人涎腺腺样囊性癌中RUNX3的表达、甲基化状态及与肿瘤临床病理因素和预后之间的相关性研究
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摘要
第一章：5-氮唑-2'-脱氧胞苷(5-Aza-dc)对ACC-2、ACC-3和ACC-M细胞株RUNX3表达的影响
腺样囊性癌(adenoid cystic carcinoma，ACC)是涎腺中最为常见的恶性肿瘤之一，约占所有涎腺上皮性肿瘤的10％，其临床特点是生长缓慢，但侵袭性较强，易侵犯神经及血管，远处转移率高达40％。然而目前对该肿瘤的高侵袭性和远处转移的具体机制仍缺乏了解。人类Runt相关转录因子-3(human runt-related transcriptionfactor-3，RUNX3)是在胃癌中新发现的抑癌基因，由该基因甲基化引起的表达减少或缺失与胃癌的高侵袭性和远处转移密切相关，而在一些肿瘤细胞株中这种甲基化状态可以被DNA甲基化的逆转录酶5-氮唑-2'-脱氧胞苷(5-Aza-dc)在体外环境中逆转。基于RUNX3基因与肿瘤转移的相关性，因而对RUNX3基因及蛋白在涎腺腺样囊性癌细胞株中的表达情况作一探讨。
实验一分别用RT-PCR、免疫荧光化学和Western blot检测ACC-2、ACC-3和ACC-M细胞在5-Aza-dc处理前后的RUNX3的表达
无菌条件下取腺样囊性癌ACC-2和ACC-3细胞株以及肺高转移性腺样囊性癌ACC-M细胞株，将培养瓶置于二氧化碳孵箱中培养，培养条件为37℃、5％CO_2、饱和湿度。倒置相差显微镜下观察细胞活力，传代培养后加入300nmol／L 5-Aza-dc共培养72hrs后，分别提取总RNA和蛋白质，检测RUNX3 mRNA和蛋白质的表达。Western blot结果用Image-Pro Plus软件进行灰度分析，以GAPDH蛋白的灰度值进行标准校正，计算RUNX3蛋白产物的相对量。
实验结果：RT-PCR结果显示RUNX3 mRNA在ACC-2和ACC-3细胞株中存在弱表达，而在ACC-M中无表达。经过300nmol／L 5-Aza-dc处理后，RUNX3的表达在以上三株细胞株中的表达均提高。Western blot结果显示RUNX3蛋白分子量约为45kDa，通过灰度比值，RUNX3相对表达量在5-Aza-dc处理后比处理前有所增加，Western blot和RT-PCR的结果相似。激光共聚焦结果显示RUNX3蛋白主要定位在细胞浆中，经过300nmol／L 5-Aza-dc处理72hrs后，在ACC-2和ACC-3细胞核中出现表达，而在ACC-M细胞中仍然定位在细胞浆中。
这部分实验结果初步说明RUNX3基因甲基化可能是腺样囊性癌细胞株中RUNX3表达抑制的一个相关因素，而RUNX3在肺高转移性腺样囊性癌细胞株ACC-M中的表达缺失可能与该肿瘤的远处转移有关。由于RUNX3蛋白在细胞核内表达时才能激活相应的靶基因发挥作用，RUNX3蛋白在腺样囊性癌细胞株中的细胞浆中异位表达，可能与RUNX3蛋白的功能被抑制有关，其具体机制有待于进一步阐明。
第二章：涎腺腺样囊性癌中RUNX3基因启动子CpG岛甲基化的关键位点和演进规律
一些研究表明，RUNX3基因启动子CpG岛高甲基化是该基因表达沉默的主要方式之一。前一章结果表明腺样囊性癌细胞株ACC-2、ACC-3和ACC-M细胞株在DNA甲基化转移酶抑制剂5-Aza-dc共培养后RUNX3表达有所增加，提示在该肿瘤中RUNX3基因存在甲基化。RUNX3基因作为一个新的抑癌基因，在多种恶性肿瘤中由于启动子甲基化而导致表达抑制甚至缺失。但腺样囊性癌中RUNX3基因沉默的机制目前尚未阐明。因此在本章中对人涎腺腺样囊性癌及相应的正常涎腺组织标本中RUNX3基因启动子CpG岛多位点的甲基化状态与RUNX3基因甲基化和临床病理指标之间的相互关系作一比较分析，对腺样囊性癌中RUNX3基因甲基化的关键位点和演进规律作初步探讨，以更好地了解其分子机制。
实验二以定量甲基化特异性PCR(qMSP)法检测41例涎腺腺样囊性癌及其相应的正常涎腺组织中RUNX3基因的甲基化状态以及RUNX3基因启动子CpG岛第1-10位点中各位点的甲基化状态，用Logistic ANOVA模型分析RUNX3基因及各位点甲基化在涎腺腺样囊性癌中发生的危险度，并比较RUNX3基因启动子甲基化与腺样囊性癌临床病理因素之间的相关性。同时用Western blot法检测19例经过qMSP检测的腺样囊性癌及其匹配的正常涎腺组织中RUNX3蛋白的表达，以分析RUNX3基因启动子甲基化对蛋白表达的影响。
