| Objective: Neuroblastoma(NB)is the most common extracranial malignant solid tumor in pediatric tumors.The incidence rate of NB is only 15/million,but it accounts for 15% of the pediatric tumor-related deaths primarily originates from the sympathetic nervous system.More than half of NB occurs in the adrenal gland,other common sites include the neck,chest,and pelvic cavity.Conventional anti-tumor treatment and symptomatic treatment are helpful to favorable prognosis for low-risk and medium-risk NB patients,the 5-year overall survival rate is higher than 90%.Nevertheless,even though being treated by comprehensive combined methods including chemotherapy,surgical resection,radiotherapy,autologous stem cell transplantation,and immunotherapy,the prognosis for high-risk NB patients is still poor,the five-year overall survival rate is less than 40%.Chemotherapy resistance is the difficulty in the treatment of NB for the high-risk group,and also being the major account for the final treatment failures.Therefore,it is particularly significant to detect new therapeutic drugs and targets which will have influence on the chemo sensitivity of NB and subsequently improve the prognosis of patients with NB.Previous studies have indicated that Perifosine,a small molecule inhibitor of Akt,can significantly inhibit the survival,migration and invasion of NB cells in vitro.While in NB Xenograft model,in addition to inhibiting the growth of tumor notably,Perifosine can prolong the survival time as a single drug.Moreover,in a Phase I Trial of Perifosine in patients with relapsed and refractory NB,it was found to have a low toxicity and be well tolerated as a single agent,and demonstrated favorable efficacy in the treatment of NB.A number of studies have indicated that Perifosine can also inhibit MAPK,Wnt and other signaling pathways,showing satisfactory anti-tumor effects on breast cancer,lung cancer,colorectal cancer,prostate cancer and multiple myeloma.Proteomic analysis and comprehensive analysis revealed that the expression of Spinocerebellar ataxia type 3(Ataxin-3,ATX3,ATXN3)protein was remarkably increased in NB cells by the treatment of Perifosine.As a member of spinocerebellar ataxia protein family,and furthermore one of the five major members of deubiquitylates(DUBs),ATXN3 is mainly distributed in the nucleus,cytoplasm and mitochondria,plays a role in maintenance of protein homeostasis,gene transcription,regulation of cytoskeleton stabilization,myogenesis and degradation of misfolded proteins chaperone-substrate complexes.Since the role of ATXN3 in NB has not been explored,in this study,we will explore the role of ATXN3 in the cell death induced by AKT inhibitor(Perifosine or MK-2206)or chemotherapy drugs(etoposide or cisplatin)in NB cells.Materials and methods: In this study,SK-N-AS and SK-N-BE2 cells were used.NCBI-GEO,TCGA,GTEX and R2 databases were used to analyze the relationship between the expression of ATXN3 and the clinicopathological characters and prognosis of neuroblastoma patients.Western blot was used to detect the protein expression levels of ATXN3 and BCL-2 family.We aim to investigate the effect of ATXN3 on neuroblastoma cells treated with small molecule Akt inhibitors(Perifosine and MK-2206)and chemotherapeutic drugs(etoposide and cisplatin)by transfecting small interfering RNA(si RNA)or overexpression plasmid to up-regulate or down-regulate the expression level of ATXN3.After transfection with ATXN3 si RNA,small molecule Akt inhibitors(Perifosine and MK-2206)and chemotherapy drugs(etoposide and cisplatin)were given to NB cells for 48 hours.Incucyte Zoom system was used to observe NB cell confluency curve from each group at different time points,indirectly reflect the proliferation of NB cells.CCK8 technology was used to detect the absorbance of cells in each treatment group,observe the cell viability and detect the proliferation of NB cells.Annexin V / PI staining was used to measure the apoptosis of NB cells in each treatment group by flow cytometry.The data were analyzed by Graph Pad prism 8 software.Results:1.The expression level of ATXN3 was negatively correlated with the amplification status of MYCN,risk grouping and tumor stage in NB patients(P<0.01),and ATXN3 was an independent prognostic factor of neuroblastoma patients(risk