LSD1 Promotes The Accumulation Of β-catenin In The Cytoplasm Of Neuroblastoma Cytoplasm

ObjectiveThe aim of this study was to confirm that LSD1 promotes the accumulation ofβ-catenin in the cytoplasm of neuroblastoma cells,and to confirm that LSD1 activates the Wnt pathway to induce the proliferation and metastasis of NB cells,and providing further exploration of its molecular mechanism,theoretical and experimental support.MethodsWestern Blot method was used to detect the expression of LSD1 in SH-SY5 Y cells,human epithelial cells and rat hippocampal neurons in NB cell lines.The NB cell line SH-SY5 Y cells were divided into three groups:the first group was transfected with empty plasmid and the expression of LSD1 and β-catenin in SH-SY5 Y cells was not disturbed;The second group was LSD1 overexpression group,transfected with LSD1 lentivirus carrier group,and the expression of LSD1 in SH-SY5 Y cells was up-regulated;The third group was LSD1 interference group,and the activity of LSD1 in this group was inhibited by anti phenylalanine inhibitor(TCP)in SH-SY5 Y cells.The Quantitative real-time PCR(qPCR)method was used to detect the expression of LSD1 in the three groups and the transfection efficiency of the lentivirus vector.Western blot was used to detect the expression of LSD1 and β-catenin in three groups of cells and analyze the expression relationship.Immunohistochemical method was used to detect the change of β-catenin after the expression of LSD1 in SH-SY5 Y cell line.ResultsThe gray values of Western blot for human epithelial cells,rat neurons and SH-SY5 Y cells were 133.87±10.54;135.00±12.48;192.00±13.23 and there was no significant statistical significance in single factor analysis of variance between the three groups(p>0.05).Before and after the establishment of lsd1 transfected cell line,theexpression of LSD1 in SH-SY5 Y cells was changed,and the relative amount of LSD1 gene in SH-SY5 Y cell line was detected by qPCR.The threshold cycles of qPCR in the first group,the second group and the third group were 13.216±0.137、17.330±1.166、22.003 ± 0.320,three groups were analyzed by single factor analysis of variance(ANOVA)p<0.01.The difference was statistically significant;Western Blot results showed that the gray scale values of the first group,the second group and the third group of LSD1 protein were 130.46±10.99、236.4±13.22、103.44±12.66;p<0.01.The difference was statistically significant;The gray levels of β-catenin in the first group,the second group and the third group were 137.82±10.99、218.53±13.22、118.58±12.66,p<0.01,and the difference was statistically significant;After regulating the expression of LSD1,the expression level of β-catenin in SH-SY5 Y cells was significantly changed.The expression of β-catenin was regulated up and down after the LSD1 expression was up regulated,indicating that the expression of β-catenin was positively correlated with LSD1 expression in NB cells.(data correlation test between different groups,r = 0.99 p < 0.01);Immunohistochemical staining showed that the first group of immunohistochemical staining,the cytoplasm was light brown,indicating that β-catenin expression,but the expression level was not as obvious as the LSD1 high expression group;Second groups of immunohistochemical staining,the cytoplasm was brown,indicating the high expression of β-catenin;The third group of cytoplasm was not stained,indicating that the expression of β-catenin was less.ConclusionsThe expression of LSD1 was up-regulated in the neuroblastoma cell line SH-SY5 Y.Our preliminary experiments in this experimental group showed that the expression level of LSD1 in NB cells was higher than that in normal neurons and epithelial cells.Further speculate its biological activity,we found that β-catenin changes with the change of the expression of LSD1,and there is a positive correlation between the amount of β-catenin in SH-SY5 Y cells.Β-catenin is a key factor in the Wnt pathway,and LSD1 affects the biological activity of NB cells by activating the Wnt pathway.MeaningIn this experiment,the lsd1 transfected SH-SY5 Y cell line was successfully constructed and used for further experiments.It was confirmed that LSD1 was highly expressed in the NB cell SH-SY5 Y cell line.After verifying its biological activity,it wasfound that there was a positive correlation between β-catenin and LSD1 expression in NB cell SH-SY5 Y cell line.It can be inferred that LSD1 further affects the biological activity of NB by up-regulating the expression of β-catenin in cells.