实验结果：实时定量qMSP结果显示腺样囊性癌及其相应正常涎腺组织中其RUNX3基因甲基化阳性率分别为42．69％-85．69％和22．17％-59．43％。RUNX3基因在第1、2位点(5’区)甲基化程度最高，随着向转录起始点方向演进，甲基化程度逐渐减弱，在转录起始点位置(第7-8位点)甲基化阳性率最低，向3’方向甲基化又略微升高。另外，在正常涎腺组织中RUNX3基因各位点甲基化定量值显著低于腺样囊性癌(P＜0．001)，同时在转录起始点位置甲基化阳性率最低。Logistic ANOVA模型分析结果进一步说明RUNX3基因启动子CpG岛的甲基化是人涎腺腺样囊性癌发生的一个重要因素，RUNX3基因CpG岛的甲基化从5’区向转录起始点方向演进，转录起始点区可能是RUNX3基因甲基化的关键位点。Western blot结果显示腺样囊性癌中RUNX3蛋白的表达量显著低于匹配的正常涎腺组织中的表达(P＜0．001)，而腺样囊性癌中RUNX3基因启动子CpG岛的甲基化阳性率显著高于正常涎腺标本(P＜0．001)，在一定程度上说明RUNX3基因甲基化是引起RUNX3蛋白表达下调的原因之一，也与肿瘤的发生相关。虽然RUNX3基因CpG岛各位点甲基化程度与各临床病理因素之间未发现有显著相关性，检测RUNX3基因转录起始点区域的甲基化状态在肿瘤的诊断和风险评估中仍有参考价值。
基因甲基化是引起肿瘤发生一个重要因素，本实验中人涎腺腺样囊性癌RUNX3基因启动子CpG岛甲基化的发生率显著高于正常涎腺组织，并且是导致RUNX3蛋白表达下调的原因之一，说明RUNX3基因甲基化与腺样囊性癌的发生存在密切联系。通过定量qMSP法检测各位点的甲基化状态说明各位点的甲基化程度存在差异，其甲基化现象是从5’区向转录起始点不断演进，而启动子区转录起始点可能是RUNX3基因甲基化的关键位点，在检测RUNX3基因甲基化状态时，应选择转录起始点周围的区域进行检测，如本实验中的第6-8位点。另外，本实验中未发现RUNX3基因的甲基化与肿瘤临床病理因素之间的相关性，这可能需要增加临床样本进行进一步评估。
第三章：RUNX3在涎腺腺样囊性癌和正常涎腺中的表达及与患者预后的相关性
涎腺腺样囊性癌患者的远期预后较差。目前的一些研究认为外科切缘阳性、临床分期、病理类型、神经侵犯等是腺样囊性癌患者的一个预后因素。同时，在分子水平一些研究者认为一些增殖或凋亡相关基因与腺样囊性癌患者的预后相关。RUNX3作为在胃癌中新发现的一种抑癌基因，其表达的沉默与胃癌的发生和发展及远处转移有着高度的相关性，而且也影响着胃癌的治疗效果和预后，因此检测恶性肿瘤中RUNX3的表达可能提供一个比较好的预后指标。由于腺样囊性癌患者出现远处转移是导致其预后差的一个主要因素，虽然在涎腺腺样囊性癌中没有关于RUNX3表达的报道，基于RUNX3与肿瘤远处转移的相关性，探讨RUNX3在ACC中的表达对判断该肿瘤的发生进展和预后都将有一个参考作用。
塞验三分别用荧光定量qRT-PCR、Western blot和免疫组织化学检测RUNX3在正常涎腺和腺样囊性癌中的表达
从液氮中取出9例正常涎腺(包括3例腮腺、2例舌下腺及2例颌下腺)和7例腺样囊性癌组织，分别以荧光定量RT-PCR和Western blot检测RUNX3 mRNA和蛋白的表达情况。qRT-PCR结果以标准曲线法测定RUNX3 mRNA的相对表达量。Western blot结果用Image-Pro Plus软件进行灰度分析，以GAPDH蛋白的灰度值进行标准校正，计算RUNX3蛋白产物的相对量。同时用免疫组织化学检测73例石蜡包埋的腺样囊性癌组织中RUNX3蛋白在肿瘤细胞中的定位表达情况。
实验结果：荧光定量qRT-PCR结果显示，与正常涎腺组织中RUNX3 mRNA的表达量(0．19±0．08)相比，RUNX3 mRNA在腺样囊性癌中的表达水平(0．09±0．05)下调了2．2倍。Western blot结果显示在人正常涎腺和腺样囊性癌中均存在分子量约为45kDa的RUNX3蛋白的表达。通过灰度值显示：腺样囊性癌组织中RUNX3蛋白的相对表达量(0．79±0．03)低于正常涎腺组织中的相对表达量(0．88±0．03)。免疫组化显示在正常涎腺组织中，RUNX3蛋白主要表达在腺泡细胞和导管细胞的细胞浆和细胞核中；在腺样囊性癌中则均表达在细胞浆中，在一些实体型ACC中则无表达。
实验四分析RUNX3表达与腺样囊性癌临床病理指标和预后的相关性
以Image-Pro Plus图像分析软件检测RUNX3蛋白在73例腺样囊性癌中的表达水平，并以Tertile法将其分为高表达组(≥14．91％)、中表达组(6．94％-14．91％)和低表达组(≤6．94％)，并分析RUNX3的表达水平与73例腺样囊性癌的临床病理指标的相关性。同时以Cox回归模型评价各临床病理指标与预后的相关性，以Kaplan-Meier生存曲线检测RUNX3表达水平对患者预后的影响，从而评估RUNX3在涎腺腺样囊性癌中的表达对评价患者的预后的临床价值。
