Background
Multiple myeloma (MM) originates in neoplastic plasma cell disorder, and it is characterized by the clonal proliferation of malignant plasma cells in the bone marrow. As the second most general hematological cancer, the incidence of MM worldwide is about 1.5/100,000 new case [
1]. It is also found that the incidence of MM is higher in men than in women, as well as in black than white in the USA [
2]. MM is always associated with organ dysfunction, especially osteolysis [
3]. Although the survival rate of MM patients is increased due to the advanced medicines [
4], MM continues to be considered as an incurable disease, and further study is required to fully understand the potential molecular mechanisms of osteolysis in MM patients.
To adopt a volume appropriate for the local environment, the bone continuously remodels to keep the balance between bone formation and resorption mediated by osteocytes, osteoblasts, and osteoclasts [
5]. Osteocyte represents the most abundant cell type in the bone and is actively involved in bone turnover. Dickkopf 1(
DKK1) levels increase in the peripheral blood and bone marrow plasma of MM patients and relate to the development of osteolytic lesions [
6,
7]. Tumor necrosis factor-related apoptosis-inducing ligand (
TRAIL) produced by myeloma cells is positively correlated with osteolytic markers (such as urinary deoxypyridinoline and serum calcium), indicating that
TRAIL may function in osteolysis of MM patients [
8]. Via promoting the expression of receptor activator of NF-kB (
RANK) in osteoclast precursors, c-Akt (
AKT) plays a role in the osteoclast formation and bone osteolysis induced by MM [
9]. Giuliani et al. found that MM cells promoted osteocyte death and altered the transcriptional profile in osteocyte [
10]. However, they did not further perform comprehensive bioinformatics analysis to investigate the internal causes. In spite of the above researches, the molecular mechanism of the increased osteocyte death in MM patients is still unclear.
In our study, in order to investigate the molecular mechanism of osteocyte death in MM patients, we reanalyzed the gene expression profile in Giuliani et al. study and identified the differentially expressed genes (DEGs) between normal osteocytes and osteocytes affected by MM cells. The enriched functions and pathways of DEGs were further identified. In addition, we constructed the protein-protein interaction (PPI) network of DEGs. Then, the most significant sub-network was screened out.
Discussion
To gain insight into the molecular mechanisms of the myeloma-induced osteocyte death, gene expression profiles in osteocytes co-cultured with or without myeloma cells were systematically analyzed here. A total of 226 up-regulated genes and 167 down-regulated genes were identified. Then, PPI network was constructed, and sub-network was identified.
TFs like
KLF4 and
IRF8 were up-regulated in osteocytes co-cultured with myeloma cells in comparison with osteocytes cultured alone. Previous study reported that
KLF4 expression is essential for blocking cell cycle and increasing the resistance of MM cells to alkylating agents [
22]. As one of the Yamanaka reprogramming factors and TFs,
KLF4 can promote the expression of autophagy-related genes [
23]. In addition, silencing of the interferon consensus sequence-binding protein (
ICSBP/IRF8) gene may be induced by DNA methylation or other mechanisms and correlates with the malignant phenotype of MM [
24]. In our study,
KLF4 and
IRF8 were up-regulated TFs, and
IRF8 was a tumor suppressor gene. These suggested that
KLF4 and
IRF8 might play a role in osteolysis in MM patients.
In this study, EGF and EGR1 had high connectivity degrees in PPI network. A heparin-binding factor in EGF family, amphiregulin (
AREG) is overexpressed in primary myeloma cells and can promote growth of bone marrow stromal cell [
25].
EGR-1 induces apoptosis in MM via interacting with
JUN, and decreased
JUN/EGR-1 can enhance resistance of MM cells to bortezomib [
26]. Functional enrichment indicated that
EGF was involved in circulatory system development. It has been reported that the substances of the circulatory system can induce the apoptosis of tumor cells [
27]. EGF could interact with EGR1 in the PPI network, indicating that
EGF and
EGR1 might be involved in osteocytes apoptosis induced by MM cells through interacting with each other.
Enrichment analysis suggested that
S1PR1,
C3AR1, and
NPY1R in sub-network were enriched in the pathway of neuroactive ligand-receptor interaction, meanwhile
S1PR1 and
NPY1R were involved in the function of circulatory system development. S1PR1 is a G-protein-coupled receptor which binds to the bioactive signaling molecule sphingosine 1-phosphate (S1P) [
28]. It is reported that
S1PR1 and
S1PR2 regulate osteoclast precursor migration between the bone marrow cavities and the circulation [
29]. It is shown that component 3 (C3A) and its receptor C3AR play a role in osteoclast formation [
30,
31], implying a potential role of
C3AR in osteocyte death. Thus,
S1PR1 and
C3AR1 may promote the death of osteocytes, and this is consistent with the finding of Giuliani et al. [
10].
NPY1R is a receptor of neuropeptide Y (
NPY), which is a neurotransmitter. There is evidence that
NPY regulates bone homeostasis via actions in peripheral tissues [
32].
NPY1R is the only Y receptor which is robustly expressed in bone marrow stromal cells [
33] and osteoblasts [
34], implying a direct role of
NPY1R in bone remodeling. In addition, the absence of peripheral Y1 receptor will lead to pronounced anabolic effects on the bone [
35]. Here,
NPY1R was significantly down-regulated in osteocytes co-cultured with myeloma cells, inhibiting bone remodeling. In the sub-network, S1PR1, C3AR1, and NPY1R had interactions with each other, suggesting that
S1PR1,
C3AR1, and
NPY1R might function in osteocytes apoptosis induced by MM cells via interactions.
Conclusions
In conclusion, a total of 393 DEGs were identified between osteocytes co-cultured with and without myeloma cells, and KLF4, IRF8, EGF, EGR1, S1PR1, C3AR1, and NPY1R might be involved in osteocyte cell apoptosis induced by MM cells. Here, we studied the potential molecular mechanism of the increased osteocyte death, which may provide a theoretical basis for understanding and treatment of osteolysis in MM patients. However, the present study analyzed gene expression data generated from not primary cells isolated from myeloma patients and healthy controls but cell lines, and the sample size was small. Thus, confirmation of results by quantitative real-time polymerase chain reaction (qRT-PCR) in cell lines followed by the investigation of the roles and functions of candidate genes in cells isolated from MM patients and healthy controls is still needed.
Abbreviations
DEGs, differentially expressed genes; DKK1, Dickkopf 1; ECM, extracellular matrix; EGF, epidermal growth factor; KEGG, Kyoto Encyclopedia of Genes and Genomes; MM, multiple myeloma; NPY1R, neuropeptide Y1 receptor; PPI, protein-protein interaction; RMA, robust multi-array average; S1P, sphingosine 1-phosphate; S1PR1, sphingosine-1-phosphate receptor 1; STRING, Search Tool for the Retrieval of Interacting Genes; TAG, tumor-associated gene; TAGs, tumor-associated genes; TFs, transcription factors; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand
Acknowledgements
This work was supported by special fund for medical service of Jilin finance department (No.: SCZSY201507).