The Preliminary Study About The Mechanism Of The Wnt1 Signal Pathway In The Development Of Neuroblastoma

Backgrounds and purpose:Neuroblastoma(NB) is a kind of embryonal tumor.Its development is closely related to the abnormal of adrenal gland in the course of embryonic development.The classic Wnt signal pathway can control the balance of proliferation and differentiation of the neural precursor cell.Now,if the classic Wnt signal pathway is the essential factor in the development of embryonic neural crest cells that is the original cell of neuroblastoma is not clear.Therefore,in our experiment,I detected the expression of Wntl,β-catenin and CyclinD1 protein in neonatal adrenal gland,＞2-year-old children's adrenal gland and neuroblastoma tissue using immunohistochemical method. And the above gene expression in neuroblastoma and adrenal gland tissue adjacent to the tumor was detected using real-time PCR method.Martial and methods:Martial:Formalin-fixed,paraffin-embedded tissue sections of adrenal glands from autopsy and neuroblastoma were taken from department of pathology,Children's Hospital of Fudan University.Fresh neuroblastoma tissue and adrenal glands tissue nearby were harvested during the operation and transferred to -80℃for storage.Wnt1 specific antigen(Aoweiya corporation,Beijing),β-catenin and CyclinD1 specific antigens (Sant Cruz corporation,USA) were used for the staining of Immunohistochemical. RT-PCR kit(Qiagen corporation,Germany) was used for the RT-PCR test.Methods:Immunohistochemical staining for Wntl,β-catenin,CycinD1 were performed in adrenal glands collected in both neonates and children more than two years old, together with neuroblastoma tissue divided according to the different stage(INSS, Shimade).Expression of the mRNA motioned above was detected by RT-PCR.Results:Immunohistochemical:1.Positive staining of Wntl specific antigen were found in 23 adrenal glands of neonate,20 adrenal glands of children more than two years old,and 34 neruoblastoma without chemotherapy.The IOD values of 34 neuroblastomas without chemotherapy were much higher than in adrenal glands of neonates(p=0.002).IOD value in neonate adrenal was higher than in children more than two years old(p=0.014).There existed an augmentation of IOD in neuroblastoma grouped inⅢ-Ⅳstages comparing with those inⅠ-Ⅱstages(p=0.02).Among neuroblastoma inⅢ-Ⅳstages,IOD value was much higher in neuroblastoma without chemotherapy before operation than those underwent chemotherapy before operation(p=0.014).Neuroblastoma grouped in unfavored histology(UH) had higher IOD value than those in favored histology(FH) (p=0.038).2.Positive immunohistochemical staining forβ-catenin specific antigen was detected in all of the samples just like Wntl.The IOD value in 34 neuroblastomas without chemotherapy before operation was extensively higher than in adrenal glands of neonates(p=0.000).IOC value in adrenal glands of neonates was higher than those of children more than two years old(p=0.031).Progressive neuroblastoma ofⅢ-Ⅳstages had higher IOD value than those ofⅠ-Ⅱgrades.(p=0.039).Among neuroblastoma withⅢ-Ⅳstages,it had a higher IOD value in neuroblastoma without chemotherapy comparing with those underwent chemotherapy before operation (p=0.038).IOD value in UF was higher than those in FH(p=0.035).3.Immunohistochemical staining for CyclinD1 specific antigen was negative in adrenal glands both in neonates and children more than two years old.There were no significant difference between adrenal glands of neonates and those of children more than two years old(p=0.874).Among neuroblastoma grouped inⅢ-Ⅳstages,IOD value was much higher in neuroblastoma underwent chemotherapy before operation comparing with those without chemotherapy before operation(p=0.001).IOD value in UH group was higher than FH group(p=0.029).4.In 34 neuroblastoma without chemotherapy before operation,significant correlation of IOD value was found between Wnt1 andβ-catenin(R=0.764,P＜0.01). Both between Wnt1 and CyclinD1 and betweenβ-catenin and CyclinD1 had significant correlation according to IOD value(R=0.448,P＜0.01,R=0.513,P＜0.01).Real-time PCR:1.26 neuroblastoma without chemotherapy before operation had higher expression of mRNA comparing with 16 adrenal glands nearby without apparent invasion of neuroblastoma(p=0.012).Neuroblastoma without chemotherapy before operation grouped inⅢ-Ⅳstages had higher expression of mRNA than neuroblastoma inⅠ-Ⅱstages(p=0.026).There exited a significant evaluation of Wnt1 mRNA expression in neuroblastoma without chemotherapy before operation grouped inⅢ-Ⅳgrades comparing with those receiving chemotherapy before operation(p=0.027). Neuroblastoma grouped in UH showed higher expression of Wnt1 mRNA expression than those grouped in FU.2.There was no significant difference in mRNA expression ofβ-catenin between neuroblastoma underwent chemotherapy and adrenal glands nearby without invasion of neuroblastoma(p=0.519).There was no diffience between gradesⅠ-ⅡandⅢ-Ⅳ(p=0.667).Among neuroblastoma grouped inⅢ-Ⅳgrades,it showed no significant difference between those underwent chemotherapy and without chemotherapy before operation(p=0.683).There was also no difference between FU and UH(p=0.261).3.The expression of CyclinD1 mRNA in 26 neuroblastomas without chemotherapy before operation was extensive higher than those in adrenal glands(p=0.000).No significant evaluation of expression of CyclinD1 mRNA between progressive stages (Ⅲ-Ⅳ) without chemotherapy and neuroblastoma inⅠ-Ⅱstages.(p=0.033) Neuroblastoma inⅢ-Ⅳstages underwent chemotherapy had lower expression of CyclinD1 mRNA comparing with those receiving chemotherapy before operation (p=0.033).There showed significant evaluation between FH and UH(p=0.017).4.There was no significant difference between Wnt1 andβ-catenin,betweenβ-catenin and cyclinD1 according to the expression of mMRA.(R=-0.101,P＞0.05, R=0.244,P＞0.05).But Wnt1 and CyclinD1 had significant correlation on the expression of mRNA(R=0.488,P＜0.05).Conclusion:1.Wnt1,β-catenin and Cyclin D1 protein in Wnt1 signal pathway will reduce in the developement of adrenal gland,which indicats that Wnt1 signal pathway may have a certain degree of regulation in the differentiation and maturation of the adrenal cells.2.Wnt1,β-catenin and Cyclin D1 protein in neuroblastoma unusual increase and the expression have correlation,which suggests that Wnt1 signal pathway may be one of the mechanisms leading to neuroblastoma.3.Wnt1,β-catenin-positive staining IOD value are closely related to neuroblastoma stage and histological type,which indicats that excessive activation of Wnt1 signal pathway may be involved in neuroblastoma differentiation and the regulation of invasion.4.Wnt1,cyclinD1 in mRNA levels increase in neuroblastoma tissue,which further prompts that the Wnt1 signal pathway activates in neuroblastoma. 5.In development of neuroblastoma,high expression of Wnt1 had no relationship with activation ofβ-catenin,abnormal expression ofβ-catenin probably caused by dysfunctional regulation after mRNA transcription.