实验结果：免疫组化半定量结果显示RUNX3高、中和低表达组中RUNX3的阳性表达率分别为19．60±4,26％、11．19±2．42％和4．78±1．59％。而筛孔型、导管型和实性型腺样囊性癌中RUNX3的表达率分别为：13．69±6．46％、11．78±4．40％和8．864±5．20％，RUNX3低表达与腺样囊性癌的组织病理类型(实性型)密切相关(P=0．025)，同时RUNX3低表达的腺样囊性癌具有高度的远处转移可能(P=0．009)，这些结果提示RUNX3的表达抑制与肿瘤的分化及进展存在密切联系。在T1-3期腺样囊性癌中RUNX3的表达率为13．65±6．44％，在T4期中其表达率为10．24±5．19％。统计学结果显示RUNX3低表达与肿瘤临床分期存在弱相关(P=0．061)。RUNX3的低表达与患者的年龄、性别、肿瘤部位、神经浸润和淋巴转移无统计学差异。预后分析显示18例RUNX3高表达、37例RUNX3中表达及18例RUNX3低表达腺样囊性癌患者的5年生存率分别为90％、75．4％和48．27％。单因素Cox回归模型分析显示RUNX3低表达(P=0．012)，淋巴结转移(P=0．007)，远处转移(P＜0．001)与患者预后显著相关。Kaplan-Meier生存曲线结果显示RUNX3高表达组的生存率显著高于RUNX3低表达组(P=0．022)。同时临床分期(T4期)(P=0．003)，淋巴转移(P=0．004)和远处转移(P＜0．001)与患者的预后显著相关。然而腺样囊性癌的病理类型(实体型)(P=0．771)和神经浸润(P=0．554)与患者的预后无统计学差异。而多因素Cox回归模型显示只有远处转移是患者预后的独立指标(P=0．043)，而T4分期与患者的预后存在弱相关(P=0．063)。
目前的研究认为RUNX3作为一个抑癌基因，其表达水平与患者预后和生存期显著相关，可能是影响恶性肿瘤预后的独立指标。RUNX3高表达的恶性肿瘤中，其5年生存率显著高于RUNX3低表达者，并且与细胞分化程度有关，高分化的恶性肿瘤中RUNX3表达水平高于低分化者，且RUNX3低表达与肿瘤的远处转移能力显著相关。本实验的结果表明RUNX3蛋白的表达可能与人涎腺腺样囊性癌的病理分化和转移相关，提示RUNX3蛋白的表达下调可能对ACC的发生发展具有重要作用，检测该肿瘤中RUNX3蛋白的表达情况可能有助于腺样囊性癌的筛查、临床诊断和预后判断。
结论：
1．RUNX3在腺样囊性癌细胞株ACC-2和ACC-3中存在表达，而在肺高转移腺样囊性癌细胞株ACC-M中则无表达。经过300nmol／L的5-Aza-dc处理72小时后能提高RUNX3在这些细胞株中的表达。
2．人涎腺腺样囊性癌中RUNX3基因甲基化的概率显著高于正常涎腺组织，提示RUNX3基因启动子CpG岛甲基化是人涎腺腺样囊性癌发生的一个重要因素。
3．RUNX3基因CpG岛的甲基化的规律是从5’区向转录起始点方向演进，转录起始点的甲基化程度最低，提示该位点为RUNX3基因甲基化的关键位点。
4．RUNX3基因CpG岛的甲基化状态与肿瘤临床病理指标之间无显著相关性。
5．RUNX3在人涎腺腺样囊性癌中存在表达。RUNX3蛋白低表达与肿瘤的分化和远处转移及患者的预后存在显著关系，对临床预后的判断具有诊断价值。
Part one: Effect of 5-Aza-dc on RUNX3 expression in salivary gland adenoidcystic carcinoma cell lines ACC-2, ACC-3 and ACC-M
Adenoid cystic carcinomas (ACC)are one of the most common malignancies insalivary gland, and comprise approximately 10% of all epithelial salivary neoplasms. Itis characterized by slow growth, followed by high neural and blood vessal invasion. Thedistant metastasis rate is appropriately 40% which results in a poor long term survival.Surgery followed by postoperative radiation therapy is the treatment of choice. It isreported that alternation of the antioncogen and cell adhesion molecules has beenproposed as factors associated with the aggressive behavior in salivary