Association of miRNA-196b-5p and miRNA-99a-5p with autophagy and apoptosis in multiple myeloma cells / 中华血液学杂志

Objective: To investigate the relationship between miRNA-196b-5p and miRNA-99a-5p expression and autophagy and apoptosis in multiple myeloma cells. Methods: Human myeloma cell line U266 and normal CD138+ plasma cells were selected as the research objects. The subjects were divided into 45 cases of multiple myeloma patients and 40 healthy controls. The expression of miRNA-196b-5p and miRNA-99a-5p was measured by real-time quantitative PCR, and Western blot was used to determine the expression of autophagy related protein LC3-Ⅱ, LC3-Ⅰ, P62, Beclin-1 expression, apoptosis related protein CL caspase3, CL caspase7, Bcl-2, Bax, and TGF-β/Smad pathway associated proteins TGF-β1, Smad2/3, p-Smad3 and Smad7. The cell apoptosis rate was determined by flow cytometry. The correlation between miRNA expression level and clinical characteristics of multiple myeloma patients was analyzed. Results: Compared with normal plasma cells, the expression of miRNA-196b-5p in myeloma cells increased significantly (0.43±0.15 vs 2.44±0.63 or 2.02±0.85, all P<0.001), the expression of miRNA-99a-5p was significantly decreased (1.87±0.61 vs 0.62±0.15 or 0.80±0.33, P<0.001), LC3-Ⅱ/LC3-Ⅰ increased significantly (P<0.05), Beclin-1 expression increased significantly (P<0.05), P62 expression decreased significantly (P<0.05). The expression of Bax, CL caspase3 and CL caspase7 decreased significantly (P<0.05), and the expression of Bcl-2 increased significantly (P<0.05) and apoptosis rate significantly decreased (P<0.05). After transfected with miRNA-196b-5p mimic or miRNA-99a-5p inhibitor, the LC3-Ⅱ/LC3-Ⅰ of CD138+ plasma cells increased significantly (P<0.05), the expression of Beclin-1 increased significantly (P<0.05), P62 expression decreased significantly (P<0.05), and the apoptosis rate significantly decreased (P<0.05). However, after autophagy inhibitor of 3-MA was administered, the apoptotic rate of the above reaction system did not change significantly (P>0.05). The expression of miRNA-196b-5p and miRNA-99a-5p was significantly correlated with DS and ISS stage in multiple myeloma patients (P<0.05). Conclusion: miRNA-196b-5p and miRNA-99a-5p are closely related to the clinical characteristics of patients with multiple myeloma. The overexpression of miRNA-196b-5p and down regulation of miRNA-99a-5p could inhibit the apoptosis of myeloma cells by up regulation of autophagy, and the mechanism is related to the activation of the TGF-β/Smad signaling pathway.

Effect of Expression Levels of MiRNA-132, -125, -143 and -145 on Autophagy and Apoptosis of Multiple Myeloma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate relationship of miRNA-132, miRNA-256, miRNA-143 and miRNA-145 level with antophagy and apoptosis of multiple mgeloma cells.@*METHODS@#Human myeloma cell line U266 and normal CD138 plasma cells were selected and used for study and detection, the 45 cases of MM were enrolled in MM group, and 40 normal persons were sellectod in control group. The expression of miRNA-132, miRNA-125b, miRNA-143 and miRNA-145 were measured by using qPCR, the expressions of autophagy-related protein (LC3-Ⅱ, LC3-Ⅰ, P62, beclin-1) and apoptosis-related molecules (cleaved-Caspase3, cleaved-Caspase7, BCL-2, BAX) were measured by using Western blot, respectively. The rate of apoptosis was measured by using flow cytometry. The correlation of miRNA expression level with clinical-related indexes including M protein, hemoglobin, β2-MG, lactate dehydrogenase, albumin, creatinine and serum calcium was analyzed.@*RESULTS@#Compared with normal plasma cells, the expression of miRNA-132 and miRNA-125b in myeloma cells increased significantly (P0.05). The expressions of miRNA-132, miRNA-125b, miRNA-143 and miRNA-145 were significantly different between DS and ISS staging group, also between the patients with abnormal and normal chromosome karyotype (P<0.05). The miRNA-125b and miRNA-143 significantly correlated with the levels of β2-MG, albumin and hemoglobin (P<0.05).@*CONCLUSION@#The expressions of miRNA-132, miRNA-125b, miRNA-143 and miRNA-145 in patients with multiple myeloma closely relate with the clinical characteristics. Both over-expression of miRNA-125b and down-expression of miRNA-143 inhibit the apoptosis of myeloma cells by up-regulation of autophagy.

