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Objective To explore the effect of microRNA( miR) -193a on the apoptosis of mouse podocytes in diabetic nephropathy (DN) and its mechanism. Methods The DN model was replicated by culturing podocytes with high glucose in vitro and intraperitoneal injection of streptozocin (STZ) in mice in vivo. The cells or 60 mice were randomly divided into normal control (NC) group, model control group, and miR-NC inhibitor group, miR-193a inhibitor group, miR-NC mimic group and miR-193a mimic group. Flow cytometry, immunohistochemistry, TUNEL, Real-time PCR, Western blotting were used to examine the apoptosis of DN mice and mouse podocytes. Results The expression of Nephrin and Podocin in podocytes was weakened in DN mice and renal podocytes induced by high glucose, the apoptotic rate increased significantly, miR-193a was highly expressed, the levels of cleaved-Caspase-3 and Bax protein increased significantly, the level of Bcl-2 protein decreased significantly, and miR-193a inhibitor could improve this process. Wilms' tumor gene 1 ( WT1 ) mRNA and protein expression levels were significantly reduced in DN mice and podocytes cultured with high glucose. WT1 protein expression increased significantly after miR-193a inhibitor intervention, and WT1 protein expression was significantly reduced after miR-193a mimic transfection. Up-regulating WT1 could reduce the effect of miR- 193a on the apoptosis of mouse podocytes induced by high glucose. The dual luciferase reporter experiment confirmed the targeting relationship between miR-193a and WT1. Conclusion MiR-193a down-regulates the expression of WT1 and promotes apoptosis of DN podocytes.
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Objective@#To probe the potential utility of Wilms tumor 1 (WT1) as a marker of minimal residual disease (MRD) in acute myeloid leukemia (AML) to estimate the relapse-predicting cut-off value.@*Methods@#Quantitative assessment of bone marrow WT1 mRNA level was preformed using real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) assay. The expression levels of WT1 dynamically measured with RQ-RT-PCR were retrospectively analyzed in 121 AML cases (not including acute promyelocytic leukemia) achieving complete remission (CR) after induction therapy followed by consolidation therapy. By comparing WT1 levels of patients with different post-therapy outcomes, the investigators used the receiver operating characteristic (ROC) curve to determine WT1 threshold so as to predict their clinical relapses. Then prognoses and the significance of intervention were analyzed between WT1 positive and negative patients according to the cut-off value of WT1.@*Results@#According to ROC curve, WT1 level higher than 2.98% predicted the possibility of relapse. For simplicity and clinical application, 3.00% was used as the cut-off value of WT1 level for relapse. WT1 levels in 41 patients at diagnosis were detected, meanwhile 3 patients whose WT1 levels at diagnosis below 3.00% were excluded, then the median WT1 level of the rest 38 patients at diagnosis was 44.09% (range 7.19%-188.06%) . The median WT1 level in remission was 0.48% (352 samples, range 0-8.41%) . The median WT1 level at diagnosis was higher than that in remission. Excluding the 3 patients with WT1 level at diagnosis under 3.00%, the relapse rate of WT1 positive group (>3.00% during consolidation phase and follow-up) and WT1 negative group (≤3.00%) was 70.0% (14/20) and 12.2% (12/98) respectively (P<0.001) . The median time from WT1 positivity to clinical relapse was 58 days.@*Conclusions@#WT1 expression level above 3.00% was associated with markedly high risk of relapse, which could be as a useful marker for monitoring MRD following consolidation therapy.
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Objective To investigate the feasibility of Wilms’tumor gene 1 (WT1)-specific CD8+T cells from periph?eral blood for the treatment of breast cancer by detecting the killing activity of WT1 specific CD8+T cells on breast cancer cells. Methods Flow cytometry was used to detect WT1-specific CD8+T cells in the peripheral blood of 20 samples from HLA-A2 seropositive healthy donors, which were isolated by WT1/MHC streptamer magnetic beads and cultured. The func?tion of WT1-specific CD8+ T cells were analysis by cytotoxicity assay. Results Twelve of 20 healthy donors had naive WT1-specific CD8+T-cell frequencies of>0.5%, and 4 of 20 even>1.0%of all CD8+T cells. After positive selection by magnetic cell separation, a purity of up to 80%can be achieved. WT1 specific CD8+T cells can specifically kill breast can?cer cell line with WT1 polypeptide. Conclusion WT1 specific CD8+T cells can be detected in peripheral blood of healthy volunteers. WT1 specific CD8+T cells have killing effect on breast cancer cells, suggesting the feasibility of adoptive immu?notherapy for breast cancer.
ABSTRACT
Background: Wilms’ tumor (WT1) gene expression has been reported in the majority of acute leukemia patients at diagnosis and has been evaluated as a prognostic and minimal residual disease (MRD) marker but its role is still controversial. Methods: Real-time quantitative polymerase chain reaction was used on bone marrow samples from 100 newly diagnosed adults and pediatrics acute leukemia patients (50 AML and 50 ALL patients). WT1 expression were examined at diagnosis and at the end of induction. Results: WT1 was expressed in (14%) ALL and in (36%) AML patients. We found no statistically significant impact of WT1 expression at diagnosis on response p= 0.054, 0.057, DFS (P = 0.591, 0.858), or OS (p= 0.339, p= 0.331) in ALL and AML patients respectively. Persistence of WT1 expressions at the end of induction didn't show any effect on relapse rate in AML however, it showed significant results in ALL p=0.045. Conclusion: Our results suggest that WT1 expression in patients with acute leukemia doesn't have any implication on response or survival however, significant association was found in predicting ALL relapse but the small sample size should be considered.