半乳糖凝集素-1在神经母细胞瘤组织中的表达及其对细胞增殖和侵袭的影响

Expression of Galectin-1 in neuroblastoma tissues and its effect on cell proliferation and invasion

目的探讨半乳糖凝集素-1(Galectin-1)在神经母细胞瘤(NB)组织中的表达以及下调该基因对细胞增殖和侵袭能力的影响。方法选取2010年1月至2018年1月在潍坊医学院附属益都中心医院儿内科初次治疗且资料完整的NB患儿62例,采用免疫组织化学法检测患儿NB组织中Galectin-1的蛋白表达。培养NB细胞株SH-SY5Y,分为siRNA-Gal1组(转染Galectin-1干扰序列siRNA-Gal1)、siRNA-NC组(转染阴性对照序列siRNA-NC)和空白组(不作任何处理),采用实时荧光定量PCR法检测3组Galectin-1的表达,细胞计数试剂盒-8(CCK-8)法检测3组细胞的增殖活性,Transwell法检测3组细胞的迁移和侵袭力,Western blot法检测3组Galectin-1、上皮钙黏着蛋白(E-cadherin)和波形蛋白(Vimentin)的蛋白表达水平。结果62例患儿组织中,Galectin-1蛋白阳性表达率为69.35%,且在国际神经母细胞瘤分期系统分期Ⅲ~Ⅳ期、危险度分层高危组及发生骨转移的患儿NB组织中Galectin-1蛋白阳性表达率(78.57%、78.05%、84.00%)较Ⅰ~Ⅱ和Ⅳs期、低中危组、无骨转移者(50.00%、52.38%、59.46%)高,差异均有统计学意义(均P<0.05);siRNA-Gal1组与siRNA-NC组和空白组相比,细胞中Galectin-1 mRNA(0.23±0.06、1.04±0.05、1.00±0.08)和蛋白表达量(0.23±0.05、0.86±0.06、0.84±0.05)均降低,差异均有统计学意义(均P<0.05);siRNA-Gal1组24 h、48 h、72 h和96 h时的细胞吸光度值均低于siRNA-NC组和空白组,差异均有统计学意义(均P<0.05);siRNA-Gal1组与siRNA-NC组和空白组比较,细胞迁移能力(101.55±5.56、137.24±5.14、132.76±7.46)和侵袭能力(78.21±5.08、114.46±7.31、120.06±6.47)均明显降低,差异均有统计学意义(均P<0.001);siRNA-Gal1组与siRNA-NC组和空白组比较,Vimentin蛋白表达量(0.24±0.03、0.69±0.07、0.70±0.06)均降低,差异均有统计学意义(均P<0.05);而E-cadherin蛋白表达量(0.77±0.09、0.29±0.05、0.33±0.04)均升高,差异均有统计学意义(均P<0.05)。结论Galectin-1蛋白在NB组织中阳性表达率为69.35%,可能参与了NB的恶性化过程,沉默Galectin-1的基因表达可抑制NB细胞的增殖、迁移和侵袭力,其机制可能与抑制上皮-间质转化过程有关。

Objective To investigate the expression of Galectin-1 in neuroblastoma(NB)tissues and the effects of down-regulating this gene on cell proliferation and invasion.Methods A total of 62 cases of NB children who had complete data and were initially treated at the Department of Pediatrics,Yidu Central Hospital,Weifang Medical College from January 2010 to January 2018 were enrolled.The expression of Galectin-1 protein in NB tissues was detected by immunohistochemistry.NB cell line SH-SY5Y was cultured and divided into the siRNA-Gal1 group(with Galection-1 transfected interference sequence siRNA-Gal1),siRNA-NC group(negative control sequence siRNA-NC was transfected)and blank group(without any treatment).The expression of Galectin-1,cell proliferation activity,and cell migration and invasiveness in 3 groups were detected by real-time quantitative PCR,cell counting kit-8(CCK-8)assay and Transwell assay,respectively.Western blot were used to detect the expressions of Galectin-1,E-cadherin and Vimentin proteins in 3 groups.Results Among 62 cases,the positive expression rate of Galectin-1 protein was 69.35%.The positive expression rates of Galectin-1 protein in cells with grade of clinicopathological indexes of International Neuroblastoma Staging System stage ofⅢ-Ⅳ,cells at high risk and cells with bone metastasis(78.57%,78.05%and 84.00%)were significantly different from stage ofⅠ-ⅡandⅣs,cells at low risk and cells without bone metastasis(50.00%,52.38%,59.46%)(all P<0.05).Compared with the siRNA-NC group and the blank group,the expression of Galectin-1 mRNA(0.23±0.06 vs.1.04±0.05 and 1.00±0.08)and protein(0.23±0.05 vs.0.86±0.06 and 0.84±0.05)in the siRNA-Gal1 group was significantly decreased(all P<0.05).The cell optical density(OD)values at 24 h,48 h,72 h and 96 h in the siRNA-Gal1 group were significantly lower than those in the siRNA-NC group and the blank group(all P<0.05).Compared with the siRNA-NC group and the blank group,the siRNA-Gal1 group showed a significant decrease in cell migration(101.55±5.56 vs.137.24±5.14 and 132.76±7.46)and invasion(78.21±5.08 vs.114.46±7.31 and 120.06±6.47,all P<0.001).The expression level of Vimentin protein in the siRNA-Gal1 group was significantly lower than that in the siRNA-NC group and the blank group(0.24±0.03 vs.0.69±0.07 and 0.70±0.06)(all P<0.05),while the expression level of E-cadherin protein in the siRNA-Gal1 group was significantly higher than that in other 2 groups(0.77±0.09 vs.0.29±0.05 and 0.33±0.04)(all P<0.05).Conclusions The positive expression rate of Galectin-1 protein in NB tissues is 69.35%,which indicates that Galectin-1 might be involved in the malignant process of NB.Silencing the expression of Galectin-1 gene can inhibit the proliferation,migration and invasion of NB cells,and the mechanism may be related the inhibition of epithelial-mesenchymal transition.