DA-EPOCH Chemotherapy Combined with G-CSF Effectively Mobilizes Autologous PBHSC in NHL Patients / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the effect of the DA-EPOCH chemotherapy combined with G-CSF and the CTX therapy with G-CSF on mobilizing and collecting the peripheral blood hematopoietic stem cells and the later hematopoietic recovery.</p><p><b>METHODS</b>Forty patients accepted mobilization and collection of peripheral blood stem cells(PBSC) after treated by CTX+G-CSF and DA-EPOCH+G-CSF therapy respectively, and were treated by auto-transfusion after BEAM pre-regimen. The mobilization efficacy, adverse effects and hematopoietic recovery after autologous transplantation were analyzed retrospectively.</p><p><b>RESULTS</b>During the CTX+G-CSF mobilization, only one patient achieved the white blood cell(WBC) at 0.8×10/L, while the others were with the lowest WBC level above 2.0×10/L. The platelet counts were all normal with the exception of 3 cases at 80×10/L. The median percentage of CD34cells in one period of collection was 0.99(0.35-1.30)%. The median MNC was (3.80±2.05)×10. The cumulative total of mononuclear cell was (5.84±2.48)×10/kg, and the median CD34cell count was 3.84(3.91-6.5)×10/kg. During the DA-EPOCH+G-CSF mobilization, the peripheral WBC count of patients were decreased to the lowest level at (0.2-1.4)×10/L. The platelet counts were all above 40×10/L except for 1 case in which the platelet count was reduced to 8×10/L. The median percentage of CD34cells in one period of collection was 0.85(0.34-1.2)%. The median MNC was (3.68±1.56)×10. The cumulative total of mononuclear cells was (6.01±2.26)×10/kg, and the median CD34cell count was 4.44(2.7-7.10)×10/kg. There were no statistical differences between the 2 groups in the median percentage of CD34cells, the median MNC, the cumulative total of mononuclear cells and the median CD34cell counts (P>0.05). The average acquired time for granulocyte engraftment was 10.00(9.00-11.00) days, and for platelet engraftment was 12.50(11.00-17.25) days, with no statistical difference(P>0.05). No death occurred during the process of transplantation.</p><p><b>CONCLUSION</b>DA-EPOCH therapy combined with G-CSF can effectively mobilize the peripheral blood hematopoietic stem cells in NHL patients with higher safety and lower price, and proves to be worth recommending in clinical use.</p>

Effect of bortezomib combined with bisphosphonates on bone metabolism index in multiple myeloma / 中国实验血液学杂志

This study was aimed to investigate the effect of bortezomib combined with bisphosphonates on serum levels of DKK-1 and RANKL in multiple myeloma patients, and to evaluate its role in the therapy of osteolytic lesion. Fourty-three patients with newly diagnosed and relapsed myeloma were divided into 2 groups. Twenty-three patients were treated with bortezomib combined with bisphosphonates (A group) and 20 patients were treated with bisphosphonates combined with traditional chemotherapy (B group). Serum levels of DKK-1 and RANKL were measured by ELISA before and after 4 cycles of chemotherapy. The results indicated that serum DKK-1 level significantly decreased in patients of A group (43.2 µg/L before vs 30.4 µg/L after 4 cycles of chemotherapy), and so did for serum RANKL level in A group (0.83 pmmol/L before vs 0.45 pmmol/L after 4 cycles of chemotherapy). While there was no significant differences in DKK-1 and RANKL serum level before therapy between A and B groups, but there was significant differences in DKK-1 and RANKL levels after 4 cycles of chemotherapy (P < 0.05). It is concluded that bortezomib combined with bisphosphonates obviously reduce the serum levels of DKK-1 and RANKL, thus has beneficial effect on osteolytic lesion.