gland adenoidcystic carcinoma. However the underlying mechanism for the aggressive nature and thefatal course of this tumor are pooly investigated. The human runt-related transcriptionfactor-3 (RUNX3) is a tumor suppressor gene in gastric cancer. The inhibition orsilence of this gene expression is supposed to be highly associated with invasion anddistant metastasis of a variety of malignant tumor cell lines, whilst, the promotermethylation of RUNX3 gene is the main cause. However, after using the DNAmethyltransferase enzyme inhibitor, 5-Aza-dc, the expression of this gene can berestored in vitro. Promoter methylation is a common mechanism to inactivate tumorsuppressor genes in tumorigenesis, which plays an important role in the development ofsalivary gland tumors; however, the expression of RUNX3 in salivary tumor cell lines israre. Thus, it is necessary to investigate the expression of RUNX3 gene and protein insalivary gland adnoid cystic carcinoma cell lines, namely ACC-2, ACC-3, and ACC-M.
Experiment one: in this part, RT-PCR, laser scanning confocal microscope (LSCM),and western blot were used to detect the expression of RUNX3 gene and protein insalivary gland adenoic cystic carcinoma cell lines, ACC-2, ACC-3, and ACC-M,before/after treated with 5-Aza-dc, restropectively.
Three adenoid cystic carcinoma cell lines (ACC-2, ACC-3, and ACC-M) were usedin this vitro experiment. Those cell lines were cultured in RPMI-1640 with 10% fetalbovine serum, and incubated them at 37℃in a humidified atmosphere containing5%CO_2. 5-Aza-dc (300nmol/L) was added into the medium, and the cell lines were cultured together with 5-Aza-dc for 72 hours. After that the total RNA and proteinswere isolated from the cultured cells with or without treated with 5-Aza-dc. RT-PCRand western blot were used to detect the RUNX3 mRNA and protein expression in thesethree cells lines, respectively. In western blot analysis, the relative level of RUNX3protein was decided by the ratio between RUNX3 gradation and GAPDH gradationwith using Image-Pro Plus software.
