| Background & ObjectivesMultiple myeloma (MM) is a plasma cell clonal malignancy.And it is a kind of molecular cloning disease with low proliferative and apoptotic activity. Invasion and metastasis is an important feature of malignant tumors. However, the migration of tumor cells is the basis for its invasion and metastasis.Cell migration refers to the cells that received signal or feel the migration of certain substances after the concentration gradient arising from the movement. In this process, cells are constantly stretching out their front foot, and then pull the cycle of cell body. Cytoskeleton and its binding protein are the material basis for this process. In addition, a variety of substances are on the precise adjustment.HSP90 is a highly conserved cytoplasmic proteins in the process of biological evolution. the tumor cells angiogenesis and mobility were inhibited after HSP90 was inhibited. And HGF/ SFMET signaling pathway was also inhibited.The cell's athletic ability and invasion capacity was diminished too.Studies have shown that HSP90 inhibitors could induce MM cell apoptosis. HSP90 has two types:HSP90a and HSP90β. HSP90a may regulate the cell cycle to promote cell proliferation, HSP90βcan inhibit cell apoptosis and differentiation.Bortezomib is the most promising new drug for treatment of relapsed or refractory MM. It induced cell apoptosis in many ways, one of which is to activate the classical stress response proteins such as heat shock proteins, HSP27, HSP70 and HSP90. Large number of datas indicate that, HSP90 was overexpressed in malignant cells. And especially the expression of HSP90a was obviously increased. In our experiment, human multiple myeloma cell line U266 cells were studied to examine the expression changes of heat shock protein HSP90 after Bortezomib interaction. And Transwell chamber was used was observed the cell migration results after Bortezomib effect. At last,the correlation between HSP90 and human multiple myeloma cell migration ability were analyzed. The experiment will guide the development of new drugs and provide a theoretical basis for clinical treatment.Methods1. Cell culture MM cells were resuscitation from frozen state. And then were cultured in RPMI 1640 medium containing 10% of fetal calf serum, at 37℃,5% CO2 and 95% air humidity conditions, collecting the cells when they were growing well.2. Migration experiment 8μm pore size of Transwell Chambers were used in this experiment. Well incubated U266 cells were added to the upper chambers. The culture medium separatly with 0,50,100,150,200 nmol/L bortezomib concentration were placed in lower Chambers.Then incubated them at 37℃, collected cells in the lower Chambers after 4h of drug effect, cell counting was determined by using cell counting plate in an inverted microscope.3. RT-PCR with and without bortezomib role, the HSP90αand HSP90βexpression in MM cells in vitro were detected by RT-PCR4. Statistical analysis all datas are available on SPSS 13.0 statistical package processing, measurement datas used x±S for statistical description; Multiple sets of datas used the single-factor analysis of variance. Comparision between the two groups was the SNK method; Correlation analysis was used to evaluate the relationship between HSP90α,HSP90βexpression and human multiple myeloma cell migration capacity. All results will have statistical significance which based on P<0.05.ResultsThrough the study above, we found that1. the expression of HSP90mRNA in Human multiple myeloma cell linesWith increasing concentrations of bortezomib, HSP90αof mRNA expression in MM cells gradually increased. The HSP90αQuantitative results from low concentrations to high concentrations were corresponding 0.343±0.017,0.505±0.039,0.640±0.029,0.760±0.059,0.963±0.054; And there are statistical difference between each group (P<0.05) However, the HSP90βquantitative results in Onmol/L concentration of Bortezomib (0.610±0.022) have statistical difference between 50,150,200nmol/L groups (P<0.05).HSP90βquantitative results in 50 (0.765±0.050) and 100nmol/L (0.645±0.052) nmol/L groups are different (P<0.05).Compared with 100nmol/L concentration of Bortezomib group, statistical difference also exists in 150 (0.770±0.059) and 200nmol/L (0.790±0.027) groups (P<0. 05).Although there is no obvious increase in the mRNA expression of HSP90βfrom the chart, statistical difference existed in the whole datas (P<0.05)2. effects of Bortezomib in human multiple myeloma cell migration ability of U266 cells Cells migrate from the upper chambers to lower chambers under the influence of chemokines, but at last the number of cells in lower chambers is different due to the drug concentrations of bortezomib differences, With the increase of drug concentration, Cells migrate into the lower chambers were 98.25±9.639; 87.75±17.154; 73.50±7.594; 70.25±19.973; 52.75±8.500. The SNK method was used to Compare results between groups. Finally,we got the following results, Onmol/L group and 100,150,200 nmol/L are different (P<0.05); 50nmol/L group and 200nmol/L also have statistical difference (P<0.05).Therefore, with increased Bortezomib concentration, the number of U266 cell migration gradually reduced (P<0.05)3. correlation analysis on HSP90α,HSP90βexpression and human multiple myeloma cell migration abilityWith concentration of Bortezomib gradually increased, the ability of cell migration gradually weakened, but the expression of HSP90 in U266 cells showed increased,and mainly HSP90a increased. The Pearson correlation analysis that HSP90 expression and human multiple myeloma cell migration were negatively correlated (HSP90a r-value of-0.818, P<0.05; HSP90βr-value of-0.591, P<0.05).Conclusion1. With the increased of Bortezomib concentration, the HSP90αmRNA expression gradually increased in human multiple myeloma cells. The HSP90βmRNA expression are different overall, but compared with the HSP90a,the increasing trend is not very obvious.2. With the increased of Bortezomib concentration,human multiple myeloma cell migration gradually weakened.3. There are some relationship between the HSP90 mRNA expression and the ability of multiple myeloma cell migration... |
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