miR‑654‑5p inhibits autophagy by targeting ATG7 via mTOR signaling in intervertebral disc degeneration

Abstract

Intervertebral disc degeneration (IDD) is a common chronic disease characterized by the loss of extracellular matrix (ECM) in the nucleus pulposus (NP). Accumulating evidence has revealed that abnormal expression of microRNAs (miRs) is closely associated with IDD development. The present study aimed to investigate the precise role and possible mechanism underlying the effects of miR-654-5p in the pathogenesis of IDD. NP cells were isolated from patients with IDD. Monodansylcadaverine staining was conducted to reveal cell autophagy, while western blotting was performed to detect the expression of ECM-related proteins in NP cells. Luciferase reporter and RNA immunoprecipitation assays were conducted to identify the binding between RNAs. The results demonstrated that miR-654-5p was significantly upregulated in degenerated NP tissues from patients with IDD and high miR-654-5p expression was positively associated with disc degeneration grade. Functional assays suggested that miR-654-5p facilitated ECM degradation by increasing the expression levels of MMP-3, MMP-9 and MMP-13, as well as decreasing collagen I, collagen II, SOX9 and aggrecan expression by inhibiting autophagy. Furthermore, autophagy-related gene 7 (ATG7) was verified as a direct downstream target gene of miR-654-5p. miR-654-5p could bind to the 3' untranslated region of ATG7 to inhibit its mRNA expression and further reduce its translation. Notably, ATG7 knockdown abrogated the effects of the miR-654-5p inhibitor on ECM degradation and autophagy regulation. Furthermore, miR-654-5p inhibited autophagy in NP cells by increasing the protein expression levels of phosphorylated (p)-PI3K, p-AKT and p-mTOR in an ATG7-dependent manner. In conclusion, the results of the present study revealed that miR-654-5p may enhance ECM degradation via inhibition of autophagy by targeting ATG7 to activate the PI3K/AKT/mTOR signaling pathway. These findings may provide novel insights into the treatment of IDD.

Regulatory roles of noncoding RNAs in intervertebral disc degeneration as potential therapeutic targets (Review)

“…miR-654-5p has been reported to be involved in various biological activities, such as cancer progression, liver regeneration and cardioprotection ( 30 , 31 ). Wang et al ( 32 ) showed that the expression of miR-654-5p is dysregulated in degenerated NP tissues obtained from patients with IDD. Further assays indicated that miR-654-5p promotes ECM degradation by upregulating expression levels of matrix metalloproteinase (MMP)-3, MMP-9 and MMP-13, while downregulating collagen I, collagen II, SRY-box transcription factor 9 (SOX9) and aggrecan via autophagy suppression by binding to autophagy-related (ATG)7 to activate the PI3K/AKT/mTOR pathway ( 32 ).…”

“…Wang et al ( 32 ) showed that the expression of miR-654-5p is dysregulated in degenerated NP tissues obtained from patients with IDD. Further assays indicated that miR-654-5p promotes ECM degradation by upregulating expression levels of matrix metalloproteinase (MMP)-3, MMP-9 and MMP-13, while downregulating collagen I, collagen II, SRY-box transcription factor 9 (SOX9) and aggrecan via autophagy suppression by binding to autophagy-related (ATG)7 to activate the PI3K/AKT/mTOR pathway ( 32 ). Du et al ( 33 ) explored the role of miR-16 in the inflammatory response in NP cells stimulated by lipopolysaccharide (LPS); miR-16 upregulated the expression of ECM genes (aggrecan and collagen II) in NP cells, while it downregulated genes associated with ECM-degrading enzymes [MMP3, MMP13, A disintegrin and metallopeptidase (ADAM) with thrombospondin type 1 motif (ADAMTS)4 and ADAMTS5].…”

Mammalian Target of Rapamycin as the Therapeutic Target of Vascular Proliferative Diseases: Past, Present, and Future

“…58 Furthermore, mTORC1 activation directly inhibits the expression of autophagy-related proteins, such as ATG7, which is important for the initiation of autophagy. [59][60][61] The process of autophagy inhibition mediated by mTOR is useful for studying the mechanisms of many diseases. [62][63][64][65] mTORC1 activation also promotes lipid synthesis.…”

Targeting Autophagy for Developing New Therapeutic Strategy in Intervertebral Disc Degeneration

“…Similarly, miR-210 suppresses autophagy in human degenerated NP cells by directly targeting ATG7 and upregulating MMP-3 and -13 expression, leading to enhanced degradation of Col II and aggrecan [ 285 ]. Another functional assay suggests that miR-654-5p inhibits autophagy and facilitates ECM degradation by boosting MMP-3, -9, and -13 expression and lowering collagen I, collagen II, SOX9, and aggrecan expression via increased levels of phosphorylated (p)-PI3K, p-AKT, and p-mTOR [ 286 ]. Furthermore, expression of MMP-14 is induced by miR-193a-3p downregulation, which accelerates the loss of type II collagen and, hence, promotes IVDD [ 287 ].…”