Identification and validation of serum autoantibodies in children with B-cell acute lymphoblastic leukemia by serological proteome analysis.

BACKGROUND: B-cell acute lymphoblastic leukemia (B-ALL) is the most common malignancy of childhood. Even though significant progresses have been made in the treatment of B-ALL, some pediatric B-ALL have still poor prognosis. The identification of tumor autoantibodies may have utility in early cancer diagnosis and immunotherapy. In this study, we used serological proteome analysis (SERPA) to screen serum autoantibodies of pediatric B-ALL, aiming to contribute to the early detection of B-ALL in children. METHODS: The total proteins from three pooled B-ALL cell lines (NALM-6, REH and BALL-1 cells) were separated using two-dimensional gel electrophoresis (2-DE), which was followed by Western blot by mixed serum samples from children with B-ALL (n=20) or healthy controls (n=20). We analyzed the images of 2-D gel and Western blot by PDQuest software, and then identified the spots of immune responses in B-ALL samples compared with those in control samples. The proteins from spots were identified using mass spectrometry (MS). The autoantibodies against alpha-enolase (α-enolase) and voltage-dependent anion-selective channel protein 1 (VDAC1) were further validated in sera from another 30 children with B-ALL and 25 normal individuals by the use of enzyme-linked immunosorbent assay (ELISA). The protein expression levels of the candidate antigens α-enolase and VDAC1 in B-ALL were thoroughly studied by immunohistochemical analysis. RESULTS: Utilizing the SERPA approach, α-enolase and VDAC1 were identified as candidate autoantigens in children with B-ALL. The frequencies of autoantibodies against α-enolase and VDAC1 in children with B-ALL were 27% and 23% by using ELISA analysis, respectively, which were significantly higher than those in normal controls (4% and 0, p<0.05). Immunohistochemical analysis showed the expression of α-enolase and VDAC1 was positive in 95% and 85% of B-ALL patients, respectively, but negative expression levels were showed in the control group. CONCLUSIONS: This study incidated that α-enolase and VDAC1 may be the autoantigens associated with B-ALL. Therefore, α-enolase and VDAC1 autoantibodies may be the potential serological markers for children with B-ALL.

NDRG2 and TLR7 as novel DNA methylation prognostic signatures for acute myelocytic leukemia.

Acute myelocytic leukemia (AML) is an aggressive malignant tumor and typically fatal without treatment. Identification and development of novel biomarkers could be beneficial for the diagnosis and prognosis of AML patients. Here, we aimed to identify the accurate DNA methylation prognostic signatures for AML patients. The DNA methylation data of AML patients and corresponding clinical information were retrieved from The Cancer Genome Atlas database. CPG sites that correlates closely with the survival of the AML patients were identified and further combined into CPG sites pairs to screen the survival-related pairs. The prognostic signatures were identified by the C-index and forward search algorithms and validated by the verification group. Finally, the functional enrichment analysis was performed on these CPG sites. As a result, a total of 498 CPG sites associated with the overall survival of AML patients was obtained. A prognostic signature composed of 10 CPG sites pairs was obtained and validated. The functional enrichment analysis showed prognostic genes were mainly enriched in tumor protein processing, cell differentiation, blood leukocyte immunity, and platelet growth factor pathways. In summary, we identified two accurate prognostic methylation signatures (NDRG2 and TLR7), which would be served as a novel therapy target for AML.

MicroRNA-33b regulates sensitivity to daunorubicin in acute myelocytic leukemia by regulating eukaryotic translation initiation factor 5A-2.

