Circular RNA circ-4099 is induced by TNF-α and regulates ECM synthesis by blocking miR-616-5p inhibition of Sox9 in intervertebral disc degeneration

Abstract

Circular RNAs (circRNAs) play important roles in the initiation and development of different diseases. Here, we detected their role in intervertebral disc (IVD) degeneration. An Arraystar human circular RNA microarray assay was used to detect circRNAs in normal and degenerated human IVD nucleus pulposus (NP) tissues. The role of circ-4099 in IVDD and its mechanism were evaluated by qRT-PCR and gain-of-function/loss-of-function studies. Interaction networks for competing endogenous RNAs (ceRNAs), miRNAs, and miRNA target gene were detected by bioinformatics analysis, RNA immunoprecipitation and luciferase assay. Expression of seventy-two circRNAs were increased by more than twofold in degenerated NP tissues. qRT-PCR showed that the expression of circ-4099 in NP tissues was consistent with that of the array screening. Over-expression of circ-4099 increased the expression of Collagen II and Aggrecan and decreased the secretion of the pro-inflammatory factors IL-1β, TNF-α, and PGE2. TNF-α treatment increased circ-4099 expression in NP cells. NF-κB/MAPK inhibitors or shRNAs abolished the inductive effects of TNF-α on circ-4099 expression. We further demonstrated that circ-4099 was able to function as a “sponge” by competitively binding miR-616-5p, which reversed the suppression of Sox9 by miR-616-5p. We used DNA pull-down and spectrometry experiments to show that TNF-α can promote circ-4099 transcription through upregulation of GRP78. We provide the first evidence that shows circRNAs are differentially expressed in degenerated and normal NP tissues. Circ-4099 may play a role in a protective mechanism and be part of a compensatory response that maintains the synthesis and secretion of the extracellular matrix in NP cells and might be a protective factor in IVD degeneration as well as restore NP cell function.

The CircRNA-ACAP2/Hsa-miR-21-5p/ Tiam1 Regulatory Feedback Circuit Affects the Proliferation, Migration, and Invasion of Colon Cancer SW480 Cells

“…Biotin-coupled circRNA and miRNA capture Biotin-coupled miRNA and circRNA pull-down assays were performed as described previously [11,12]. Briefly, 3′ end biotinylated circRNA-ACAP2 (digoxin-5′-TCACACAGGCAGCTTTGGAAGAAG-3′-Digoxin; RiboBio, Guangzhou, China) was transfected into SW480 cells at a final concentration of 20 nM for 1 day.…”

CircGLCE alleviates intervertebral disc degeneration by regulating apoptosis and matrix degradation through the targeting of miR-587/STAP1

“…38 Recently, it has been reported that specific circRNAs have been identified as significant regulators and biomarkers for degenerative disc disease. 39-42 The underlying mechanism mostly relies on their abilities to counteract the inhibitory effects of miRNAs on the target genes. Due to the development of microarray profiling, bioinformatic approaches and sequencing techniques, the identification of novel circRNAs as effective regulatory factors and potential therapeutic targets for IDD is expected.…”

Circular RNAs in nucleus pulposus cell function and intervertebral disc degeneration

“…A recent microarray study by Wang and colleagues also reported that human NP cells isolated from degenerated and non‐degenerated discs showed distinct circRNA expression patterns, with 21 downregulated and 51 upregulated by more than twofold in the degenerated NP cells. The upregulation of circ‐4099, circ‐2069, circ‐92328 and circ‐68610 was further confirmed by RT‐qPCR …”