唾液腺腺样囊性癌中Runx3的表达及临床意义

2016-02-27 12:10许正元何剑锋
中国现代医生 2015年25期
关键词:唾液腺

许正元+何剑锋

[摘要] 目的 探讨唾液腺腺样囊性癌(SACC)中Runx3的表达及与临床病理因素的相关性。 方法 采用荧光定量RT-PCR检测Runx3在6例SACC组织及正常唾液腺组织中的表达。免疫组化检测58例SACC和正常唾液腺组织中Runx3蛋白的表达。采用SPSS17.0软件包分析Runx3的表达程度与临床病理因素之间的相关性。 结果 qRT-PCR的测量显示唾液腺腺样囊性癌中Runx3 mRNA表达明显下调。免疫组化结果显示,Runx3在SACC中的表达与病理类型(实体型)、T临床分期及淋巴结转移显著相关,而与肿瘤远处转移存在弱相关。 结论 Runx3表达下调与SACC发生发展相关,可能是临床诊断和治疗SACC的重要生物标志物。

[关键词] Runx3;唾液腺;腺样囊性癌;RT-PCR

[中图分类号] R739.8 [文献标识码] A [文章编号] 1673-9701(2015)25-0001-03

Expression and clinical significance of Runx3 in salivary adenoid cystic carcinoma

XU Zhengyuan1 HE Jianfeng2

1.Department of Stomatology, Changxing Hospital of Traditional Chinese Medicine in Zhejiang Province, Changxing 313100, China; 2.Department of Stomatology, the First Affiliated Hospital of Medical College in Zhejiang University, Hangzhou 310000, China

[Abstract] Objective To investigate the correlation between the expression of Runx3 and the clinical pathologic factors in salivary adenoid cystic carcinoma (SACC). Methods The fluorescent quantitation RT-PCR was used to detect the expression of Runx3 in 6 cases of SACC tissues and normal salivary gland tissues. The immunohistochemistry was used to detect the expression of Runx3 protein in 58 cases of SACC and normal salivary gland tissues. The SPSS 17.0 software package was used to analyze the correlation between the expression degree of Runx3 and the clinical pathological factors. Results The qRT-PCR measurement showed that the expression of Runx3 mRNA reduced significantly in SACC. The immunohistochemical results showed that the expression of Runx3 in SACC was significantly correlated to the pathological pattern(entity type), T clinical stage and lymph node metastasis, but was slightly correlated to the distant metastasis of tumors. Conclusion Runx3 expression downregulation is correlated to the occurrence and development of SACC, which can be an important biomarker for the clinical diagnosis and treatment of SACC.

[Key words] Runx3; Salivary gland; Adenoid cystic carcinoma; RT-PCR

人类Runt相关转录因子-3(runt related transcription factor 3,Runx3)位于人染色体1p36上,该基因或蛋白的表达缺失是多种恶性肿瘤发生和发展的内在原因[1],也是肿瘤侵袭和远处转移的重要调控因子[2-6],在肿瘤的早期发生中具有重要的调控作用[7,8]。唾液腺腺样囊性癌(salivary adenoid cystic carcinoma,SACC)约占唾液腺上皮性肿瘤的10%,具有局部高侵袭性和易沿神经侵犯的临床特点,并且容易侵犯血管,导致较高的远处转移率。因此,探讨Runx3在唾液腺腺样囊性癌中的表达对判断肿瘤发生和进展及预后将有一定的参考作用。本实验中,我们通过分别运用qRT-PCR和免疫组化测量涎腺腺样囊性癌(SACC)和正常唾液腺组织中Runx3 mRNA和蛋白的表达,并通过比较Runx3的表达水平与临床因素和病理之间的相关性,从而探讨Runx3在SACC中的临床价值和诊断价值。

1 材料及方法

1.1 材料

选取2007年1月~2013年12月档案库中58例石蜡包埋的唾液腺腺样囊性癌标本,并根据WHO腺样囊性癌诊断标准重新确认病理结果(SACC组)。其中男40例,女18例;年龄25~83岁,平均51.5岁。选取6例配对-80℃保存的腺样囊性癌组织和正常腺体组织进行基因水平的检测。

1.2 方法

1.2.1 RNA的提取和实时定量RT-PCR检测 利用Trizol(Invitrogen)提取腺样囊性癌和正常腺体组织中的总RNA,实时定量RT-PCR(qRT-PCR)按照TaKaRa的SYBR green试剂盒说明说进行操作。Runx3及GAPDH引物由上海生工公司设计并生产。Runx3的上游引物为:5-CACTGGCGCTGCAACAAGA-3,Runx3的下游引物为:5-CACGAAGCGAAGGTCGTTGA-3。GAPDH的上游引物为:5-GAAGGTGAAGGTCGGAGTC-3,GAPDH的下游引物为:5-GAAGATGGTGATGGGATTTC-3。