In this study, we aimed to study the effect of miR-33b in regulating sensitivity to daunorubicin (DNR) in acute myelocytic leukemia (AML). We used quantitative real-time polymerase chain reaction and Cell Counting Kit-8 assay to detect the level of miR-33b and cell viability. Cell apoptosis and the expression of eIF5A-2 and MCL-1 protein were detected by flow cytometry analysis and Western Blot analysis, respectively. MiR-33b mimic increased sensitivity of AML cells against DNR, while miR-33b inhibitor had the opposite effect. Furthermore, the results showed that the eIF5A-2 gene was a direct target of miR-33b, and miR-33b regulated eIF5A-2 mRNA and protein expression. Silencing of eIF5A-2 by RNA interference increased the sensitivity of AML cells against DNR. We also found that MCL-1 contributed to the regulation of DNR sensitivity, which was dependent on downregulation of eIF5A-2. Finally, knockdown of eIF5A-2 eliminated the effects of miRNA-33b mimic or inhibitor on DNR sensitivity. These findings indicate that miR-33b maybe as a new therapeutic target in AML cells.

MicroRNA-9 suppresses cancer proliferation and cell cycle progression in acute lymphoblastic leukemia with inverse association of neuropilin-1.

Acute lymphoblastic leukemia (ALL) is one of the most common and most malign childhood cancers. In this work, we investigated the expression and function of human mature microRNA-9 (miR-9) in ALL. In ALL in vitro cell lines and in situ clinical specimens, gene expression of miR-9 was tested by qRT-PCR. MiR-9 was overexpressed in CEM/C1 and Molt-3 cells to investigate its possible anti-cancer effects on ALL in vitro proliferation, cell-cycle progression, and in vivo explant growth. The possible downstream target of miR-9, neuropilin-1 (NRP1), was examined by dual-luciferase activity assay, qRT-PCR, and Western blot. NRP1was upregulated in miR-9-overexpressed CEM/C1 and Molt-3 cells to investigate the functional involvement of NRP1 in miR-9-mediated regulation on ALL in vitro proliferation and cell-cycle progression. MiR-9 was downregulated in ALL cell lines and leukemic T-cells of ALL patients. Lentivirus-mediated miR-9 overexpression inhibited ALL in vitro proliferation, cell-cycle progression, and in vivo explant growth. NRP1 was confirmed be the downstream target of miR-9, and inversely modulated by miR-9 in ALL. NRP1 upregulation reversed the anti-cancer regulations of miR-9 on ALL in vitro proliferation and cell-cycle progression. MiR-9 is downregulated in ALL. Overexpressing miR-9 may inhibit ALL development, possible through its downstream target of NRP1.

iTRAQ-Based Proteomic Analysis Reveals Potential Serum Biomarkers for Pediatric Non-Hodgkin's Lymphoma.

Non-Hodgkin's lymphoma (NHL) is the third most common malignant tumor among children. However, at initial NHL diagnosis, most cases are at an advanced stage because of nonspecific clinical manifestations and currently limited diagnostic methods. This study aimed to screen and verify potential serum biomarkers of pediatric NHL using isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analysis. Serum protein expression profiles from children with B-NHL (n=20) and T-NHL (n=20) and healthy controls (n=20) were detected by utilizing iTRAQ in combination with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) and analyzed by applying Ingenuity Pathway Analysis (IPA). The candidate biomarkers S100A8 and LRG1 were further validated by using enzyme-linked immunosorbent assays (ELISAs). Receiver operating characteristic (ROC) analysis based on ELISA data was used to evaluate diagnostic efficacy. In total, 534 proteins were identified twice using iTRAQ combined with 2D LC-MS/MS. Further analysis identified 79 and 73 differentially expressed proteins in B-NHL and T-NHL serum, respectively, compared with control serum according to our defined criteria; 34 proteins were overexpressed and 45 proteins underexpressed in B-NHL, whereas 45 proteins were overexpressed and 28 proteins underexpressed in T-NHL (p < 0.05). IPA demonstrated a variety of signaling pathways, including acute phase response signaling and liver X receptor/retinoid X receptor (LXR/RXR) activation, to be strongly associated with pediatric NHL. S100A8 and LRG1 were elevated in NHL patients compared to normal controls according to ELISA (p < 0.05), which was consistent with iTRAQ results. The areas under the ROC curves of S100A8, LRG1, and the combination of S100A8 and LRG1 were 0.873, 0.898 and 0.970, respectively. Our findings indicate that analysis of the serum proteome using iTRAQ combined with 2D LC-MS/MS is a feasible approach for biomarker discovery. Serum S100A8 and LRG1 are promising candidate biomarkers for pediatric NHL, and these differential proteins illustrate a novel pathogenesis and may be clinically helpful for NHL diagnosis in the future.

