| Background:Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, accounting for ～25% of cancers in children<15 years of age. The incidence of childhood leukemia has increased yearly since the past few years, and 30/1,000,000-40/1,000,000 children<10 years of age are diagnosed with ALL annually in China. Most childhood ALLs arise from a B-cell precursor (B-ALL) (-85%), whereas the remaining～15% of cases are accounted for by T-cell acute lymphoblastic leukemia. Survival outcomes have greatly improved with combination chemotherapy and treatment intensification; however, relapsed patients often develop resistance to chemotherapy and experience very poor prognosis, which has prompted us to further explore the molecular mechanisms underlying ALL.B-ALL is a malignancy characterised by progressive accumulation of immature clonal B-cell precursors in the bone marrow. Numerous transcriptional regulators have critical functions in this malignant process and dysregulated expression of genes whose products affect the development and maturation of lymphoid cells may be potentially important contributors to the pathogenesis of B-ALL. The IKAROS family of proteins is a prime example of such factors which encode zinc-finger DNA-binding proteins. AIOLOS, the second IKAROS family member identified, is an important regulator of B-cell differentiation, proliferation and maturation to an effector state. Disruption of AIOLOS increases the number of pre-B and immature B cells, and significantly reduces the number of circulating B cells. In addition, Aiolos null mutation in mice causes B-cell hyperproliferation, elevates serum antibody levels, promotes auto-antibody formation and triggers lymphoma development, thereby demonstrating tumour suppressor function. Deregulated AIOLOS expression has been associated with adult B-ALL and chronic lymphocytic leukaemia in human patients. In addition, aberrant AIOLOS expression levels have been reported in cases of lymphoma. However, the function of AIOLOS in childhood B-ALL is not fully understood, and the molecular mechanisms through which AIOLOS exerts its effect on leukemia cells remain to be determined.Objective:1. To initially explore the role of AIOLOS in the process of ALL by investigating the expression of AIOLOS in pediatric patients with ALL.2. To construct the AIOLOS expression lentivirus vector and transfect it into Nalm-6 cells for further research.3. To study the effects of AIOLOS overexpression on biological behaviors of leukemia cells.4. To illustrate the mechanisms through which AIOLOS exerts its effect on biological behaviors of leukemia cells.Methods:1. Expression of AIOLOS in B-ALL samples and leukemia cell lines1.1 Bone marrow samples from 36 B-ALL patients and 6 normal donors were obtained after informed consent. Mononuclear cells (MNCs) were isolated from bone marrow aspirates by density-gradient centrifugation using Ficoll-Paque Plus and then B lymphocytes were sorted using CD 19 magnetic beads. Expression of AIOLOS mRNA was examined using RT-PCR and real-time quantitative PCR.1.2 Leukemia cell lines Ball-1、Nalm-6、Jurkat、Molt-4 and K562 were selected as research objects, and B lymphocytes of normal donors were selected as the control. The mRNA and protein levels of AIOLOS were examined using real-time quantitative PCR and Western blotting, respectively.2. Construction and identification of AIOLOS overexpression lentivirus vectors2.1 The AIOLOS gene was amplified from MNCs of normal donors by RT-PCR. The expression vector named pWPT-PURO-AIOLOS was constructed by linking the AIOLOS gene and pWPT-PURO-GFP both cut by EcoR I and Mlu I. AIOLOS lentivirus expressive vectors were packaged by co-transfecting 293T cells with 4-plasmid system and the titer of lentivirus particle was detected by titration dilution.2.2 The AIOLOS lentivirus particle was infected into Nalm-6 cells and the expression of AIOLOS was detected by flow cytometry, real-time quantitive PCR and Western blotting.3. Effect of AIOLOS overexpression on the biological characteristics of Nalm-6 cells3.1 Nalm-6 cells were divided into three groups:the experimental group (Nalm-6 cells transfected with pWPT-PURO-AIOLOS; Nalm-6-AIOLOS), the negative