Studies On The Mechanisms Of Chemo-resistance,cell Migration And Invasion,And Cell Proliferation In Neuroblastoma

Objective: Neuroblastoma(NB)is the most common extracranial malignancy solid tumor in childhood.According to the clinical manifestation and the biological characteristics of tumor,patients with NB can be divided into low-risk group,moderate-risk group and high-risk group.Patients with NB have heterogeneity phenotypes,patients with low-risk and moderate-risk diseases have much better response to clinical therapy strategies,some of them can spontaneous,the event-free long-term survival rate can reach more than 90%.Patients with high-risk disease,even treated with multi-modal therapy using surgery,radiotherapy,chemotherapy and stem cell transplantation,the long-term survival rate is still less than 50%.Most of the patients are diagnozied with high-risk disease.Therefore,to improve long-term survival rate of patients with high-risk disease is the important thing and chanllege for treatment of NB.The clinical studies found that about 50% to 60% of patients with high-risk NB over-expressed BDNF(brain-derived neurotrophic factor)and/or Trk B(tropomyosin receptor kinase B),these patients usually have poor prognosis.It is found that BDNF/Trk B induces chemoresistance of NB cells via PI3 K signaling pathway and MAPK signaling pathway,but the role and mechanisms of BDNF/Trk B in the migration and invasion ability of NB cells and mechanisms regulated chemoresistance still need further study,and there have no reports show whether or not PI3 K pathway or MAPK pathway mediate the effect of BDNF/Trk B on NB cell migration and invasion.The first part of this study focus on the role of BDNF/Trk B in the migration and invasion of NB cells,and the mechanisms of BDNF/Trk B in the chemoresistance of NB cells.In the maturation stage of sympathetic nervous system development,maintaining the expression of MYCN oncogene can induce the occurrence of NB.MYCN amplification is another diagnostic index of patients with high-risk NB.About 20% of patients with NB have MYCN amplification,40% to 50% of patients with high-risk NB show MYCN amplification,and these patients usually have poor prognosis.Therefore,to explore the related mechanisms of MYCN in NB is one of the research directions to find out the therapy targets of NB.Protein arginine methyltransferase 1(PRMT1)is one of the members of the protein arginine methyltransferase(PRMT)family,and it is found that event-free survival rate of patients with over-expression of PRMT1 is significantly lower than patients with low-expression of PRMT1.The expression level of PRMT1 is obviously correlated with the expression level of MYCN,and PRMT1 can methylate MYCN.In the second part of this study,the role of PRMT1 in the cell proliferation of NB cells and its related mechanisms were studied.Methods:In the first part of this study,Tetracycline(TET)regulated Trk B-expressing TB3 and TB8 cell lines were used.Scratch wound healing assay and boyden chamber migration and invasion assays were used to detect NB cell migration and invasion.Western Blot assay was used to detect the expressions of P-Akt(Ser473),P-Erk(Thr202/Tyr204)and P-m TOR(Ser2481).The expressions of P53 and BCL2 family members were detected by Western Blot at protein level and RT-q PCR at m RNA level.The expression of P53 or PUMA was down regulated by transfection si RNA into cells.Activated PUMA lentiviral infection was used to over-express PUMA.MTS assay was used to detect cell survival and Incucyte ZOOM Living cell imaging system was used to detect the percentage of cell confluence by collecting images at different time points.In the second part of this study,MYCN-amplified BE2 C cell line and Kelly cell line,as well as Doxycline(Dox)regulated PRMT1-knockdown BE2 C cell line and Kelly cell line were used.Cell proliferation was detected by counting cell number with trypan blue staining and Alamar Blue assay.The expressions of PRMT1,PARP and ATF5 at protein level were detected by Western Blot.Virus infection was used to overexpress ATF5.Caspase-Glo 3/7 Assay was used to detect the activity of Caspase3/7.Results:Part I:1.BDNF/ Trk B increased the migration and invasion ability of NB cells: The cell migration ratio was detected by scratch wound healing assay,and after 30 hours of BDNF treatment,the cell migration ratio was 93%,without BDNF treatment the cell migration ratio was 39.2%.The boyden chamber migration assay showed that the number of migrating cells increased to 1.5-fold after BDNF treatment in the Trk B-expressing cells(P < 0.01),and the boyden chamber invasion assay showed that the invading cell number was 54-fold higher in the BDNF-treated cells compared to that in the control group(P<0.05).In non-Trk B-expressing TB3 cells,the results of scratch wound healing assay and boyden chamber migration and invasion assays showed no statistical difference in the cell migration and invasion ratio treated with or without BDNF(P>0.05).2.PI3 K pathway and MAPK pathway mediated BDNF /Trk B-increased cell migration and invasion in NB cells: PI3 K inhibitor LY294002 or MAPK inhibitor PD98059 can inhibit BDNF/Trk B-induced increased expressions of P-Akt(Ser473)or P-Erk(Thr202/Tyr204)in NB cells.The cell migration ratio was detected by scratch wound healing assay,the cell migration ratio of control group was 54.90%,BDNF treatment group was 93.00%,and pretreatment with LY294002 or PD98059,the cell migration ratio was 38.10% or 30.80%.The boyden chamber migration assay showed that the migrating cell number increased to 1.67-fold