ratio: 0.74,95%confidence interval: 0.57-0.86,P = 0.0383).2.Down-regulation of ATXN3 enhances the sensitivity of NB to small molecule Akt inhibitors(Perifosine and MK-2206).Downregulation of ATXN3 with Perifosine or MK-2206 treated for 48 hours,by the analyzing the confluence in Incucyte Zoom system,we found that,with Perifosine or MK-2206 treated,the cell confluency curve of silencing ATXN3 was significantly lower than that of Ctrl si RNA group.After analyze the cell confluency of each group,we found that: with Perifosine or MK-2206 treated,the cell confluency of ATXN3 si RNA#1 and ATXN3 si RNA#2 was significantly lower than Ctrl si RNA group.For Perifosine treated,in SK-N-AS cells,ATXN3 si RNA#1 and ATXN3 si RNA#2 vs ATXN3 Ctrl si RNA: 51.6% and 30.6% vs 77.5%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA vs Ctrl si RNA: 38.1% vs 60.8%,P<0.01.For MK-2206 treated,in SK-N-AS cells,ATXN3 si RNA#1 and ATXN3 si RNA#2 vs Ctrl si RNA: 48.9% and 42.6% vs 65.3%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA vs Ctrl si RNA: 46.9% vs 75.2%,P<0.01.For the CCK8 assay,under the condition of Perifosine or MK-2206 treated,the survival rate of NB cells with ATXN3 si RNA #1 and ATXN3 si RNA #2 transfection were significantly lower than ATXN3 Ctrl si RNA transfection.For Perifosine treated,in SK-N-AS cells,ATXN3 si RNA#1 and ATXN3 si RNA#2 vs Ctrl si RNA: 46.3% and 39.9%vs 68.9%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA vs Ctrl si RNA: 33.6% vs 59.2%,P<0.01.For MK-2206 treated,in SK-N-AS cells,ATXN3 si RNA#1 and ATXN3 si RNA#2 vs Ctrl si RNA: 51.2% and 41.5% vs 69.5%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA vs Ctrl si RNA: 44.5% vs 69.3%,P<0.01.For Annexin V/PI flow cytometry,under the condition of Perifosine or MK-2206 treated,the apoptotic rate of NB cells with ATXN3 si RNA #1 and #2 transfection was significantly higher than ATXN3 Ctrl si RNA transfection.For Perifosine treated,in SK-N-AS cells,ATXN3 si RNA#1and ATXN3 si RNA#2 vs Ctrl si RNA: 24.7% and 29.8%vs 16.8%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA#2 vs Ctrl si RNA: 27.7% vs15.9%,P<0.01.For MK-2206 treated,in SK-N-AS cells,ATXN3 si RNA#1 and ATXN3 si RNA#2 vs Ctrl si RNA: 31.4% and 37.0% vs 16.1%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA vs Ctrl si RNA: 23.0% vs 16.3%,P<0.01.3.BIM mediates the process that down-regulation of ATXN3 promotes the cell death induced by Akt inhibitor(Perifosine and MK-2206).ATXN3 si RNA was transfected into NB cells SK-N-AS and SK-N-BE2,and then treated with Perifosine and MK-2206 respectively.Western blot showed that the down-regulation of ATXN3 expression combined with Perifosine or MK-2206 treatment could significantly promote the expression of BIM.Downregulation of ATXN3 and BIM with Perifosine or MK-2206 treated for 48 hours,by the analyzing the confluence in Incucyte Zoom system,we found that,with Perifosine or MK-2206 treated,the cell confluency curve of ATXN3 si RNA-BIM si RNA was significantly higher than that of ATXN3 si RNA group.After analyzing the cell confluency of each group,we found that: For Perifosine treatment,in SK-N-AS cells,ATXN3 si RNA-BIM si RNA vs ATXN3 si RNA: 75.5% vs 51.9%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA-BIM si RNA vs ATXN3 si RNA: 56.6% vs 38.1%,P<0.01.For MK-2206 treated,in SK-N-AS cells,ATXN3 si RNA-BIM si RNA vs ATXN3 si RNA:55.5% vs 41.0%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA-BIM si RNA vs ATXN3 si RNA: 64.8%% vs 52.6%,P<0.01.For the CCK8 assay,under the condition of Perifosine or MK-2206 treatment,the survival rate of NB cells with ATXN3 si RNA-BIM si RNA transfection was significantly higher than ATXN3 si RNA transfection.For Perifosine treatment,in SK-N-AS cells,ATXN3 si RNA-BIM si RNA vs ATXN3 si RNA: 66.2% vs 48.8%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA-BIM si RNA vs ATXN3 si RNA: 60.8% vs 33.6%,P<0.01.For MK-2206 treated,in SK-N-AS cells,ATXN3 si RNA-BIM si RNA vs ATXN3 si RNA:60.3% vs 45.0%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA-BIM si RNA vs ATXN3 si RNA: 62.7%% vs 44.5%,P<0.01.4.Down-regulation of ATXN3 decreased the sensitivity of NB cells to chemotherapy drugs(etoposide and cisplatin).Downregulation of ATXN3 with etoposide or cisplatin-treated for 48 hours,by the analyzing the cell