miR-328-5p Induces Human Intervertebral Disc Degeneration by Targeting WWP2

Abstract

Intervertebral disc degeneration (IDD) development is regulated by miRNA, including inflammatory reactions, cell apoptosis, and degradation of extracellular matrix. Nucleus pulposus cells apoptosis has a absolute influence in the development of IDD. This experiment explores the mechanism of miR-328-5p regulating IDD. Through the analysis of miRNA and mRNA microarray database, we screened the target genes miR-328-5p and WWP2. We verified the expression of miR-328-5p, WWP2, and related apoptotic genes in normal and degenerative nucleus pulposus tissues by qRT-PCR. The expressions of WWP2, Bcl-2, and Bax were detected by qRT-PCR and western blot after transfection to nucleus pulposus cell. The nucleus pulposus cell proliferation and apoptosis after transfection were confirmed by CCK8 and flow cytometry. Luciferase reporter assay and bioinformatics analyzed the targeting relationship between miR-328-5p and WWP2. Firstly, the qRT-PCR experiments confirmed the significant increase of miR-328-5p expression, while significant reduction of WWP2 in a degenerative tissues compared to the normal tissues. Surprisingly, miR-328-5p expression was positively, while that of WWP2 negatively correlated with the degeneration grade of IDD. And we also identified the high expression of Bax and Caspase3, while low expression of Bcl-2 in a degenerative tissues. After miR-328-5p mimic transfected into nucleus pulposus cell, qRT-PCR and western blot confirmed that WWP2 and Bcl-2 expressions were downregulated, while Bax and Caspase3 expressions were upregulated, and the same results were obtained by knocking down WWP2. CCK8 and flow cytometry confirmed that miR-328-5p inhibited the proliferation and induced apoptosis of nucleus pulposus cells. WWP2 is a target gene of miR-328-5p by bioinformatics and luciferase reporter assay. In summary, miR-328-5p targets WWP2 to regulate nucleus pulposus cells apoptosis and then participates in the development of IDD. Furthermore, this study may provide new references and ideas for IDD treatment.

Cellular Senescence in Intervertebral Disc Aging and Degeneration: Molecular Mechanisms and Potential Therapeutic Opportunities

“…Direct and indirect regulation of senescence pathways by miRNAs have also been reported [ 128 ]. Several miRNAs are upregulated [ 130 , 131 ] or downregulated [ 60 , 132 , 133 , 134 , 135 ] during IDD. In addition, circRNAs and LncRNAs, at times acting in concert with miRNAs regulate NP cell function and IDD [ 118 , 136 ].…”

The potential mechanisms and application prospects of non-coding RNAs in intervertebral disc degeneration

“…In addition, silencing of miR-141 had a protective effect on IDD mice, while upregulating miR-141 accelerated the development of IDD. Similar to miR-141, a large body of studies demonstrated that miRNAs were up-regulated in DD and aggravated NP cells apoptosis, such as miR-96/FRS2 ( 20 ), miR-4478/MTH1 ( 21 ) and miR-328-5p/WWP2 ( 22 ). On the contrary, there were substantial numbers of miRNAs were down-regulated and most likely exert anti-apoptotic effects in IDD, mainly involving miR-129-5p/BMP2 ( 23 ), miR-623/CXCL12 ( 24 ) and miR-155-3p/KDM3A/HIF1α ( 25 ).…”

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

“…According to a recent study, miR-328-5p is abnormally expressed in numerous types of cancer (colorectal cancer and lung cancer) and is associated with apoptosis of cancer cells ( 72 ). Yan et al ( 73 ) found that the expression of miR-328-5p is upregulated significantly, while the expression of its target gene WW domain containing E3 ubiquitin protein ligase 2 (WWP2) is downregulated in IDD tissues compared with normal tissues. miR-328-5p upregulation is positively, whereas WWP2 downregulation is inversely, linked with the degeneration grade of IDD.…”