許正元+何劍鋒
[摘要] 目的 探討唾液腺腺樣囊性癌(SACC)中Runx3的表達及與臨床病理因素的相關(guān)性。 方法 采用熒光定量RT-PCR檢測Runx3在6例SACC組織及正常唾液腺組織中的表達。免疫組化檢測58例SACC和正常唾液腺組織中Runx3蛋白的表達。采用SPSS17.0軟件包分析Runx3的表達程度與臨床病理因素之間的相關(guān)性。 結(jié)果 qRT-PCR的測量顯示唾液腺腺樣囊性癌中Runx3 mRNA表達明顯下調(diào)。免疫組化結(jié)果顯示,Runx3在SACC中的表達與病理類型(實體型)、T臨床分期及淋巴結(jié)轉(zhuǎn)移顯著相關(guān),而與腫瘤遠處轉(zhuǎn)移存在弱相關(guān)。 結(jié)論 Runx3表達下調(diào)與SACC發(fā)生發(fā)展相關(guān),可能是臨床診斷和治療SACC的重要生物標志物。
[關(guān)鍵詞] Runx3;唾液腺;腺樣囊性癌;RT-PCR
[中圖分類號] R739.8 [文獻標識碼] A [文章編號] 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
人類Runt相關(guān)轉(zhuǎn)錄因子-3(runt related transcription factor 3,Runx3)位于人染色體1p36上,該基因或蛋白的表達缺失是多種惡性腫瘤發(fā)生和發(fā)展的內(nèi)在原因[1],也是腫瘤侵襲和遠處轉(zhuǎn)移的重要調(diào)控因子[2-6],在腫瘤的早期發(fā)生中具有重要的調(diào)控作用[7,8]。唾液腺腺樣囊性癌(salivary adenoid cystic carcinoma,SACC)約占唾液腺上皮性腫瘤的10%,具有局部高侵襲性和易沿神經(jīng)侵犯的臨床特點,并且容易侵犯血管,導致較高的遠處轉(zhuǎn)移率。因此,探討Runx3在唾液腺腺樣囊性癌中的表達對判斷腫瘤發(fā)生和進展及預后將有一定的參考作用。本實驗中,我們通過分別運用qRT-PCR和免疫組化測量涎腺腺樣囊性癌(SACC)和正常唾液腺組織中Runx3 mRNA和蛋白的表達,并通過比較Runx3的表達水平與臨床因素和病理之間的相關(guān)性,從而探討Runx3在SACC中的臨床價值和診斷價值。
1 材料及方法
1.1 材料
選取2007年1月~2013年12月檔案庫中58例石蠟包埋的唾液腺腺樣囊性癌標本,并根據(jù)WHO腺樣囊性癌診斷標準重新確認病理結(jié)果(SACC組)。其中男40例,女18例;年齡25~83歲,平均51.5歲。選取6例配對-80℃保存的腺樣囊性癌組織和正常腺體組織進行基因水平的檢測。
1.2 方法
1.2.1 RNA的提取和實時定量RT-PCR檢測 利用Trizol(Invitrogen)提取腺樣囊性癌和正常腺體組織中的總RNA,實時定量RT-PCR(qRT-PCR)按照TaKaRa的SYBR green試劑盒說明說進行操作。Runx3及GAPDH引物由上海生工公司設計并生產(chǎn)。Runx3的上游引物為:5-CACTGGCGCTGCAACAAGA-3,Runx3的下游引物為:5-CACGAAGCGAAGGTCGTTGA-3。GAPDH的上游引物為:5-GAAGGTGAAGGTCGGAGTC-3,GAPDH的下游引物為:5-GAAGATGGTGATGGGATTTC-3。
1.2.2 免疫組織化學檢測 采用超敏S-P法染色,一抗為兔抗人Runx3多克隆抗體(Abcam,美國),抗兔二抗試劑盒購自福州邁新公司,按試劑盒說明書進行免疫組織化學操作,石蠟組織切片經(jīng)過脫蠟、水化,3%過氧化氫室溫孵育10 min,0.01 M枸櫞酸鹽緩沖液高壓鍋抗原修復,正常山羊血清封閉30 min,滴加1∶100抗人Runx3一抗,濕盒中4℃過夜,滴加生物素化二抗室溫30 min,DAB顯色,蘇木精復染,脫水、透明、封片,以細胞核或細胞漿內(nèi)的棕黃色顆粒為陽性染色結(jié)果。以Image-Pro Plus(version 5.1,美國)分析高倍鏡下(400×)陽性細胞數(shù),陽性細胞數(shù)<10%為低表達組,≥10%為Runx3高表達組。
1.3 統(tǒng)計學方法
采用SPSS17.0統(tǒng)計學分析軟件,Runx3的表達水平與腫瘤的臨床病理因素相關(guān)性采用χ2檢驗,以單因素方差分析Runx3 mRNA表達水平在腫瘤和配對的正常組織中的差異,P<0.05為差異有統(tǒng)計學意義。
2 結(jié)果
