4 Discussion
Through previous studies, it is known that osteoarthritis is a degenerative bone and joint disorder. Although aging and obesity are closely related to the onset of OA [32, 33], the specific mechanism has not yet been clarified. This study investigated the role of RNA-binding protein GNL3 in the pathogenesis of osteoarthritis. It is found that GNL3 indirectly affected the pathogenesis of osteoarthritis by affecting the transcription of downstream genes. So this study has provided new therapeutic targets of osteoarthritis.
GNL3 (Nucleolar GTP-binding Protein 3), also known as nucleostemin, plays an important role in cell proliferation, cell cycle regulation and other aspects [12, 13]. Although previous studies showed that the expression of GNL3 gene increased in OA rats and Chinese Han population [16], the specific molecular mechanism has not been clarified. We considered multiple possibilities of the potential mechanism of GNL3 in OA. There have been few previous studies on GNL3 as an RNA-binding protein, and some studies have confirmed that GNL3 interacts with the susceptibility gene loci of osteoarthritis [34]. Therefore, we speculated that RNA-binding protein GNL3 affects the transcription and expression of downstream genes thus affecting the incidence of osteoarthritis.
In this experiment, high-throughput RNA sequencing (RNA-seq) was performed on the experimental and control groups after GNL3 gene was knocked down in Hela cells to obtain complete transcriptome information. Unlike traditional hybridization methods, RNA-seq has several advantages. First of all, RNA-seq is not limited to detecting transcripts corresponding to existing genome sequences. For example, 454-based RNA-seq was used to sequence the transcriptome of Glanville fritillary butterfly [35]. RNA-seq also revealed changes in the sequence of transcriptional regions [36, 37]. RNA-seq has no quantification upper limit, which is associated with the number of sequences obtained. So it can detect expression levels over a large dynamic range. A greater than 9,000-fold range was measured in a study that analyzed 16 million mapped reads in Saccharomyces cerevisiae [38], and a range spanning five orders of magnitude was measured for 40 million mouse sequence reads [39]. RNA-seq is controlled at very precise quantifying expression levels using quantitative PCR (q-PCR) and spike-in RNA of known concentrations [38] making the results highly reproducible for both technical and biological replicates [36, 38].
It was observed that during GNL3 gene knock-down, a large number of genes showed differential expression, indicating that GNL3 extensively regulated gene expression. The functions of these genes were clustered into GO and KEGG databases, and interestingly the results showed that the functions of down-regulated genes were mainly in signal transduction, apoptotic, transcription, cell proliferation and primary immunodeficiency. The DEGs were comprised in pathways such as Jak-STAT signality pathway, p53 pathway, p53-Akt pathway, transcription misregulation in cancer, NF-kappa B signality pathway and cell adhesion molecules. Notably the two down-regulated genes, IL24 gene and PTN gene, have been closely associated with the development of osteoarthritis in previous researches. It is obviously that PTN is upregulated in our eight sets of experiments. To draw the heat-map, we only selected two appropriate sets of results where PTN gene up-regulation was not significant.
In this work, it is confirmed that RNA-binding protein GNL3 affected the expression of downstream genes, including IL24 and PTN, by affecting RNA transcription. IL24 and PTN play an important role in promoting the development of osteoarthritis. To further prove the role of GNL3 in the pathogenesis of osteoarthritis, the local tissues of OA patients and the control groups were selected for q-RT-PCR. The results are shown as the mean ± standard deviation of three genes. It indicates that there is a significant difference in gene expression between the OA groups and the control groups. The expression level of IL24 gene in OA patients was nearly twice higher than control group insisting that it mainly aggravates the inflammatory response at the joint site in osteoarthritis. IL24 is an immune-regulating cytokine with a wide range of tumor-specific inhibitory effects [40, 41], and it induces apoptosis through intracellular and extracellular signaling pathways [42]. It has also been observed that IL24 induces metaphase chromosome protein 1 (MCP1) production in synovial mononuclear cells, which aggravates the inflammatory response and promotes the development of spinal joint arthritis and rheumatoid arthritis [43]. Similarly, as PTN is responsible for promoting angiogenesis, the expression level of PTN gene in OA patients was nearly seen to be seven times higher than that in control group. PTN is a secretory polypeptide that plays an important role in cell growth, migration and angiogenesis [44, 45]. In addition to directly promoting angiogenesis, PTN also influences the behavior of other cells by promoting the proliferation and migration of endothelial progenitor cells [46], and promotes the formation of angiogenic microenvironment [47]. Angiogenesis is one of the earliest histopathological changes in chronic non-communicable arthritis [48]. GNL3 was also expressed about seven times higher than that in control group. Hence, from the results observed in HeLa cells and in the specimens of OA patients, GNL3 developed osteoarthritis by acting on both of these two downstream genes.
GNL3 affects the JAK-STAT pathway through its interaction with Signal Transducer And Activator Of Transcription 3 (STAT3) [49]. It has been shown that when STAT3 was activated, the expression level of downstream gene IL24 increased [50]. Binding anaplastic lymphoma kinase (ALK) which induces MAPK pathway activation is an important step in the anti- apoptotic signaling of PTN and regulation of cell proliferation [51]. STAT3 inhibitor can inhibit the function of ALK, thereby reducing the expression of PTN gene [52]. Therefore, we believe that after knocking down GNL3, expression levels of IL24 and PTN genes in the JAK-STAT pathway are reduced through the interaction with STAT3 gene.