I2PP2A在神经母细胞瘤组织中的表达及其降表达后SH-SY5Y细胞迁移、侵袭能力观察

Expression of I2PP2A in neuroblastoma and effects of down-expression of I2PP2A on migration and invasion of SH-SY5Y cells

目的探讨蛋白磷酸酶2A抑制剂2(I2PP2A)在神经母细胞瘤组织中的表达变化及其降表达对SH-SY5Y细胞迁移和侵袭能力的影响。方法采用免疫组化法检测66例份神经母细胞瘤组织及配对的20例份癌旁正常组织中的I2PP2A蛋白表达,并分析不同临床病理参数患儿的I2PP2A蛋白表达情况。将对数生长期的人神经母细胞瘤细胞SH-SY5Y分为空白组(正常培养不转染)、阴性对照组(转染无义随机干扰序列质粒)、沉默组(转染I2PP2A的shRNA干扰质粒),采用Western blotting法检测三组细胞I2PP2A及基质金属蛋白酶2(MMP-2)蛋白表达,细胞划痕实验及Transwell侵袭实验分别观察细胞迁移和侵袭能力。结果神经母细胞瘤组织中I2PP2A高表达41例份(62.1%),癌旁组织为7例份(35.0%),二者比较P<0.05。不同性别、年龄、核分裂—核碎裂指数(MKI)及分化程度的神经母细胞瘤患儿癌组织I2PP2A蛋白高表达率比较均无统计学差异(P均>0.05);危险度为中危+高危的患儿癌组织I2PP2A高表达率高于低危患儿,临床分期为Ⅲ期+Ⅳ期的患儿癌组织I2PP2A高表达率高于Ⅰ期+Ⅱ期+4S期患儿(P均<0.05)。与空白组、阴性对照组比较,沉默组细胞I2PP2A、MMP-2蛋白相对表达量及划痕愈合率、穿膜细胞数均降低(P均<0.05),空白组、阴性对照组细胞上述指标比较均无统计学差异(P均>0.05)。结论神经母细胞瘤组织中I2PP2A蛋白呈高表达,并与患儿危险度和临床分期密切相关;降表达I2PP2A会减弱SH-SY5Y细胞的迁移和侵袭能力,其机制可能与调控MMP-2表达有关。

Objective To investigate the expression of inhibitor of protein phosphatase2 A-2(I2PP2A)in the neuroblastoma tissues and effects down-expression of I2PP2A on the migration and invasion of SH-SY5Y cells.Methods Immunohistochemistry was applied to detect the expression of I2PP2A protein in 66 cases of neuroblastoma tissues and 20 matched cases of adjacent normal tissues,then the relationship between I2PP2A expression and clinicopathological features of patients was analyzed.Human neuroblastoma SH-SY5Y cells in the logarithmic growth phase were divided into the blank group(normal culture without transfection),negative control group(transfected with nonsense random interference sequence),and silence group(transfected with the siRNA targeted I2PP2A shRNA).The expression levels of I2PP2A and matrix metalloproteinase-2(MMP-2)protein were observed by Western blotting.The migration and invasion were detected by cell Scratch test and Transwell invasion assay.Results The high expression of I2PP2A was found in 41 cases(62.1%)in the neuroblastoma tissues and 7 cases(35.0%)in the adjacent normal tissues,with statistically significant difference(P<0.05).Whereas,no statistically significant difference was found in the high expression rate of I2PP2A protein between neuroblastoma tissues with different sex,age,MKI,and the degree of differentiation(all P>0.05).The high expression rate of I2PP2A in neuroblastoma children was higher in intermediate-risk/high-risk group than that in low-risk group,and the high expression rate of I2PP2A in children with clinical stageⅢ/Ⅳwas higher than that in children with stageⅠ/Ⅱ/4S.Compared with blank group and negative control group,the relative expression levels of I2PP2A and MMP-2 protein,the wound healing rate and the number of transmembrane cells in the silence group decreased(all P<0.05).There were no significant differences in the above indexes between blank group and negative control group(all P>0.05).Conclusions I2PP2A is highly expressed in neuroblastoma tissues,which is closely related to the risk stratifica-tion and clinical stage.Meanwhile,down-expression of I2PP2A may decrease the migration and invasion of SH-SY5Y cells by regulating the expression of MMP-2.