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.