[Screening Serum Differential Proteins in Children with Acute Promyelocytic Leukemia Based on iTRAQ Technique].

OBJECTIVE: To screen the serum differentially expressed proteins of APL in children. METHODS: Serum protein expression profiles from 20 cases of normal healthy controls, and 20 cases of APL patients were detected by iTRAQ (isobaric tag for relative and absolute quantification)labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry(2DLC-MS/MS), and analyzed by bioinformatics software. S100A8, LRG1 and SPARC were validated by ELISA. ROC was built by SPSS 20.0 software. RESULTS: Analysis identified 83 differentially expressed proteins in APL serum compared with control according to our defined criteria, of which 33 proteins were up-regulated and 50 proteins were down-regulated (P<0.05)．IPA analysis revealed that these differentially expressed proteins were related to the function of Cellular Movement, Immune Cell Trafficking, Hematological System Development and Function, Cell-To-Cell Signaling and Interaction, Tissue Development, and involved in a variety of signalling Pathways, the most representative pathways including LXR/RXR Activation and Acute Phase Response Signaling. S100A8 and LRG1 were found to be elevated and SPARC was markedly down-regulated in serum of childhood APL when compared to the normal controls as examined by ELISA (P<0.05), which was consistent with the iTRAQ result. The overall predictive accuracy of each protein was reflected by the area under the ROC curve(AUC), S100A8,LRG1 and SPARC with ROC areas of 0.841,1.000 and 0.944 respectively. CONCLUSION: S100A8,LRG1 and SPARC may be serve as serum candidate biomarkers for pediatric APL.

[Expression and Clinical Significance of C/EBPα Gene in Elderly Multiple Myeloma].

OBJECTIVE: To investigate the expression of C/EBPα gene in elderly patients with multiple myeloma （MM） and its prognostic significance. METHODS: Sixty-nine olderly patients with multiple myeloma (MM) treated in our hospital from February 2015 to October 2017 were selected and enrolled in the MM group, 38 healthy persons received physical examination were selected and enrolled in the control group. The bone marrow of 2 groups was collected and the mononuclear cells were isolated.The mRNA expression level of C/EBPα gene in mononuclear cells was determined by RT-PCR, the Western blot was used to detect the protin expression level of PBMNC C/EBPα, and the protein level of C/EBPα in bone marrow was detected by immunohistochemistry. The correlations of C/EBPα gene expression with the clinical characteristics and survival time in MM patients were analyzed. RESULTS: The expression level of mRNA and protein of C/EBPα in MM patients was significantly lower than that in the control group (P<0.05). The expression level of C/EBPα gene significantly correlated with the ISS stage, CRP, Calcium, ß2-MG, LDH and the percentage of myeloma cells in MM patients (P<0.05). The expression of C/EBPα gene was not correlate with sex, age, immunoglobulin typing, Hb in MM patients (P>0.05).Immunohistochemical staining showed that the bone marrow samples of the control group were stained more deeply, and the staining intensity in bone marrow samples of MM patients with CR, PR and relapse was successively descended. The protein level of C/EBPα in CR patients with MM was significantly higher than that in PR and relapsed patients by Western blot (P<0.05). Kaplan-Meier survival analysis showed that OS and DFS in the patients with high expression of C/EBPα gene were higher than those in low expression group (P<0.05). Multivariate Cox regression analysis showed that CRP,ratio of myeloma cells and C/EBPα gene were independent factors affecting OS and PFS (P<0.05). CONCLUSION: The expression level of C/EBPα gene in MM patients is low that may stimulate the genesis of MM, and the expression of C/EBPα gene closely relates with the development of MM disease.

iTRAQ-based quantitative protein expression profiling of biomarkers in childhood B-cell and T-cell acute lymphoblastic leukemia.

