| Myeloma bone disease is one of the most common complications of MM. The key to understanding the biology of MBD is due to increased osteoclast activity and reduced osteoblast function. Osteolytic bone destruction results from classic signaling pathways such as RANKL/RANK pathway,and local production of an osteoclast stimulatory factors(OSFs) that is secreted by the interactions between myeloma cells and bone marrow stromal cells.Chemokine macrophage inflammatory protein-1 alpha(MIP-1alpha) is the critical role of OSFs. The early stage of the study and abroad recent research reports not only increasing osteoclast activity, but also reducing bone formation by inhibiting osteoblast(OB) function. But he specific mechanism of MIP-1alpha inhibiting osteoblast(OB) function is not clear.Sclerostin is a product of the sclerostin gene(SOST), which is a Wnt antagonist, osteocyte-specific protein and a potent suppressor of bone formation. Some studies have demonstrated that activation of Wnt/β- signaling is a pivotal pathway in the differentiation of mesenchymal stem cells(MSCs) into osteoblasts. Recent studies have shown that sclerostin levels is elevated in the serum of patients with active myeloma, which correlated with advanced disease features including severe bone disease.But the specific mechanism of the increasing of sclerostin and the relationship with clinical characteristics are not clear. In this paper,we try to investigate the expression of MIP-1 alpha and sclerostin in bone marrow of patients with myeloma bone disease(MBD), and discuss the possible association of the sclerostin and MIP-1α with MBD and the clinical characteristics such as the stage of MBD, the ISS stage, the clinical and biochemical characteristics and the prognosis of patients.We may come to the conclusion the relationship with these two protein by the tools of bioinformatics,which through data mining and literature. To further validate the relationship of these two protein in vitro, for exploreing new ideas and provide theoretical basis for prevention and cure patients with MBD.We finished this study with three parts as follows.Part 1 protein-1 alpha(MIP-1 alpha) and Sclerostin in bone marrow plasma of myeloma bonedisease: relationship with bone disease and clinical characteristicsObject:The aim of the study was to investigate the expression of MIP-1 alpha and sclerostin in bone marrow of patients with myeloma bone disease(MBD), the possible association of concentrations of the sclerostin and MIP-1α with MBD including the presence of lytic bone disease, the disease stage, and patients’ survival,the clinical characteristics and thses two kinds of protein.Method: 53 patients(24 F, 29 M), median age 64 years, human Myeloma Cell Lines RPMI-8226,H929 and 30 controls patients with leukopenia and immune thrombocytopenia purpura were studied. MIP-1 alpha and sclerostin levels were quantified using an enzyme-linked immunosorbent assay(ELISA). Sclerostin and MIP-1 alpha m RNA expression was determined by RT-PCR. PTH and 1, 25(OH) 2D3 levels were measured with an electrochemiluminescence immunoassay. Conventional biochemical indicators of patients were measured with Automatic biochemical analyzer. All data were show by(x±s) and were analyzed using SPSS18.0.Results:Among the patients and healthy control subjects, the levels of MIP-1α(153.35±81.09 pg/ml)and sclerostin(539.42±201.86pg/ml) in MBD patients bone marrow were obviously highe than those in the control group, there was statistically significant difference between these two groups(t=6.85 and 8.26,p=0.00<0.01). In vitro, the levels of MIP-1α and sclerostin in supernatant of human Myeloma Cell Lines RPMI-8226,H929 were obviously highe than those in the control group(t=23.52,13.42;P=0.00<0.01). The results of RT-PCR also confirmed that the levels of MIP-1α and sclerostin protein of bone marrow mononuclear cells(BMMNCs) in most of the patients with MBD and the human Myeloma Cell Lines RPMI-8226,H929 more were obviously highe than those in the control group(P<0.01).But the levels in different cell lines were different. The levels of sclerostin(0.61±0.20 ng/ml)and MIP-1 alpha(173.81±97.74 pg/ml)in patients with ISS stage III disease were significantly higher than those in patients with ISS stage II disease(MIP-1α117.73±54.35pg/ml, sclerostin 0.42±0.18 ng/ml)(p=0.01 and 0.06).The sclerostin(0.60±0.02 pg/ml)and MIP-1 alpha(170.37±103.69 pg/ml)levels in patients with BMD in group C were significantly higher than those in group A+B(MIP-1α 119.82±38.15 pg/ml, sclerostin 0.45±0.19 ng/m)(both P<00.01). There was positive correlation between sclerostin levels and MIP-1 alpha(r=0.720,p=0.00<0.001),beta2-microglobulin(r=0.467,p=0.00<0.001)LDH(r=0.453,p=0.001)and a Ca levels(r=0.313,p=0.023). A negative association was seen between sclerostin levels and b ALP(r=-0.578,p=0.00<0.001),HB(r=-0.412,p=0.002),and ALB(r=-0.388,p=0.004)Levels. There was positive correlation between the levels of sclerostin and MIP-1 alpha with the grade of BMD(r=0.439 and 0.358, p=0.001 and 0.008) and the clinc ISS stage(r=0.436 and 0.323, p=0.001 and 0.018).The MBD patients with high sclerostin levels(>0.54 ng/ml) had significantly shorter median survival than those with low sclerostin levels(≤0.54 