| ObjectiveTo investigate the expression of Asef in neuroblastoma tissues and to a nalyze the relationship among them in different pathological types.Then,observe the relationship of Asef and ?-catenin proteins in SH-SY-5Y cell line to support the exploration of mechanisms for identifying the occurrence and metastasis of neuroblastoma.MethodsSurgical specimens of 54 children with NB and 11 children with ganglion cells neuroma(GN)(30 females,35 males)in the Affiliated Hospital of Qingdao University from 2012 to 2017 were picked as experimental groups(NB group,GN group)and 15 peritumoral tissues were chosen from NB patients as a control group.All patients are ranging in age from 4 months to 9 years old with an average age of 3 years and 9 months.Subsequently retrospective analysation were made to those samples.We used Immunohistochemical staining SP method to determine the expression of Asef.Then the relationship of the expression of Asef with Shimada typing were also analyzed.In the NB cell line SH-SY-5Y,the cells were divided into blank(group A),Asef overexpression(group B)and inhibition group(group C)by lentivirus transfection,and Asef was detected in the cell line by qRT-PCR.The expression of ?-catenin in group A,B and C was determined by Western Blot,and the relationship between Asef expression and ?-catenin was analyzed.ResultsThere are Asef expression in 50 cases of NB,3 cases of peritumoral tissue and 5 cases of GN.Most of Asef were found in the cytoplasm of tumor cell and a small amount of it in the nucleus.In the three groups,the positive expression rate of Asef is 92.6%(NB),20%(peritumoral tissue)and 45.5%(GN).There were statistically significant differencesin NB,peritumoral tissue groups(P<0.05)and in NB,GN groups(P<0.05).And the positve rate of Asef expression were 74.1% in good prognosis(FN)and 89.5% in poor prognosis(UFN),which showed no statistically significant difference(P>0.05).After establishing a stable SH-SY-5Y cell line,the expression of Asef in A,B and C groups were detected by qRT-PCR,which were 0.997±0.028,1.443±0.066,0.773±0.035,respectively.There were significant differences(P<0.05);Western Blot was performed on the transfected SH-SY-5Y cells.The expressions of Asef in groups A,B and C were 1.099±0.101,1.474±0.251,0.601±0.062,respectively.There was a significant difference(P<0.05).At the same time ?-catenin detected at group A,B and C were 1.129±0.023,1.511±0.170 and 0.543±0.007,respectively.There were also significant differences in expression among the three groups(P<0.05).Along with the expression of Asef-regulated in SH-SY-5Y cells,?-catenin protein also changed,and there was a linear correlation between them(r=0.948,P<0.05).ConclusionThe expression intensity of Asef in NB tissues and control group is significantly different between benign and malignant tumors.In NB SH-SY-5Y cell line,the expression of ?-catenin in cytoplasm is positively correlated with Asef,so the expression of Asef has certain reference significance for the pathological diagnosis of NB.Asef may also regulate the biological activity of NB by regulating the expression of ?-catenin.SignificanceIn this study,the expression of Asef in different pathological tissues of was detected,and it was found that the expression of Asef was higher in tumors with high malignancy,which may be a reference factor for clinically judging the degree of malignancy in NB.At the same time,by constructing the NB cell line transfected with lentivirus and regulating the expression of Asef in the cells,it was found that the accumulation of ?-catenin in the cytoplasm was positively correlated with Asef.According to the results of this experiment,it can be concluded that Asef may affect the development of NB by regulating ?-catenin,which may link Asef protein with Wnt signaling pathway and pro vide clues for the study of NB mechanism. |
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