Expression And Function Of Ik6 In Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common one of the childhood hematologic malignancies. Despite the cure rate has been as high as 80% owing to the constant improvement of chemotherapy, some children have poor outcome including chemotherapy resistance and relapse. Clinical studies have shown that recurrence arises not only in high-risk groups but also in low-risk or medium risk group, which is no doubt a challenge to the current criteria of rick classifications. Therefore, it is very important that continuously identify new prognostic factors, improve the classification standard and give individual chemotherapy to improve the prognosis of ALL children.Dominant negative Ikaros isoform 6, Ik6, with a deletion of coding exons 3 through 6, is the most common form of IKZF1 deletion. It is able to suppress the function of DNA-binding isoforms of Ikaros and is the strongest transcriptional repressor in Ikaros family. It has shown that Ik6 appears high frequently in BCR/ABL-positive ALL and is associated with poor prognosis of ALL including increased proportion of primitive cells, abnormal cytogenetics, chemotherapy resistance, difficulty to achieve complete remission and so on. Ik6 is also involved in the pathway of cell apoptosis. It can protect cells from apoptosis caused by external stimuli. Accordingly, we hypothesized that Ik6 may play a role in the progress of acute lymphoblastic leukemia.It is a preferred method that introducing a foreign gene into suspension cells by lentiviral vector because of the high transduction efficiency, lasting stability of gene expression and low cytotoxicity. Lentiviral vector-mediated gene overexpression or silence is a classic method to study the function of new genes.In this study, we examined the expression of Ik6 in children with ALL and analyzed its relationship with clinical features. We still studied the effect of Ik6 on the growth, invasion and chemosensitivity of ALL cell line Nalm-6 by lentiviral vector-mediated overexpression to further understand the role and significance of Ik6 gene in ALLPart IExpression and clinical significance of Ik6 in pediatric patients with acute lymphoblastic leukemiaObjective:The purpose of this study is to investigate the expression and clinical significance of IKZF1, especially Ik6 in pediatric patients with ALL, to initially explore the role of Ik6 in the process of ALL.Methods:We analyzed the mRNA expression of Ik6 in 88 children with previously untreated ALL by nested RT-PCR. Sequencing analysis of samples was performed to confirm correct isoforms definition. Fusion genes expression was measured by multiplex RT-PCR. Analyze the relation between Ik6 expression and clinical characteristics.Results:Among 88 cases, the expression of Ik6 was observed in 11.43%(8 /70) cases of B-lineage ALL and 5.56%(1 /18) cases of T-lineage ALL patients. In Ik6+ B-lineage ALL patients, three carried BCR/ABL1 fusion gene and one expressed HOX11. Ik6 was independent of age, WBC count at diagnosis, risk group and expression of the fusion genes currently measured in China (P>0.05) except BCR/ABL1 (P<0.01). It was strongly associated with elevated levels of minimal residual disease detected after induction chemotherapy (P<0.01).Conclusion:Ik6 is positively expressed in some ALL patients. It may be evaluated as a high-risk factor at diagnosis to identify patients with high-risk leukemia.PartⅡConstruction and identification of Ik6 expression lentivirus vectorsObjective:To construct the Ik6 expression lentivirus vector pHR-CSIGW-Ik6 and obtain the virus particle. Nalm-6 cells were transfected and expressed Ik6 protein for further study on the role of Ik6 in ALLMethods:The Ik6 gene was amplified from a human ALL cell line Sup-B15 by RT-PCR and inserted into plasmid pEASY-T to construct the expression vector pEASY -Ik6. The expression vector named