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RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Published on: April 10, 2018

Knockdown of Long Non-Coding RNA RP11-445H22.4 Alleviates LPS-Induced Injuries by Regulation of MiR-301a in

Taitao Sun¹, Jian Yu², Liang Han³

¹Department of Orthopedics, Jining No.1 People's Hospital, Jining, China.

Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology

|February 8, 2018

Abstract

Background/Aims:

Several long non-coding RNAs (lncRNAs) play vital roles in osteoarthritis (OA), whereas the role of lncRNA RP11-445H22.4 in OA remains unclear. The study aimed to investigate the effect of lncRNA RP11-445H22.4 on lipopolysaccharide (LPS)-induced cell viability, apoptosis and inflammatory injury of OA.

Methods:

The expression of RP11-445H22.4, miR-301a and CXCR4 in human cartilage ATDC5 cells were altered by transfection, and then cells were exposed to 5 µg/ml LPS for 12 h. Then cell viability, apoptosis, apoptosis-related factors and inflammatory cytokines were analyzed by CCK-8, flow cytometry, western blot, RT-qPCR and ELISA, respectively. Dual-luciferase reporter assay was performed to assess the binging sites of RP11-445H22.4 and miR-301a. The signal pathways of NF-κB and MAPK/ ERK were determined by western blot.

Results:

LPS reduced cell viability, increased apoptosis and stimulated release of IL-1β, IL-6, IL-8 and TNF-α. However, RP11-445H22.4 inhibition significantly rescued LPS-induced injuries by promoting cell viability, suppressing apoptosis and inflammatory cytokines secretions in ATDC5 cells. In addition, miR-301a directly bound to RP11-445H22.4, and suppression of miR-301a inversed the effects of RP11-445H22.4 inhibition. Furthermore, CXCR4 was a direct target of miR-301a, and CXCR4 silencing increased cell viability, decreased apoptosis and inflammatory cytokines secretions in LPS-treated ATDC5 cells. Besides, we found that CXCR4 silencing blocked LPS-activated NF-κB and MAPK/ERK pathways.

Conclusions:

The study indicated that lncRNA RP11-445H22.4-miR-301a-CXCR4 axis played an important role in cartilage ATDC5 cells and provided a theoretical basis of lncRNA RP11-445H22.4 in OA.

Keywords:

Apoptosis CXCR4 Cell viability Inflammatory injury LncRNA RP11-445H22.4 MiR-301a

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