ng/ml)(χ2=7.376, p=0.007). But the MM patients with high MIP-1 alpha levels(>153.71pg/ml) had no significantly difference with those low MIP-1 alpha levels(X2=2.94, p=0.086). Dual linear regression results suggest ISS, sclerostin protein, β2-MG, MIP-1 alpha protein were independent prognosis factors in patients with MBD.Conclusion:1. The levels of MIP-1α and sclerostin protein were increased in MBD patients, and MM cell lines may express these two protein, but the levels in different cell lines was not the same.2.The level of MIP-1α, sclerostin were positively correlated with the grade of BMD, the clinic ISS stage,and refecting tumor load such as the levels of β2-MG and LDH,but were negative correlated with the levels of b ALP and HB.3. The MBD patients with high sclerostin levels(>0.54 ng/ml) had significantly shorter median survival than those with low sclerostin levels.4. The level of MIP-1α was positively correlated with sclerostin.Part2 :Identification and bioinformatic analysis of myeloma bone disease related genesObject: Identification myeloma bone disease related genes applying bioinformatic related software and online tools,and mining data and literature MBD related genes,in order to concluding the relationship between related genes.Method :Studing gene expression profiling datasets of GSE754(139multiple myeloma with osteolytic bone destruction) and GSE755(34 multiple myeloma with no osteolytic bone destruction).Using Gene Spring GX 11.5 software to screen differential genes between MBD and MM with no bone destruction.Using biological methods including GATHER to analyze these differential genes possible molecular function, participating in a variety of signal pathway and enrichment,Using Cytosine software constructing the relationship of associated protein of BMD related genes.Results:435 differentially expressed genes were identified by the Gene Spring GX11.5 software, of which 286 genes were over-expressed, 149 genes underexpressed. Imputing the differentially genes into GATHER software found that these genes associated with the following biological pathways:cell proliferation, calcium phosphorus metabolism, cell cycle, Cell adhesion and migration, cell apoptosis, protease, MAPK, chemokine signaling pathways, Erb B, cell reduction division, Wnt, T and B cell signaling pathways, Jak-STAT, Rho A/ROCK signaling pathway and so on. Imputing the differentially genes into Cytosine software found that these related genes coding protein are mainly concentrated in the Nf-kappa B, PTK2 B, ADD3, SPP1, IGF2, HSF2, HSF2, NTRK2, ADD3, GST6, CCL3, IL-6, Rh OA and sclerostin gene.Conclusion: Through bioinformatics analysis of the gene expression profiling datasets related MBD,we found that MIP-1 alpha(CCL3) and sclerostin(SOST) has a certain relevance, the related MBD genes participate in mainly involves the proteasome, MAPK, Erb B, cell cycle, Wnt, Jak-STAT etc.signaling pathways.Part 3 Validation relationship of MIP-1 alpha and sclerostin by cell culture in vitroObject: In vitro cell culture experiment, by using specific MIP-1α,s and anti-sclerostin antibody to increase or neutralize MIP-1α and sclerostin, which secreted from RPMI8226 and H929 myeloma cells,then we observed the levels changes of sclerostin/MIP-1 alpha in the two kinds of cells,in order to validate the relationship of MIP-1 alpha and sclerostin.Method: human Myeloma Cell Lines RPMI-8226, H929 were studied. MIP-1 alpha and sclerostin levels were quantified using an enzyme-linked immunosorbent assay(ELISA). Sclerostin and MIP-1 alpha m RNA expression was determined by RT-PCR.Results:In vitro, by contrasting before and after adding MIP-1α, we found that MIP-1α and sclerostin levels were higher than the control groups, there was statistically significant difference between these two groups(P<0.01).Bycontrasting before and after adding anti MIP-1α antibody, we found that MIP-1α and sclerostin levels were lower than the control groups, there was statistically significant difference between these two groups(P<0.01); Furthermore, compared with the adding sclerostin and anti-sclerostin antibody,the levels of MIP-1α had no significant difference in these groups(P>0.05),but the levels of sclerostin had significant difference in these groups(P<0.01).Conclusion: MIP-1 alpha may mediate myeloma cells or bone marrow microenvironment secretion sclerostin through various pathways.In conclusion, this study we verified the MIP-1 alpha and sclerostin protein, two protein high expression in the bone marrow of patients with MBD and most of myeloma cells. There were positive correlation between the levels sclerostin and MIP-1 alpha of these two kinds of protein with the tumor load, MBD classification and ISS stage, more interesting, and a significant positive relationship was observed between these two kinds of protein and two kinds of proteins.Furthermore, we found MIP-1 alpha and sclerostin has a certain relevance Through bioinformatics software analysis the large gene chip data download from GEO. In vitro, MIP-1 alpha may mediate myeloma cells or bone marrow microenvironment secretion sclerostin through various pathways.These reveals that MIP-1 alpha suppress OB function in MBD which may be mediate by sclerostin mechanism. |
PDF Full Text Request