after the BDNF treatment(P <0.01),and LY294002 or PD98059 could inhibit the increase of cell migration induced by BDNF.The boyden chamber invasion assay showed that the invading cell number was 59.5-fold higher in the BDNF-treated cells compared to that in the control cells(P<0.01),and LY294002 or PD98059 could inhibit the increased cell invasion induced by BDNF.3.Akt and m TOR mediated BDNF/Trk B-induced increased cell migration and invasion in NB cells: Akt inhibitor Perifosine or m TOR inhibitor Rapamycin could inhibit BDNF/Trk B-induced increased expression of P-Akt(Ser473)or P-m TOR(Ser2481).The cell migration ratio was detected by scratch wound healing assay,the cell migration ratio of control group was 54.90%,BDNF treatment group was 85.30%,and pretreatment with Perifosine or Rapamycin,the cell migration ratio was 30.70%or 26.00%.The boyden chamber migration assay showed that the migrating cell number increased to 1.76-fold after the BDNF treatment(P < 0.01),and LY294002 or PD98059 could inhibit the increase of cell migration induced by BDNF(P < 0.01).The boyden chamber invasion assay showed that the invading cell number was31.63-fold higher in the BDNF-treated cells compared to that in the control cells(P<0.01),and Perifosine or Rapamycin could inhibit the increased cell invasion induced by BDNF(P < 0.01).4.BDNF/Trk B inhibited etoposide-induced increased expression of P53 and induced chemoresistance of NB cells: BDNF/Trk B obviously inhibited the increased expression of P53 induced by etoposide.Transfection with P53 si RNAs inhibited the increased expression of P53 induced by etoposide.After etoposide treatment,compared with the control group,the survival ratio of TB3 cells transfected with P53 si RNAs increased 17.62%-27.39%(P<0.01).In TB8 cells after etoposide treatment,compared with the control group,the survival ratio of cells transfected with P53 si RNAs increased 16.40-23.71%(P<0.01).5.The effect of etoposide or BDNF on the expressions of BCL2 family members:After treatment with etoposide,the expressions of PUMA,NOXA,BAX,and BAK(pro-apoptotic)increased,and the increase of PUMA was the most significant,and the expression of MCL-1(anti-apoptotic)decreased,and there were no significant changes in the expressions BID(pro-apoptotic)and BCL2 and BCL-XL(anti-apoptotic).After treatment with BDNF,the expression of PUMA decreased,and the expressions of MCL1 and BCL-XL increased,and there were no significant changes in the expressions of NOXA,BAX,BAK,BID and BCL2.6.BDNF/Trk B inhibited Etoposide or virus-infection-induced increased expression of PUMA and induced chemoresistance of NB cells: Pretreatment with BDNF inhibited etoposide-induced increased expression of PUMA in NB cells.Transfection with PUMA si RNAs inhibited increased expression of PUMA induced by etoposide.After etoposide treatment,compared with the control group,the survival ratio of TB3 cells transfected with PUMA si RNAs increased 10.33%-22.42%(P<0.01).In TB8 cells after etoposide treatment,compared with the control group,the survival ratio of cells transfected with PUMA si RNAs cells increased 9.92-23.46%(P<0.01).BDNF/ Trk B inhibited the increased expression of PUMA induced by infection with Active PUMA virus.BDNF/ Trk B inhibited NB cell death induced by infection with Active PUMA virus.After 72 hours of Active PUMA virus infection,the survival ratio of the TB3 cells was 19.52%(P<0.01),and after BDNF treatment,the survival ratio increased to 45.87%(P<0.01).In TB8 cells,the survival ratio of cells infected with Active PUMA virus was 27.58%(P<0.01),and after BDNF treatment,the survival ratio increased to 56.19%(P<0.01).Part II:1.The role of PRMT1 in cell proliferation of NB cells: After knockdown the expression of PRMT1 by transfection with sh RNA,the cell proliferation ratios were33.4%(sh PRMT1 #1)and 14.8%(sh PRMT1 #2)in BE2 C cells,which were significantly lower than the control group(100%)(P<0.01).In Kelly cells,the cell proliferation ratios were 42.8%(sh PRMT1 #1)and 41.08%(sh PRMT1 #2),which were significantly lower than the control group(P<0.01).The inhibitor Decamidine(Dec)blocked the enzyme activity of PRMT1,and inhibited NB cell growth: the Alamar Blue assay results showed that the cell viability of BE2 C and Kelly decreased along with the concentration of Dec.2.The role of PRMT1 in cell apoptosis of NB cells: Knockdown the expression of PRMT1 by transfection with sh RNA significantly increased the expression of cleaved-PARP and the activity of Caspase3/7(P<0.01).PRMT1 inhibitor Dec significantly increased the expression of cleaved-PARP and the activity of Caspase3/7(P<0.01).The results indicated that PRMT1 regulated NB cells proliferation and cell apoptosis.3.ATF5 mediated the regulation of PRMT1 in cell proliferation and cell apoptosis of neuroblastoma NB cells: Knockdown the expression of PRMT1 by transfection with sh RNA,the expression of ATF5 significantly decreased.Overexpression of ATF5 by infection with Flag-ATF5 virus significantly blocked the inhibition of cell proliferation(P<0.05)and cell apoptosis(P<0.05)induced by PRMT1 knockdown.The results indicated that ATF5 mediated the regulation of PRMT1 in NB cells proliferation and cell apoptosis.Conclusions:1.BDNF/Trk B increased cell migration and invasion ability of NB cells.PI3K/Akt/m TOR signaling pathway and MAPK signaling pathway mediated BDNF/Trk B-induced increased cell migration and invasive ability of NB cells.2.P53 and PUMA mediated BDNF/Trk B-induced chemo-resistance in NB cells.3.PRMT1 regulated cell proliferation and apoptosis in NB cells via ATF5.