CircPAN3 contributes to drug resistance in acute myeloid leukemia through regulation of autophagy.

The aim of this study was to investigate the role and underlying mechanism of circular RNA (circRNA) circPAN3 in mediating drug resistance in acute myeloid leukemia (AML). We first established two doxorubicin (ADM)-resistant AML cell lines and then utilized high-throughput RNA sequencing (RNA-seq) to compare their circRNA expression profiles with those of the parental cell lines. With bioinformatic analysis, we identified key circRNA molecules involved in drug resistance and validated our findings in clinical specimens. The target microRNAs (miRNAs) and downstream mRNAs were also explored bioinformatically. Using RNA interference technique, the potential mechanism was further investigated. Twenty-nine circRNAs were identified to be differentially expressed between ADM-resistant and sensitive cells. We found that circPAN3 is most likely a key mediator in the development of AML drug resistance, evidenced by the increased expression in ADM-resistant cell lines and BM samples from relapsed patients. Additionally, downregulation of circPAN3 by small interfering RNA (siRNA) significantly restored drug sensitivity to ADM in the two ADM-resistant cell lines, but lentivirus-mediated circPAN3 overexpression had the opposite effect. Subsequent bioinformatic analysis and mechanistic experiments revealed that circPAN3 may facilitate AML drug resistance through regulating autophagy and influencing expression of apoptosis-related proteins, while AMPK/mTOR signaling plays a key role in the regulation of circPAN3 on autophagy. These findings may provide new important insights into the role of circRNAs in mediating AML drug resistance, and suggest that circPAN3 might be a potential target for treatment of drug-resistance AML, which merits further investigation and validation.

CircPAN3 mediates drug resistance in acute myeloid leukemia through the miR-153-5p/miR-183-5p-XIAP axis.

The contribution and role of circular RNAs (circRNAs) in mediating chemoresistance in acute myeloid leukemia (AML) are still poorly understood and need further investigation. In this study, we established a doxorubicin (ADM)-resistant THP-1 AML cell line (THP-1/ADM). A high-throughput microarray was used to identify circRNA expression profiles of THP-1/ADM cells and naive THP-1 cells. The identified potential functional circRNA molecule was further validated in THP-1/ADM cells and bone marrow (BM) specimens from 42 AML patients. The interactions with target microRNAs (miRNAs) and downstream messenger RNAs (mRNAs) were also explored. As a result, 49 circRNAs that are significantly differentially expressed between THP-1/ADM and THP-1 cells were identified. Of these circRNAs, downregulation of circPAN3 by small interfering RNA significantly restored ADM sensitivity of THP-1/ADM cells. Furthermore, BM samples from patients with refractory and recurrent AML showed increased expression of circPAN3. A detailed circRNA/miRNA/mRNA interaction network was predicated for this circRNA. Subsequent mechanistic experiments showed that downregulation of circPAN3 could decrease the expression of X-linked inhibitor of apoptosis protein (XIAP), but this effect was counteracted by miR-153-3p or miR-183-5p specific inhibitors. Luciferase experiments further demonstrated that these molecules are involved in the circPAN3 regulatory network. Our results revealed that circPAN3 may be a key mediator for chemoresistance of AML cells, which may depend on the circPAN3-miR-153-5p/miR-183-5p-XIAP axis. Our findings provide evidence that circPAN3 can be a valuable indicator for predicting clinical efficacy of chemotherapy in AML patients and also can serve as a potential target for reversing drug resistance in AML.

[Effect of Expression Levels of MiRNA-132, -125, -143 and -145 on Autophagy and Apoptosis of Multiple Myeloma Cells].

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, ß2-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 (P<0.05), and the expression of miRNA-143 and miRNA-145 decreased significantly (P<0.05), but the expression of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 increased significantly (P<0.05). The expression of P62. BAX, cleaved-Caspase3 and cleaved-Caspase7 decreased significantly (P<0.05), the BCL-2 expression increased significantly (P<0.05), but the rate of apoptosis decreased (P<0.05). After transfection with miRNA-125b mimic or miRNA-143 inhibitor by using the cationic liposomes, the LC3-Ⅱ /LC3-Ⅰ of normal plasma cells increased significantly (P<0.05), the expression of Beclin-1 significantly increased (P<0.05), the expression of P62 decreased significantly (P<0.05), and the apoptosis rate decreased (P<0.05). However, the apoptosis rate was not significantly changed after addition of the autophagic inhibitor 3-MA in the reaction system(P>0.05). 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 ß2-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].

OBJECTIVE: 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. METHODS: 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. RESULTS: During the CTX+G-CSF mobilization, only one patient achieved the white blood cell(WBC) at 0.8×109/L, while the others were with the lowest WBC level above 2.0×109/L. The platelet counts were all normal with the exception of 3 cases at 80×109/L. The median percentage of CD34+ cells in one period of collection was 0.99(0.35-1.30)%. The median MNC was (3.80±2.05)×1010. The cumulative total of mononuclear cell was (5.84±2.48)×108/kg, and the median CD34+ cell count was 3.84(3.91-6.5)×106/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)×109/L. The platelet counts were all above 40×109/L except for 1 case in which the platelet count was reduced to 8×109/L. The median percentage of CD34+ cells in one period of collection was 0.85(0.34-1.2)%. The median MNC was (3.68±1.56)×1010. The cumulative total of mononuclear cells was (6.01±2.26)×108/kg, and the median CD34+ cell count was 4.44(2.7-7.10)×106/kg. There were no statistical differences between the 2 groups in the median percentage of CD34+ cells, the median MNC, the cumulative total of mononuclear cells and the median CD34+ cell 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. CONCLUSION: 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.

[Effect of bortezomib combined with bisphosphonates on bone metabolism index in multiple myeloma].

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].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Effect of emodin on induction of apoptosis in jurkat cells and its possible mechanisms].

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.