LSD1在神经母细胞瘤细胞中促进β-catenin在胞质中堆积

LSD1 promotes the accumulation of β-catenin in the cytoplasm of neuroblastoma cytoplasm

目的本研究旨在证实在神经母细胞瘤细胞（neuroblastoma，NB）中赖氨酸特异性去甲基化转移酶1（1ysine specific demethylase 1，LSD1）促进β-联蛋白（β-catenin）在胞质中堆积，证实LSD1通过激活Wnt通路，从而引起NB细胞的增殖和转移，并探索其分子机制。方法将对数生长期的293T细胞，接种在6孔培养板上，感染时细胞密度控制在60%～80%，每孔2×106个细胞。分别配置溶液A：cDNA 10 μl＋cDNA质粒转染培养基90 μl；溶液B：空载质粒转染试剂5 μl＋cDNA质粒转染培养基95 μl，混合溶液A和溶液B，室温放置30 min形成转染复合物，在含有细胞的无抗生素培养基中加入转染复合物使终体积为2 ml。Quantitative real-time PCR（qPCR）检测LSD1在NB细胞株SH-SY5Y细胞中的表达。LSD1慢病毒过表达载体的构建上调LSD1在NB中的表达。Western blot方法检测在SH-SY5Y细胞株调控LSD1表达后β-catenin的变化。免疫组织化学方法检测在SH-SY5Y细胞株调控LSD1表达后β-catenin的变化。结果利用Western bolt方法检测人上皮细胞、大鼠神经元细胞、NB细胞中LSD1的表达量，人上皮细胞、大鼠神经元细胞、NB细胞三者的灰度值分别为133.87±10.54、135.00±12.48、192.00±13.23，三组进行单因素方差分析无明显统计学意义（P〉0.05）；建立LSD1稳转细胞株前后，SH-SY5Y细胞表达LSD1量有变化，通过qPCR对SH-SY5Y细胞株中的LSD1基因的相对量进行检测，LSD1表达抑制组的阈值循环数为22.003±0.320，转染空白质粒慢病毒载体的SH-SY5Y细胞组的阈值循环数为17.330±1.166，转染LSD1过表达慢病毒载体组的阈值循环数为13.216±0.137三组进行单因素方差分析，差异具有统计学意义（P〈0.01）。结论在神经母细胞瘤细胞株SH-SY5Y细胞中，β-catenin随着LSD1的表达量的变化而变化，两者在SH-SY5Y细胞中的量呈正相关。

Objective The aim of this study was to confirm that lysine specific demethylase 1（LSD1） promotes the accumulation of β-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 to explore its molecular mechanism.MethodsThe cells in the logarithmic phase were inoculated in the 6-well culture plate.When at infected, the density of the cells was controlled at 60%-80%, with 2×106 cells per well.Prepared solution separately ： configure A： cDNA10 μl＋ cDNA plasmid transfection medium 90 μl; configure B： plasmid transfection reagent 5 μl＋ cDNAplasmid transfection medium 95 μl.After mixing the solution A with the solution B, and incubating for 30 min at room temperature, forming a transfection complex, adding transfection complex to the non antibiotic medium containing the celled, the final volume of the mixture is 2 ml.Quantitative real-time PCR （qPCR） was used to detect the expression of LSD1 in neuroblastoma cell line SH-SY5Y.Construction of LSD1 lentiviral overexpression vector up-regulated the expression of LSD1 in neuroblastoma.Western blot method was used to detect the change of β-catenin in SH-SY5Y cell line after regulating LSD1 expression.Immunohistochemical method was used to detect the expression of β-cateninin in SH-SY5Y cell line after the regulation of LSD1 expression.ResultsTesting the different levels of LSD1 expression in human epithelial cells/ rat neuron cells and neuroblastoma cells, Western bolt was performed.The gray values of the three groups were 133.87±10.54; 135.00±12.48; 192.00±13.23, the three groups were single factor analysis of variance （P〉0.05）, the difference was not statistically significant.Before and after the establishment of stable LSD1 cell lines, the expression of LSD1 in SH-SY5Y cells was changed, the relative amount of LSD1 gene in SH-SY5Y cell line was detected by qPCR.We found that the threshold number of LSD1 expression inhibition group was 22.003±0.320, The threshold number of SH-SY5Y cell line transfected with blank plasmid lentiviral vector was 17.330±1.166.The number of threshold cycles of transfection of LSD1 overexpressing lentiviral vector was 13.216±0.137.Three groups of single factor analysis of variance P〈0.01, the difference was statistically significant.ConclusionsIn the neuroblastoma cell line SH-SY5Y, the expression of β-catenin was changed with the expression of LSD1, and the amount of positively correlated with the amount of SH-SY5Y in the cells.