PURPOSE: This study screened serum proteins to identify potential biomarkers for childhood B-cell and T-cell acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Serum collected from 20 newly diagnosed B-cell ALL, 20 T-cell ALL and 20 healthy children. The peptides from these samples were subjected to iTRAQ. Differentially expressed proteins (DEPs) were further validated by ELISA in 24 B-ALL, 24 T-ALL, and 24 healthy children. RESULTS: Bioinformatics analysis revealed several pathways, including atherosclerosis signaling, interleukin signaling and production in macrophages and clathrin-mediated endocytosis signaling, that were closely related to childhood T-cell ALL. Furthermore, four selected proteins, namely LRG1, S100A8, SPARC and sL-selectin, were verified by ELISA. These results were consistent with the results of the proteomics analysis. CONCLUSION: Serum S100A8 may serve as new diagnostic biomarkers in childhood B-cell ALL and T-cell ALL.

miR-9 Enhances the Chemosensitivity of AML Cells to Daunorubicin by Targeting the EIF5A2/MCL-1 Axis.

Daunorubicin (Dnr) is at the forefront of acute myeloid leukemia (AML) therapy, but drug resistance poses a major threat to treatment success. MicroRNA (miR)-9 has been shown to have a pivotal role in AML development. However, little is known about the role of miR-9 in Dnr resistance in AML. We explored the potential role of miR-9 in Dnr resistance in AML cells and its mechanism of action. AML cell lines with high half-maximal inhibitory concentration to Dnr in vivo had significantly low miR-9 expression. miR-9 overexpresssion sensitized AML cells to Dnr, inhibited cell proliferation, and enhanced the ability of Dnr to induce apoptosis; miR-9 knockdown had the opposite effects. Mechanistic studies demonstrated that eukaryotic translation initiation factor 5A-2 (EIF5A2) was a putative target of miR-9, which was inversely correlated with the expression and role of miR-9 in AML cells. miR-9 improved the anti-tumor effects of Dnr by inhibiting myeloid cell leukemia-1 (MCL-1) expression, which was dependent on downregulation of EIF5A2 expression. These results suggest that miR-9 has an essential role in Dnr resistance in AML cells through inhibition of the EIF5A2/MCL-1 axis in AML cells. Our data highlight the potential application of miR-9 in chemotherapy for AML patients.

[ITRAQ Coupled with 2DLC-MS/MS to Screen and Identify Speci-fic Bio-markers of T-Cell Acute Lymphoblastic Leukemia].

OBJECTIVE: To screen and identify potential biomarkers specific for T-cell acute lymphoblastic leukemia (T-ALL). METHODS: Sera were collected from 20 newly diagnosed B-cell acute lymphoblastic leukemia (B-ALL) patients and 20 T-ALL patients. Proteins were extracted, purified and digested with trypsin. All specimens were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) in a data-dependent mode. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression of serum soluble L-selectin (sL-selectin). RESULTS: A total of 468 proteins were identified from distinct peptides. Compared with B-ALL group, 31 proteins were significantly differentially up-regulated while 7 proteins were significantly down-regulated in T-ALL group, sL-selectin was the higher up-regulated in these differential expression proteins. The overexpression of sL-selectin in T-ALL was verified by ELISA. CONCLUSION: There are the differentially expressed proteins between T-ALL and B-ALL, and the sL-selectin is specific for T-ALL, which can not only become a new biomarker for the diagnosis and prognosis of T-ALL, but also can be used as a potential target for therapy of this leukemia.

The growth-inhibitory and apoptosis-inducing effect of deferoxamine combined with arsenic trioxide on HL-60 xenografts in nude mice.