control group (Nalm-6 cells transfected with pWPT-PURO-GFP; Nalm-6-Mock) and the blank control group (Nalm-6 cells with no treatment; Nalm-6-Control).3.2 Cell proliferation assay:the absorbance values of Nalm-6 cells were detected using CCK-8 assay from day 7 to day 11 after transfection, and growth curves were drawn.3.3 Cell cycle analysis:cell cycle distribution of Nalm-6 cells was analyzed by flow cytometry using propidium iodide (PI) staining.3.4 Cell apoptosis assay:cell apoptosis was detected using an Annexin V and PI Apoptosis Detection Kit by flow cytometry.3.5 Sensitivity of Nalm-6 cells to chemotherapy drugs:Nalm-6 cells of the three groups were treated with serial dilutions of VCR or DNR for up to 24 hours. CCK-8 assay was used to detect live cells and IC50 values were calculated after correction for background absorbance.3.6 Cell invasiveness assay:Transwell assay was used to analyze the invasiveness of Nalm-6 cells. The capacity of the in vitro angiogenesis of HS-5, a bone marrow stromal cell line, was examined by Matrigel tube formation assay after co-cultivation with Nalm-6 cells in different groups for 48 h.4. The influence mechanism of AIOLOS overexpression on biology behaviors in Nalm-6 cells4.1 The expression of apoptosis and cell cycle-related genes:real-time quantitive RT-PCR was used to detect the mRNA levels of apoptosis and cell cycle-related genes, including BCL-2, BAX, P27, CyclinD3 and C-MYC, while the protein levels of BCL-2, P27 and CyclinD3 were examined by Western blotting.4.2 The expression of angiogenic genes:real-time quantitive RT-PCR was used to detect the mRNA levels of angiogenic genes, including VEGF, PLGF and ANG1.4.3 mRNA microarrays and functional analysis of differentially expressed genes:the Whole Human Genome Oligo Microarray was performed to identify differentially expressed genes between Nalm-6-AIOLOS and Nalm-6-Mock. Gene Ontology (GO) functional categoriesand Kyoto Encyclopedia of Genes and Genomes (KEGG) functional pathways were searched by using DAVID (The Database for Annotation, Visualization and Integrated Discovery) Bioinformatics Resources for statistically enriched clusters/groups among the differentially expressed genes identified in this study.4.4 PI3K/Akt signaling pathway:The activation of PI3K/Akt signaling pathway was detected using a FlowCellect PI3K/MAPK Dual Pathway Activation Detection Kit by flow cytometry.4.5 The effect of Akt inhibitor on proliferation, apoptosis and cell cycle distribution of Nalm-6-AIOLOS:Nalm-6 cells were divided into four groups:Nalm-6-AIOLOS, Nalm-6-Mock, Nalm-6-Control and Nalm-6-AIOLOS+Akt inhibitor. Protein levels of Akt and phosphorylated Akt (pAkt) were detected by western blotting. Cell numbers were counted to detect cell proliferation and growth curves were drawn. Cells cycle distribution was analyzed by flow cytometry using PI staining. Cell apoptosis was examined using an Annexin V and PI Apoptosis Detection Kit by flow cytometer.Results:1. Expression of AIOLOS in B-ALL samples and leukemia cell lines1.1 Analysis of healthy donors revealed the presence of 3 AIOLOS variants corresponded to AIO1, AIO2, and AIO5. No major differences in the distribution of AIOLOS isoforms were detected between healthy donors and B-ALL patients. In all cases, AIO1 was the most abundant mRNA species, although varied between different samples. Considering the AIOLOS transcripts expressed by B cells of healthy donors and patients, we observed a significant decrease of AIOLOS transcripts in B-ALL samples compared with healthy donors(P< 0.05). The expression level of AIOLOS was independent of sex, immunophenotype and risk group (P>0.05).1.2 The mRNA level of AIOLOS in Nalm-6 cells was lower than that in normal B lymphocyte (P<0.05). Accordingly, a significant decrease of AIOLOS protein was also observed in Nalm-6 cells(P<0.05).2. Construction and identification of AIOLOS expression lentivirus vectorsThe lentivirus vector expressing AIOLOS was constructed successfully by sequencing. The titer of lentivirus particle was 3.23×108 TU/ml and was sufficient for further research. When multiple of infection (MOI) was 100, the infection efficacy of Nalm-6 cells attained 90% above. The