1.2.2 免疫组织化学检测 采用超敏S-P法染色,一抗为兔抗人Runx3多克隆抗体(Abcam,美国),抗兔二抗试剂盒购自福州迈新公司,按试剂盒说明书进行免疫组织化学操作,石蜡组织切片经过脱蜡、水化,3%过氧化氢室温孵育10 min,0.01 M枸橼酸盐缓冲液高压锅抗原修复,正常山羊血清封闭30 min,滴加1∶100抗人Runx3一抗,湿盒中4℃过夜,滴加生物素化二抗室温30 min,DAB显色,苏木精复染,脱水、透明、封片,以细胞核或细胞浆内的棕黄色颗粒为阳性染色结果。以Image-Pro Plus(version 5.1,美国)分析高倍镜下(400×)阳性细胞数,阳性细胞数<10%为低表达组,≥10%为Runx3高表达组。

1.3 统计学方法

采用SPSS17.0统计学分析软件,Runx3的表达水平与肿瘤的临床病理因素相关性采用χ2检验,以单因素方差分析Runx3 mRNA表达水平在肿瘤和配对的正常组织中的差异,P<0.05为差异有统计学意义。

2 结果

2.1 Runx3 mRNA在腺样囊性癌和正常涎腺组织中的表达

RT-PCR检测不同组织中Runx3 mRNA表达结果发现:腺样囊性癌(T)中Runx3 mRNA的表达水平显著低于配对的正常腺体组织(N),差异具有统计学意义(P<0.05)。见图1。

2.2 免疫组化检测Runx3蛋白在腺样囊性癌组织中亚细胞定位表达结果

免疫组织化学检测Runx3蛋白的表达结果表明:在正常涎腺中,Runx3表达主要表达在导管上皮和腺泡细胞核中,而在SACC中,Runx3蛋白的表达水平显著降低(图2),其表达主要定位在肿瘤细胞细胞浆中,在细胞核中也存在表达。

2.3 Runx3表达水平与腺样囊性癌的临床病理因素的相关性

免疫组织化学检测结果显示:58例SACC组织中,Runx3高表达15例,Runx3低表达43例。58例腺样囊性癌患者的临床病理因素分析表明,Runx3低表达与实性型病理分型(P=0.025)、T分期(P=0.005)及淋巴结转移(P=0.040)显著相关,与远处转移存在弱相关(P=0.054)显著相关,而与发病年龄及性别无统计学意义(P>0.05)。见表1。

3 讨论

唾液腺腺样囊性癌存在局部侵袭性强和高转移等生物学特点,为口腔颌面部肿瘤远处转移率最高的恶性肿瘤之一,其浸润、转移机制是众多学者的关注焦点。作为TGF-β/SMAD诱导细胞凋亡通路中的一个重要下游调控因子,Runx3表达的下调甚或表达缺失与肿瘤的远处转移存在一定联系。而另一方面,当肿瘤中恢复RUNX3的表达能显著的抑制肿瘤细胞的迁移及侵袭能力[9]。Sakakura等[3]报道在存在腹膜转移的胃癌中Runx3的表达沉默影响一些与转移相关,比如细胞黏附、增殖、凋亡相关的因子等重要的基因的表达,并且促进了胃癌的腹膜转移。Peng等[5]通过动物实验验证了在结肠癌细胞中恢复Runx3蛋白的表达能抑制结肠癌细胞的远处转移。本研究也发现Runx3低表达的腺样囊性癌具有更强的侵袭性,并且具有更高的侵袭能力和远处转移的风险,提示Runx3的表达下调是腺样囊性癌高侵袭和远处转移的一个风险因素,这与其他一些学者的研究一致[10,11]。

在与临床病理因素的相关性比较中发现,Runx3的表达水平与肿瘤实体型病理类型显著相关(P=0.025)。由于腺样囊性癌实体型亚型具有更强的侵袭性[12],并且Runx3作为一个抑癌基因,在癌细胞的增值和分化中具有重要的调控作用[13-14],因此我们推测,Runx3蛋白在实体型腺样囊性癌中的低表达在一定程度上说明Runx3与实体型腺样囊性癌的高侵袭性存在密切联系。另外,研究指出Runx3表达率低于10%对于肺腺癌患者是一个很强的预后指标[15]。同时,Runx3低表达或沉默能显著地影响喉鳞癌患者的生存率[16],可以作为喉癌的预后分析因子[17]。有研究表明Runx3蛋白的表达降低与食管鳞癌对放疗敏感性降低显著相关,并导致患者预后水平显著下降[16]。由此,Runx3的表达可以作为腺样囊性癌患者的预后因子,并且有可能作为一个基因靶向治疗的目标基因[18,19]。

综上所述,我们发现在人正常唾液腺组织和腺样囊性癌中都存在不同程度的Runx3蛋白表达。Runx3蛋白表达下调甚或表达抑制可能是唾液腺囊性癌发生的一个重要机制。Runx3的表达与肿瘤的病理类型、淋巴结转移和远处转移显著相关,可能是临床诊断和治疗SACC的重要生物标志物。然而Lotem等[20]学者认为Runx3并非作为一个抑癌基因在调控肿瘤的发生,而是在免疫及炎症中发挥作用,从而间接调控肿瘤的发生。因此唾液腺腺样囊性癌中Runx3蛋白表达下调的调控机制及相关功能还有待深入研究,涎腺的肿瘤发生和炎症直接或许存在相关性。

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(收稿日期:2015-05-22)

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