yong and Li, Haoxuan and Chen, Xin and Liang, Mingyu and Ding, Xiaoqiang, LncRNA GAS5 Promotes Apoptosis As a Competing Endogenous RNA for miR-21 via Thrombospondin 1 in Ischemic AKI (June 17, 2019). Available at SSRN: https://ssrn.com/abstract=3405554 or http://dx.doi.org/10.2139/ssrn.3405554

Xuemei Geng

Fudan University - Division of Nephrology

China

Xialian Xu

Fudan University - Division of Nephrology

China

Nana Song

Fudan University - Division of Nephrology

China

Shuan Zhao

Fudan University - Division of Nephrology

China

Jiarui Xu

Fudan University - Division of Nephrology

China

Yong Liu

Medical College of Wisconsin

8701 Watertown Plank Road
Milwaukee, WI WI 53226
United States

Haoxuan Li

Fudan University - Division of Nephrology

China

Xin Chen

Fudan University - Division of Nephrology

China

Mingyu Liang

Medical College of Wisconsin

8701 Watertown Plank Road
Milwaukee, WI WI 53226
United States

Xiaoqiang Ding (Contact Author)

Fudan University - Division of Nephrology ( email )

China

Shanghai Medical Center of Kidney Disease ( email )

China

Fudan University - Kidney and Dialysis Institute of Shanghai ( email )

China

Kidney and Blood Purification Laboratory ( email )

Shanghai
China

Hemodialysis Quality Control Center ( email )

Shanghai
China

Shanghai Laboratory of Kidney Disease and Dialysis ( email )

China