Proliferation-inhibiting and multidrug-resistant reversing effect of bortezomib on human HL-60 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the proliferation-inhibiting and multidrug-resistant reversing effect of bortezomib on human HL-60 cells, and to explore the mechanism of bortezomib-induced proliferation inhibition in human leukemia cells.</p><p><b>METHODS</b>The multidrug resistant leukemia cell lines HL-60/DNR and HL-60/VCR cells were used as models, and sensitive HL-60 cells as a control. The cytotoxicity of bortezomib on HL-60, HL-60/DNR, HL-60/VCR cells were measured by MTT method, and the non-cytotoxicity dose was determined as reversible dose. The cells were divided into 4 experimental groups: HL-60/DNR + DNR, HL-60/DNR + DNR + bortezomib, HL-60/VCR + VCR, HL-60/VCR + VCR + bortezomib. The bortezomib resistant reversal fold was calculated. The levels of XIAP, cIAP-1, and cIAP-2 mRNA and proteins expression and the activation of NF-κB of the HL-60/DNR, HL-60/VCR cells were examined by quantitative real time RT-PCR and western blot respectively after treated with gradually increasing concentrations of bortezomib (10, 40, 80 nmol/L) for 48 hours.</p><p><b>RESULTS</b>Bortezomib inhibited the cell growth of HL-60, HL-60/DNR, and HL-60/VCR in a concentration-dependent manner. The IC(50) values were (28.90 ± 3.99), (81.19 ± 9.34), and (73.48 ± 8.94) nmol/L, respectively. After treated with 10nmol/L bortezomib for 48 hours, the IC(50) value of DNR to HL-60/DNR decreased from (12.90 ± 1.75) µmol/L to (3.54 ± 0.57) µmol/L (P < 0.01), and that of VCR to HL-60/VCR from (33.25 ± 7.28) µmol/L to (9.97 ± 1.15) µmol/L (P < 0.01). The reversal fold (RF) values were 3.32 ± 0.53 and 2.64 ± 0.28, respectively. Bortezomib down-regulated the levels of XIAP, cIAP-1, and cIAP-2 mRNA and protein expression and inhibited the NF-κB activation in a concentration-dependent manner.</p><p><b>CONCLUSION</b>Bortezomib can inhibit the proliferation of HL-60 cells and reverse multidrug-resistance in the cells. The possible mechanism is associated with down-regulation of IAPs expression.</p>

Effect of emodin on induction of apoptosis in jurkat cells and its possible mechanisms / 中国实验血液学杂志

The aim of study was to investigate the effect of a traditional Chinese medicine, emodin, on proliferation and apoptosis in T lymphocytic leukemic cell line Jurkat and its mechanisms. Cell proliferation inhibition was detected by MTT assay. Cell apoptosis was measured by DNA ladder and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The expressions of related proteins and caspase family members were determined by Western blot. The results showed that emodin inhibited proliferation in Jurkat cells, with an IC50 about 20 micromol/L and induced cell apoptosis in both time-and dose-dependent manners. The expressions of proliferation-related protein C-MYC, hTERT and apoptosis-related protein BCL-2 were down-regulated in a time dependent manner after the treatment with emodin. The expressions of procaspase-3, -8 and -9 all decreased while activated caspase-3 and PARP expressions were up-regulated. It is concluded that emodin can remarkably inhibit cell proliferation and induce apoptosis in Jurkat cells. The down-regulation of proliferation-related proteins C-MYC, hTERT and apoptosis-related protein BCL-2 expressions and activation of caspase cascade may be involved in the process of apoptosis.