confluence in Incucyte Zoom system,we found that,with etoposide or cisplatin treatment,the cell confluency curve of silencing ATXN3 was significantly higher than that of Ctrl si RNA group.After analyzing the cell confluency of each group,we found that: with etoposide or cisplatin treatment,the cell confluency of ATXN3 si RNA#1 and ATXN3 si RNA#2 was significantly higher than Ctrl si RNA group.For etoposide treated,in SK-N-AS cells,ATXN3 si RNA#1 and ATXN3 si RNA#2 vs Ctrl si RNA: 83.3% and 74.5% vs 46.2%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA vs Ctrl si RNA: 80.1%vs 58.3%,P<0.01.For cisplatin treated,in SK-N-AS cells,ATXN3 si RNA#1 and ATXN3 si RNA#2 vs Ctrl si RNA: 53.6% and 60.3% vs 29.4%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA vs Ctrl si RNA: 63.3% vs 35.2%,P<0.01.For the CCK8 assay,under the condition of etoposide or cisplatin treatment,the survival rate of NB cells with ATXN3 si RNA #1 and #2 transfected was significantly higher than Ctrl si RNA transfected.For etoposide treated,in SK-N-AS cells,ATXN3 si RNA#1 and ATXN3 si RNA#2 vs Ctrl si RNA: 48.0% and 55.1% vs 22.5%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA vs Ctrl si RNA: 75.7% vs 56.3%,P<0.01.For cisplatin treated,in SK-N-AS cells,ATXN3 si RNA#1 and ATXN3 si RNA#2 vs ATXN3 Ctrl si RNA: 41.2%and 36.8% vs 18.7%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA vs ATXN3 Ctrl si RNA:51.1% vs 30.0%,P<0.01.For Annexin V/PI flow cytometry,under the condition of etoposide or cisplatin treatment,the apoptotic rate of NB cells with ATXN3 si RNA #1 and #2 transfected was significantly lower than Ctrl si RNA transfection.For etoposide treated,in SK-N-AS cells,ATXN3 si RNA#1 and ATXN3 si RNA#2 vs Ctrl si RNA: 24.1% and 27.7% a vs 38.4%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA vs Ctrl si RNA: 19.1% vs 28.8%,P<0.01.For cisplatin treatment,in SK-N-AS cells,ATXN3 si RNA#1 and ATXN3 si RNA#2 vs ATXN3 Ctrl si RNA: 27.4% and 23.3% vs 46.1%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA vs Ctrl si RNA: 22.8% vs 34.2%,P<0.01.5.Bcl-xl mediates the process that down-regulation of ATXN3 inhibits the cell death induced by etoposide and cisplatin.Downregulation of ATXN3 and Bcl-xl with etoposide or cisplatin-treated for 48 hours,by analyzing the cell confluence in Incucyte Zoom system,we found that,with etoposide or cisplatin-treated,the cell confluency curve of ATXN3 si RNA + Bcl-xl si RNA was significantly lower than that of ATXN3 si RNA group.After analyzing the cell confluency of each group,we found that: For Etoposide treatment,in SK-N-AS cells,ATXN3 si RNA-Bcl-xl si RNA vs ATXN3 si RNA: 45.4%vs 71.8%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA-Bcl-xl si RNA vs ATXN3 si RNA: 38.2% vs 61.8%,P<0.01.For Cisplatin treatment,in SK-N-AS cells,ATXN3 si RNA-Bcl-xl si RNA vs ATXN3 si RNA:38.9% vs 57.7%,P<0.01;In SK-N-BE2 cells,ATXN3 si RNA-Bcl-xl si RNA vs ATXN3 si RNA: 40.9% vs 61.0%,P<0.01.For the CCK8 assay,under the condition of etoposide or cisplatin treatment,the survival rate of NB cells with ATXN3 si RNA-Bcl-xl si RNA transfection was significantly lower than ATXN3 si RNA transfection.After analyzing the cell survival rate of each group,we found that: For Etoposide treatment,in SK-N-AS cells,ATXN3 si RNA-Bcl-xl si RNA vs ATXN3 si RNA: 51.5% vs 68.0%,P<0.01;in SK-N-BE2 cells,ATXN3 si RNA-Bcl-xl si RNA vs ATXN3 si RNA: 50.6% vs 65.3%,P<0.01.For cisplatin treated,in SK-N-AS cells,ATXN3 si RNA-Bcl-xl si RNA vs ATXN3 si RNA: 45.6% vs60.7%,P<0.01;in SK-N-BE2 cells,ATXN3 si RNA-Bcl-xl si RNA vs ATXN3 si RNA:51.2% vs 71.7%,P<0.01.Conclusion:ATXN3 was significantly negatively correlated with MYCN amplification,risk grouping,and tumor stage in NB,and it was an independent prognostic factor in NB patients.Down-regulation of ATNX3 could enhance the cell death induced by AKT inhibitor(Perifosine and MK-2206)via up-regulation of BIM,while down-regulation of ATNX3 did not enhance,but decreased the sensitivity to chemotherapeutic drugs(etoposide and cisplatin)by up-regulation of Bcl-xl in NB cells.These findings suggested that ATXN3 was a novel potential target for NB therapy and showed great value in guiding precision therapy and medication selection during the treatment of NB patients. |
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