神经母细胞瘤中Yes相关蛋白1的表达及其与药物敏感性关系的研究

Preliminary study of Yes-associated protein 1 expression and its relationship with drug sensitivity in neuroblastoma

目的初步探讨Hippo信号通路中Yes相关蛋白1(Yes-associated protein 1,YAP1)在儿童神经母细胞瘤(neuroblastoma,NB)组织中的表达,并分析其与临床病理参数和化疗药物敏感性的关系。方法应用实时荧光定量PCR(RT-qPCR)、免疫组织化学(IHC)法检测25例NB、7例节细胞神经母细胞瘤(ganglioneuroblastoma,GNB)和8例节细胞神经瘤(ganglioneuroma,GN)组织中YAP1的表达;应用IHC或PCR扩增测序法检测其中11例NB/GNB肿瘤标本化疗药物相关性分子靶标的表达或突变。结果YAP1的mRNA在NB/GNB中的相对表达量为2.369±1.420,明显高于GN的0.829±0.122,组间比较,差异有统计学意义(P<0.05);在NB/GNB中,YAP1的mRNA相对表达量与肿瘤的分期、危险度、Shimada组织学分型、病理分级、大小和是否发生淋巴结、骨髓、骨转移等有关(P<0.05),与患儿的年龄、性别无关(P>0.05)。IHC结果提示,YAP1在Ⅲ~Ⅳ期NB/GNB中呈强阳性表达,在Ⅰ~Ⅱ期NB/GNB中呈弱阳性表达,在GN中呈阴性表达,与mRNA表达水平一致。共检测肿瘤标本化疗药物相关性分子靶标5个,包括:人切除修复交叉互补基因1(ERCC1)、DNA拓扑异构酶Ⅰ(TOPO I)、DNA拓扑异构酶Ⅱ A(TOPO Ⅱ A)、DNA修复蛋白O 6-甲基转移酶(MGMT)和CYP2C19*2。肿瘤标本化疗药物敏感性分子靶标ERCC1表达检测提示铂类敏感性较高占36.4%(4/11),TOPO I、TOPO II A、CYP2C19*2的表达或多态性分别提示伊立替康/拓扑替康、蒽环类/依托泊苷、环磷酰胺敏感性较高占27.3%(3/11),MGMT表达检测提示替莫唑胺/卡莫司汀/司莫司汀敏感性较高占63.6%(7/11)。分析YAP1的表达量与化疗药物的敏感性的关系发现:YAP1在环磷酰胺敏感性较低组的表达(3.251±1.349)明显高于敏感性较高组(1.307±0.298),组间比较,差异有统计学意义(P<0.05);但未发现YAP1的表达量与铂类、伊立替康/拓扑替康、蒽环类/依托泊苷、替莫唑胺/卡莫司汀/司莫司汀敏感性相关(P>0.05)。结论YAP1的表达与NB的分期、危险度、Shimada组织学分型、病理分级、大小和转移相关,可能参与了NB/GNB的发生发展。且YAP1的表达与环磷酰胺药物敏感性相关,故其可能是NB/GNB环磷酰胺耐药的潜在治疗靶点。

Objective To explore the expression of Yes-associated protein 1 (YAP1) in tissues of neuroblastoma in children and to examine the relationship between the expression of YAP1 and its clinicopathological parameters and sensitivity to chemotherapeutic agents. Methods Quantitative real-time polymerase chain reaction (RT-qPCR) and immunohistochemical (IHC) staining were employed for detecting the expressions of mRNA and protein in neuroblastoma (NB, n=25), ganglioneuroblastoma (GNB, n=7) and ganglioneuroma (GN, n=8) tissues. And IHC staining, PCR amplification and sequencing were utilized for examining the expression or mutation of drug-related genomic markers in 11 NB/GNB samples. Results The RNA levels of YAP1 in NB/GNB were significantly higher than those in GN (2.369±1.420 vs 0.829±0.122, P<0.05). In NB/GNB, YAP1 mRNA expression was associated with tumor staging, risk stratification, Shimada’s histological classification, differentiation, size and metastasis (P<0.05). However, there was no correlation with age or gender (P>0.05). The protein level of YAP1 was consistent with mRNA level: strongly positive YAP1 protein expression in stages Ⅲ&Ⅳ tumors, weakly positive in stages Ⅰ&Ⅱ tumors and negative in GN. Five genomic markers of anti-neoplastic agents were examined, including excision repair cross-complementing 1 (ERCC1), topoisomerase Ⅰ(TOPO I), topoisomerase Ⅱ A (TOPO Ⅱ A), O 6-methylguanine-DNA methyltransferase (MGMT) and CYP2C19*2. It showed that 36.4%(4/11) tumor samples had a high sensitivity to platinum;27.3%(3/11) high sensitivities to irinotecan/topotecan, anthracycline/etoposide and cyclophosphamide respectively;63.6%(7/11) high sensitivities to temozolomide/carmustine/semustine. YAP1 mRNA expression was significantly higher in poor sensitivity to cyclophosphamide group than high sensitivity group (3.251±1.349 vs 1.307±0.298, P<0.05). Yet there was no significant correlation with sensitivities to platinum, irinotecan/topotecan, anthracycline/etoposide or temozolomide/carmustine/semustine (P>0.05). Conclusions Associated with tumor staging, risk stratification, Shimada’s histological classification, differentiation, size and metastasis, YAP1 expression may promote tumorigenesis and progression. Furthermore, its correlation with cyclophosphamide resistance makes it a potential therapeutic target for cyclophosphamide-resistant NB/GNB.