Oxidative Medicine and Cellular Longevity
Volume 2022 (2022), Article ID 3511967, 13 pages
https://doi.org/10.1155/2022/3511967
miR-328-5p Induces Human Intervertebral Disc Degeneration by Targeting WWP2
Correspondence should be addressed to Quan Zhou
Received 17 July 2022; Revised 30 August 2022; Accepted 7 September 2022; Published 29 September 2022
Academic Editor: Sidong Yang
Copyright © 2022 Jing Yan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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.
1. Introduction
With the increasing incidence of low back pain (LBP), it has become the most important trigger to disability worldwide, which has brought a tremendous economic pressure [1–3]. The cause of LBP is very complex. And some known factors affect the advancement of LBP include genes, age, and lousy living habits (such as occupation, smoking, trauma, and mechanical loading) [4, 5]. Furthermore, it is believed that the main cause of LBP is IDD [6–8]. As a bridge between adjacent vertebral bodies, intervertebral disc includes the nucleus pulposus (NP), annulus fibrosus, and the cartilaginous endplate [9]. The most important pathological feature of IDD is the apoptosis of nucleus pulposus cells [10–13]. The apoptosis of nucleus pulposus cells triggers the progress of IDD [14–16], and this affects clearly the disc structure balance. Some studies suggested that abnormal apoptosis is associated with degenerative diseases such as osteoarthritis, IDD, and cancer [17–19].
However, the current treatment for IDD is limited to symptomatic intervention and cannot completely improve the prognosis of the disease [20]. Many studies confirmed that some regulatory genes have an essential impact in the incidence and development of IDD, such as microRNA (miRNA). The miRNA are single-stranded noncoding small RNA with 18 to 24 bp nucleotide sequences, which participates in regulating the cell proliferation and apoptosis [21–25]. And miRNA can negatively regulate the posttranscriptional gene expression in different species by either inhibiting mRNA translation or promoting mRNA degradation [25, 26]. Previous studies have also found that miRNA affects the progress of IDD by facilitating inflammatory response, cell apoptosis, and degradation of extracellular matrix [27]. Furthermore, miRNA has contributed to cardiovascular disease, cancer, leukemia, and skeletal muscle diseases [28]. Therefore, further research on the mechanism of miRNA regulation of IDD may lead to new therapeutic directions.
Some studies have found miR-328-5p is atypically expressed in lung cancer, breast cancer, and other tumors. In addition, the researcher indicated miR-328-5p was related with the proliferation and apoptosis of cancer cells [29–32]. Furthermore, studies have shown dissimilarity expression of WWP2 in various diseases such as oral cancer, endometrial cancer, ovarian cancer, glioma, and lung cancer by regulating cell apoptosis [33–38]. Both miR-328-5p and its target gene WWP2 can affect cell proliferation and apoptosis, and the mechanism of miR-328-5p mediating WWP2 regulating IDD has not been reported. This series of studies aims to research the mechanism of the above regulatory pathways and whether they are involved in the formation of IDD by inducing nucleus pulposus cell apoptosis.
2. Materials and Methods
2.1. Clinical Sample Collection
We obtained 20 human NP samples via surgical discectomy. Surgical indications: (1) failure of conservative treatment and (2) progressive neurological deficits. Patients were excluded with isthmus or degenerative spondylolisthesis, ankylosing spondylitis, and diffuse idiopathic hyperostosis. According to T2-weighted midsagittal pfirrmann disc degeneration grading criteria [39].Grade I-II are normal intervertebral discs, and grade III-V are degenerative intervertebral discs. Table 1 presents the clinical features of patient.