| Obiective: Neuroblastoma(NB)is the most common extracranial solid tumor in children,accounting for 8% of all childhood tumors,but accounting for 15% of childhood tumor-related deaths.The prominent feature of NB is its high degree of heterogeneity,with some subgroups spontaneously regressing and some subgroups showing sustained progression.About half of the cases have a high risk of recurrence.In the past few decades,the prognosis of low-risk children has improved significantly,but the longterm prognosis of high-risk children is only 40%-50% despite the combination of multiple methods.Therefore,finding markers associated with prognostic outcomes in highrisk children will be the key to further improving the overall prognosis of children with NB.Prp19 is highly expressed in various tumor tissues and participates in tumor invasion,cell cycle regulation,and inhibition of UV-induced apoptosis.CDC5 L is also highly expressed in various tumors such as liver cancer,cervical cancer,osteosarcoma,etc.,and participates in the regulation of cell cycle and the stability of chromosomes,It can affect a variety of clinical and pathological parameters of tumor patients,such as staging,tumor size and patient prognosis.However,the role of Prp19 and CDC5 L in the development of NB has not been reported.Therefore,the purpose of this study was to analyze the association between Prp19 and CDC5 L and NB clinicopathological features and prognosis,and to elucidate the effects of Prp19 on NB cell proliferation,DNA damage and related molecular mechanisms.Methods: Immunohistochemical(IHC)and Western blot(WB)were used to detect the expression of Prp19 and CDC5 L in tumor specimens of children with NB.NB were divided into several groups according to patients’ gender,age,metastasis of bone marrow,tumor clinical stage(INSS),risk rating,pathological diagnosis,Shimada pathological classification,and the expression of Prp19 and CDC5 L in each group was analyzed by chi-square test.Kaplan-Meier and COX regression analysed the effects of Prp19 and CDC5 L on the survival of children with NB,and verified the effect of Prp19 on the prognosis of children with NB in two independent databases.Small interfering RNA(si RNA)was used to detected the effects of Prp19 down-regulation on cell proliferation,invasion and DNA damage repair function on two NB cell lines.In addition,RNA-seq identified changes in gene expression profiles after down-regulation of Prp19,thereby finding proteins or pathways that interacted with Prp19,further revealing the mechanism of Prp19 on cell proliferation,invasion and DNA damage repair.Result1.The relationship between CDC5 L expression and clinicopathological features and prognosis of NBIHC showed that CDC5 L was mainly expressed in the nucleus.In 62 cases of NB patients,high and low expression of CDC5 L both had 31 cases(50%).IHC showed that the expression of CDC5 L in NB was higher than that of GNB.WB,consistent with IHC,also showed that the expression of CDC5 L in NB was higher than that of GNB.Further statistical analysis of the relationship between CDC5 L and clinicopathological features of children with NB.the analysis found that the expression of CDC5 L in the tumor bone marrow infiltration group was higher than the non-infiltration group,and the difference was statistically significant(X~2=5.833,p=0.016);The expression of CDC5 L in the stage III-IV was higher than that of I-II and IV-S(X~2=11.328,p=0.001);MYCN amplification group had higher CDC5 L expression,p=0.038;In high-risk children,the expression of CDC5 L was higher than in children with intermediate risk(p=0.024)and lower risk(X~2=6.031,p=0.042).In the pathological classification of Shimada,unfavorable histologic(UFH)had higher CDC5 L expression than favorable histologic(FH)(X~2=7.839,p=0.005).Kaplan-Meier survival analysis showed that the higher expression group of CDC5 L had shorter survival time(p=0.026).COX regression analysis showed that clinical stage(p=0.006,HR=6.785)and CDC5L(p=0.031,HR=3.605)were both poor prognostic factors in children with NB.2.The relationship between Prp19 expression and clinicopathological features and prognosis of NBIHC showed that in 62 cases of NB patients,Prp19 was highly expressed in 30 cases and low in 32 cases;Prp19 was mainly expressed in the nucleus;IHC and WB showed that Prp19 was higher in NB/GNB-N than GNB-I.Then we analysed the different expression of Prp19 between clinical and pathological features of children with NB,and found that the tumor bone marrow infiltration group had higher expression of Prp19 than the tumor non-infiltration group(X~2=6.751,p=0.009),the difference was statistically significant;In each clinical stage,the difference of Prp19 expression was not obvious(X~2=1.180,p=0.227);The high-risk children had higher expression of Prp19(X~2=5.168,p =0.023)than the intermediate and low-risk children;The unfavorable histologic(UFH)had a higher expression of Prp19 than favorable histologic(FH)(X~2=5.274,p =0.022).Kaplan-Meier survival analysis showed that the Prp19 low expression group had a longer overall survival time(p=0.002)and free-event survival time(p=0.025).COX regression analysis showed that clinical stage and Prp19 were independent risk factors for overall prognosis and free-event survival of children with NB.3.Effect of Prp19 on biological behavior of NB cells and exploration of correlative mechanismCellular experiments suggested that knockdown of Prp19 can impair the proliferation and DNA damage repair of SK-N-BE(2)and SK-N-AS cells;knockdown of Prp19 combined with doxorubicin(Dox)can further inhibit NB cell proliferation and impair DNA damage repair function.After Prp19 knockdown,the invasive ability and epithelial stromal transition(EMT)of NB cells was also significantly attenuated.Further investigation revealed that knockdown of Prp19 can down-regulate the cyclin D1 and upregulate the DNA damage marker γH2AX,it was suggested that the phenotype changes of NB cells after knockdown of Prp19 may involve cell cycle pathway and DNA damage repair pathway.RNA-seq results showed that after down-regulation of Prp19,YAP(also called YAP1),the key molecule of Hippo-YAP pathway,expression was significantly downregulated,accompanied by down-regulation of multiple target genes downstream of YAP,such as CGEF,FGF1,AFP and MYC.Therefore,Prp19 may exert biological roles through the Hippo-YAP pathway.ConclusionThis study reported for the first time that Prp19 and CDC5 L are highly expressed in NB,and the high expression of both is related to various clinicopathological features of NB.High expression of Prp19 and CDC5 L in NB also suggests a poor prognosis.In vitro studies have shown that down-regulation of Prp19 can inhibit NB cell proliferation and DNA damage repair.In addition,this study reports for the first time the association between the Prp19 and Hippo-YAP pathways,suggesting that the alteration of the biological behavior of NB cells induced by down-regulation of Prp19 may be related to the down-regulation of Hippo-YAP pathway. |
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