OBJECTIVE: The aim of this study is to investigate the growth-inhibitory and apoptosis-inducing effect of deferoxamine (DFO) combined with arsenic trioxide (ATO) on the human HL-60 xenografts in nude mice and its mechanism. METHOD: The highly tumorigenic leukemia cell line HL-60 cells were inoculated subcutaneously into nude mice to establish a human leukemia xenograft model. The HL-60 xenograft nude mice models were randomly divided into four groups: control (Normal saline, NS), 50mg/kg DFO, 3mg/kg ATO, the combined treatment (50mg/kg DFO+1.5mg/kg ATO) once HL-60 cells were inoculated. Tumor sizes, growth curves, inhibitory rates, cell apoptosis, and the expression of apoptosis related markers were measured to evaluate the tumor growth. RESULTS: Xenografted tumors were observed in all nude mice since the 5th day of inoculation. The inhibitory rates of tumor weight were 2.67%, 10.69%, and 25.57% in DFO, ATO and combination therapy groups, respectively. The combination of DFO with ATO induces significantly more tumor cell apoptosis than either agent alone (p<0.05). The expression of NF-κBp65 and survivin proteins decreased significantly while the expression of Caspase-3 and Bax increased in the combination therapy group (p<0.05). Double immunofluorescence for Caspase-3 and NFκBp65 demonstrated an inverse relationship between Caspase-3-positive areas and NFκBp65-positive areas, as well as the co-localization of Bax and survivin in xenografted tumor cells. CONCLUSIONS: Combination of DFO and ATO has synergistic effects on tumor growth inhibition and apoptosis-inducing in vivo with no significant side effects. The DFO and ATO can up-regulate the expression of Caspase-3 and Bax, and down-regulate the expression of NF-κBp65 and survivin, especially for their combination.

[The inhibition effect of DFO alone and in combination with ATO on xenograft tumor growth of HL-60 cells in nude mice and its possible mechanism].

OBJECTIVE: To investigate the effect of deferoxamine (DFO) and DFO in combination with arsenic trioxide (ATO) on inhibition of HL-60 cells xenograft tumor growth in nude mice and its mechanism. METHODS: Xenograft tumor model of HL-60 cell line in nude mice was established by inoculating HL-60 cells subcutaneously into nude mice. The tumor-bearing mice were randomly divided into four groups: 50 mg/kg DFO group (group I), 3 mg/kg ATO group (group II), combination group (50 mg/kg DFO + 1.5 mg/kg ATO (group III) and normal saline control group. The drugs were administered intraperitoneally from the day of inoculation (once a day for 10 days). The inhibitory effects on the tumor growth were compared. NF-κBp65 expression levels of the tumors were detected by immunohistochemistry (24h after the last administration). RESULTS: (1) Tumors growth could be observed in all of the nude mice on day 7 to day 8 after inoculation, 0.5 - 1.0 cm in diameter, and then grew rapidly; (2) Tumor weight of control group, group I, group II and group III were (2.62 ± 0.54) g, (2.55 ± 0.82) g, (2.34 ± 0.79) g and (1.95 ± 0.39) g respectively, and the growth inhibition rates in group I, group II and group III were 2.67%, 10.69% and 25.57% respectively. Both DFO alone and in combination with ATO could inhibit the growth of transplanted tumors, and the combination group exhibited more effects, with no vital organ damages in the tumor-bearing mice. (3) There was significant difference in mean value of NF-κBp65 expression among the three experimental groups (P < 0.05), with a descending order of control group > group II, > group I > group III. CONCLUSION: (1) Both DFO and ATO have antitumor activities on tumor-bearing mice, and their combination has an obvious and significant effect. (2) DFO combined with ATO, is well tolerated with no significant adverse effects in the nude mice. (3) Both DFO and ATO can downregulate NF-κBp65 expression of transplanted tumors, especially for their combination.