pHR-CSIGW-Ik6 was constructed by linking the Ik6 and pHR-SIN CSIGW both cut by Xho I and BamH. Ik6 lentivirus expressive vectors were packaged by co-transfecting 293T with 3 plasmid system and the titre of lentivirus particle was detected by titration dilution. The Ik6 lentivirus particle was infected into Nalm-6 cells and the Ik6 expression was detected by flow cytometry, real-time quantitive PCR and Western Blot on the level of mRNA and protein.Results:The lentivirus vector expressing Ik6 was constructed successfully by sequencing. The titre of lentivirus particle was 2×109 TU/ml and was enough for the following experiments. When multiple of infection (MOI) was 50, the infection efficacy of Nalm-6 cells can reach to 80%. The result of real-time quantitive PCR and Western Blot showed the significant up-regulation of Ik6 mRNA and protein expression in Nalm-6 after transfection (P<0.01).Conclusion:The lentivirus expressive vectors expressing Ik6 were successfully constructed and the expression of Ik6 was effectively and stable in Nalm-6-Ik6 cells. It is the experimental base for research on the role of Ik6 in leukemia.PartⅢEffect of Ik6 overexpression on the biological characteristics of acute Iymphoblastic leukemia cell line Nalm-6Objective:To investigate the effect of Ik6 overexpression on cell proliferation, cell apoptosis and chemosensitivity of Nalm-6 cell line.Methods:The proliferation and colony formation of Nalm-6, Nalm-6-mock and Nalm-6-Ik6 cells were detected by CCK-8 and colony formation assay. The sensitivity to vincristine (VCR), daunorubicin (DNR), L-asparaginase (L-Asp) and dexamethasone (Dex) was detected by CCK-8. The cell apoptosis and Ik6 expression after treatment with drugs were detected by flow cytometry and real-time quantitive PCR and Western Blot.Results:1. The advanced effect on cell proliferation (P<0.05) and colony formation (P<0.01) were showed after Ik6 overexpression by lentiviral transfection.2. The resistance to VCR, DNR and L-Asp was enhanced by Ik6 overexpression. Compared to Nalm-6-mock cells, the 24h IC5o values of Nalm-6-Ik6 were 34.94 ng/ml (vs.20.51 ng/ml) to VCR,12.25 ng/ml (vs.1.89 ng/ml) to DNR and 2.37 IU/ml (vs.0.36 IU/ml) to L-Asp (P <0.05).3. The apoptosis of Nalm-6-mock cells increased after treatment with VCR, DNR and L-Asp (P<0.05), but Nalm-6-Ik6 did not (P>0.05).4. The three drugs can decrease the mRNA and protein expression of Ik6 in Nalm-6-Ik6 cells (P<0.01).Conclusion:Ik6 overexpression by lentivirus-mediated can improve the growth and chemoresistance of Nalm-6 cells. The mechanism of resistance may be related to apoptosis pathway and the regulation of Ik6 expression.Part IVEffect of Ik6 overexpression on invasiveness of acute lymphoblastic leukemiaObjective:To explore the effect of Ik6 overexpression on angiogenesis and invasiveness of acute lymphoblastic leukemia.Methods:The mRNA expression of angiogenic genes VEGF,Flt-1,PIGF,Ang-1 and Ang-2 in 20 cases of ALL patients was detected by real-time quantitive PCR and then the relation between Ik6 and angiogenesis was analyzed. The angiogenic genes expression and invasiveness after of Nalm-6-mock and Nalm-6-Ik6 cells detected by real-time quantitive PCR and transwell assay.Results:The expression of angiogenic genes were higher in ALL group (n=20) than that in normal control (n=10) (P<0.01). There was no difference between Ik6+ ALL group (n=8) and Ik6- ALL group (n=12) (P>0.05). Though the expression was higher in Ik6 only ALL group (n=5) than that in the other (n=15), there was no statistical significance (P >0.05). Spearman analysis showed that there was no relation between Ik6 and angiogenic genes. What's more, Nalm-6-Ik6 cells showed no difference in angiogenic genes expression, migration and invasiveness compared with Nalm-6-mock cells (P>0.05).Conclusion:Ik6 may be not involved in the angiogenesis and invasiveness of acute lymphoblastic leukemia.