results of real-time quantitive PCR and western blotting showed the significant up-regulation of AIOLOS mRNA and protein expression in Nalm-6 cells after transfection (P<.0.05).3. Effect of AIOLOS overexpression on the biological characteristics of Nalm-6 cells3.1 Cell proliferation assay showed that the growth of Nalm-6-AIOLOS was significantly slower than Nalm-6-Control and Nalm-6-Mock(P<0.05).3.2 The percentage of Nalm-6 cells in G0/G1 phase increased from 70.4%±2.39% (Nalm-6-Control) to 84.1%±2.18%(Nalm-6-AIOLOS) (P<0.05), and the S-phase cells decreased from 20.3%±0.94%(Nalm-6-Control) to 11.7%±0.78% (Nalm-6-AIOLOS) (P<0.05). The difference between Nalm-6-AIOLOS and Nalm-6-Control in G2/M phase was significant (2.2%±0.56% vs.7.3%±0.81%; P<0.05). No significant difference between Nalm-6-Control and Nalm-6-Mock was observed (P>0.05).3.3 Total apoptotic cells were significantly decreased in Nalm-6-AIOLOS (10.55%±0.67%) compared with those in the Nalm-6-Mock (16.87%±0.52%) or Nalm-6-Control (18.13%±0.51%) (P<0.05). In particular, the difference between Nalm-6-AIOLOS and Nalm-6-Control in the percentage of late apoptotic cells was significant (2.83%±0.67% vs.7.86%±0.52%; P<0.05).3.4 The resistance to VCR and DNR was similar among Nalm-6 cells of the three groups. Compared to Nalm-6-Control, the 24h IC50 values of Nalm-6-AIOLOS were 19.91ng/ml (vs.18.44 ng/ml; P>0.05) to VCR and 3.62 ng/ml (vs.3.22 ng/ml; P>0.05) to DNR, respectively.3.5 Transwell assay showed no significant difference between the absorbance values of Nalm-6-AIOLOS and Nalm-6-Control at 450 nm(0.11±0.05 vs.0.14±0.06; P> 0.05). In vitro angiogenesis analysis demonstrated more network formation in HS-5 cells co-cultured with Nalm-6-AIOLOS than in other two group cells.4. The influence mechanism of AIOLOS overexpression on biology behaviors in Nalm-6 cells4.1 Real-time RT-PCR showed that the levels of pro-apoptotic gene BAX (P<0.05), and cell-cycle-associated gene CyclinD3 (P<0.05), were markedly reduced in Nalm-6-AIOLOS compared with that in the control groups, while the expression level of anti-apoptotic gene BCL-2 (P<0.05) was increased. However, minimal change was observed in the expression of C-MYC (P>0.05) and P27 (P>0.05). Decreased expressions of CyclinD3 were observed by western blot analysis (P<0.05). No significant change was observed in the expression of BCL-2 and C-MYC protein (P>0.05).4.2 The expression of angiogenic genes were higher in Nalm-6-AIOLOS group than that in Nalm-6-Control group (P<0.05).4.3 Of all the mRNAs tested in microarray analysis,102 mRNAs were significantly up-regulated in B-ALL, while 176mRNAs were down-regulated. GO categories of differentiallyexpressed genes were mainly related tobiological regulation, metabolic process, signal transduction, binding, signal transducer activity, etc. KEGG functional pathways associated with the differentially expressed genes weremainly related to Pancreatic secretion, Neuroactive ligand-receptor interaction, Small cell lung cancer, Melanoma, etc.4.4 Flow cytometry revealed a significant increase of phosphorylated Akt positive cells in Nalm-6-AIOLOS compared to the other two group cells (P<0.05). Western blot analysis revealed that Akt protein expression did not change considerably, whereas the levels of phosphorylated Akt were significantly higher in Nalm-6-AIOLOS than in the control groups. In addition, Akti-1/2 at the 5 μM concentration can effectively inhibit Akt phosphorylation.4.5 Akt inhibitor can effectively increase the percentage of apoptotic cells in Nalm-6-AIOLOS. However, in cell proliferation experiments, Akt inhibitor treatment did not improve the growth of Nalm-6-AIOLOS. Similarly, Akt inhibitor did not affect the cell cycle of Nalm-6-AIOLOS.Conclusion:1. The expression level of AIOLOS gene decreased in B lymphocytes of B-ALL patients;2. Lentivirus-mediated overexpression of AIOLOS in Nalm-6 cells could inhibit cell proliferation, suppress cell apoptosis and arrest the cell cycle at the G0/G1 phase in vitro;3. AIOLOS limited cell expansion via CyclinD3-dependent pathways and improved cell survival via PI3K/Akt-dependent processes in Nalm-6 cells. |
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