2.1 Runx3 mRNA在腺樣囊性癌和正常涎腺組織中的表達
RT-PCR檢測不同組織中Runx3 mRNA表達結(jié)果發(fā)現(xiàn):腺樣囊性癌(T)中Runx3 mRNA的表達水平顯著低于配對的正常腺體組織(N),差異具有統(tǒng)計學意義(P<0.05)。見圖1。
2.2 免疫組化檢測Runx3蛋白在腺樣囊性癌組織中亞細胞定位表達結(jié)果
免疫組織化學檢測Runx3蛋白的表達結(jié)果表明:在正常涎腺中,Runx3表達主要表達在導管上皮和腺泡細胞核中,而在SACC中,Runx3蛋白的表達水平顯著降低(圖2),其表達主要定位在腫瘤細胞細胞漿中,在細胞核中也存在表達。
2.3 Runx3表達水平與腺樣囊性癌的臨床病理因素的相關(guān)性
免疫組織化學檢測結(jié)果顯示:58例SACC組織中,Runx3高表達15例,Runx3低表達43例。58例腺樣囊性癌患者的臨床病理因素分析表明,Runx3低表達與實性型病理分型(P=0.025)、T分期(P=0.005)及淋巴結(jié)轉(zhuǎn)移(P=0.040)顯著相關(guān),與遠處轉(zhuǎn)移存在弱相關(guān)(P=0.054)顯著相關(guān),而與發(fā)病年齡及性別無統(tǒng)計學意義(P>0.05)。見表1。
3 討論
唾液腺腺樣囊性癌存在局部侵襲性強和高轉(zhuǎn)移等生物學特點,為口腔頜面部腫瘤遠處轉(zhuǎn)移率最高的惡性腫瘤之一,其浸潤、轉(zhuǎn)移機制是眾多學者的關(guān)注焦點。作為TGF-β/SMAD誘導細胞凋亡通路中的一個重要下游調(diào)控因子,Runx3表達的下調(diào)甚或表達缺失與腫瘤的遠處轉(zhuǎn)移存在一定聯(lián)系。而另一方面,當腫瘤中恢復RUNX3的表達能顯著的抑制腫瘤細胞的遷移及侵襲能力[9]。Sakakura等[3]報道在存在腹膜轉(zhuǎn)移的胃癌中Runx3的表達沉默影響一些與轉(zhuǎn)移相關(guān),比如細胞黏附、增殖、凋亡相關(guān)的因子等重要的基因的表達,并且促進了胃癌的腹膜轉(zhuǎn)移。Peng等[5]通過動物實驗驗證了在結(jié)腸癌細胞中恢復Runx3蛋白的表達能抑制結(jié)腸癌細胞的遠處轉(zhuǎn)移。本研究也發(fā)現(xiàn)Runx3低表達的腺樣囊性癌具有更強的侵襲性,并且具有更高的侵襲能力和遠處轉(zhuǎn)移的風險,提示Runx3的表達下調(diào)是腺樣囊性癌高侵襲和遠處轉(zhuǎn)移的一個風險因素,這與其他一些學者的研究一致[10,11]。
在與臨床病理因素的相關(guān)性比較中發(fā)現(xiàn),Runx3的表達水平與腫瘤實體型病理類型顯著相關(guān)(P=0.025)。由于腺樣囊性癌實體型亞型具有更強的侵襲性[12],并且Runx3作為一個抑癌基因,在癌細胞的增值和分化中具有重要的調(diào)控作用[13-14],因此我們推測,Runx3蛋白在實體型腺樣囊性癌中的低表達在一定程度上說明Runx3與實體型腺樣囊性癌的高侵襲性存在密切聯(lián)系。另外,研究指出Runx3表達率低于10%對于肺腺癌患者是一個很強的預后指標[15]。同時,Runx3低表達或沉默能顯著地影響喉鱗癌患者的生存率[16],可以作為喉癌的預后分析因子[17]。有研究表明Runx3蛋白的表達降低與食管鱗癌對放療敏感性降低顯著相關(guān),并導致患者預后水平顯著下降[16]。由此,Runx3的表達可以作為腺樣囊性癌患者的預后因子,并且有可能作為一個基因靶向治療的目標基因[18,19]。
綜上所述,我們發(fā)現(xiàn)在人正常唾液腺組織和腺樣囊性癌中都存在不同程度的Runx3蛋白表達。Runx3蛋白表達下調(diào)甚或表達抑制可能是唾液腺囊性癌發(fā)生的一個重要機制。Runx3的表達與腫瘤的病理類型、淋巴結(jié)轉(zhuǎn)移和遠處轉(zhuǎn)移顯著相關(guān),可能是臨床診斷和治療SACC的重要生物標志物。然而Lotem等[20]學者認為Runx3并非作為一個抑癌基因在調(diào)控腫瘤的發(fā)生,而是在免疫及炎癥中發(fā)揮作用,從而間接調(diào)控腫瘤的發(fā)生。因此唾液腺腺樣囊性癌中Runx3蛋白表達下調(diào)的調(diào)控機制及相關(guān)功能還有待深入研究,涎腺的腫瘤發(fā)生和炎癥直接或許存在相關(guān)性。
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(收稿日期:2015-05-22)