Results: RT-PCR and western blot analysis showed weak expression of RUNX3mRNA and protein in ACC-2 and ACC-3, and no expression of RUNX3 in ACC-3 cellline. The western blot analysis disclosed a RUNX3 protein of 45kDa in the three ACCcell lines. After treated by 300 nmol/L 5-Aza-dc for 72 hours, the expression level ofRUNX3 in ACC-2 and ACC-3 was improved, and in ACC-M was restored by analyzingthe gradation ratio of RUNX3 protein and GAPDH protein. A similar result was foundbetween RT-PCR and western blot analysis. The results of LSCM showed that theRUNX3 protein located mainly in the cytoplasm of the ACC cell lines. After treatedwith 300nmol/L 5-Aza-dc for 72 hours, both nuclear and cytoplasmic location ofRUNX3 positive signals were found in the ACC-2 and ACC-3 cells. However, the weakpositive signal was still only found in the cytoplasm of ACC-M cells
In this part, the expression of RUNX3 gene and protein in salivary gland ACC celllines, ACC-2, ACC-3, and ACC-M, before/after treated with 5-Aza-dc, was exploredrestropectively. These results indicate that the promoter methylation of RUNX3 mightplay an important role in the inhibition of RUNX3 expression in ACC cell lines. Thesilence expression of RUNX3 in high lung metastasis salivary adenoid cystic carcinomacell line, ACC-M, provide a clew that the RUNX3 inhibition is associated with distantmetastasis of ACC. Because RUNX3 can fuction as a tumor suppressor when locates inthe nuclear of cells, the cytoplasmic mislocalization of RUNX3 in ACC cells might becorrelated with the development and progression of ACC.
Part two: Promoter methylation and protein expression of the RUNX3 gene inthe clinicopathologic assessment of salivary gland adenoid cystic carcinoma
The methylation of CpG island in promoter region is the main mechanism of theRUNX3 gene silence. In part one, the results showed that the RUNX3 expression waselevated in adenoid cystic carcinoma cell lines ACC-2, ACC-3, and ACC-M after being treated by 5-Aza-dc, which indicates the association between promoter methylation inRUNX3 gene and the progression of salivary gland adnoid cystic carcinoma. As a tumorsuppressor, the RUNX3 expression was inhibited due to the promoter methylation in avariety of malignant tumors. However the mechanism of inhibition or sitence ofRUNX3 gene in salivary gland adenoid cystic carcinoma is pooly investigated. Thus, inthis chapter, the methylation status in successive ten regions ranging from the 5' regionto the transcription start site within the RUNX3 CpG island and the expression levels ofRUNX3 protein in ACC samples and compared non-neoplastic salivary glands wereexplored, and the relationship between the methylation status of RUNX3 gene andRUNX3 protein expression or the clinicopathological parameters was also analyzed, aswell as the mechanism of the critical regions and spreading of the RUNX3 CpG islandmethylation in ACC.
Experiment two: The quantitive MSP method was used to detect the methylationstatus of CpG island in various regions (No. 1-10) of RUNX3 promoter region, and theWestern blot method was used for detecting the expression of RUNX3 protein in 41salivary gland ACC samples and the corresponding non-neoplastic salivary glands. ALogistic ANOVA model is used to analysis the risk ratio between the methylation statusof CpG island in RUNX3 gene and development of salivary ACC, meanwhile, thepossible association among the methylation of RUNX3 gene, the clinicopathologicalparameters of ACCs, and RUNX3 protein expression was compared.
Results: The results of qMSP show that the hypermethylation initially occurs atthe most 5' region of the RUNX3 CpG island and spreads to the transcription start site.And the methylation rate is highest in region No. 1 and No. 2 among the successive tenregions ranging from the 5' region to the transcription start site within the RUNX3 CpGisland, and lowest in the transcription start site both in ACCs and normal salivary glands.Furthermore, there is no methylation in the transcription start site in nomal salivaryglands tissues. Together with the results of Logistic ANOVA model analysis, thoseresult indicats that the transcription start site within the RUNX3 promoter CpG island iscritical for gene silencing. Western blot results reveal that the RUNX3 protein level inACC was significantly lower than in normal salivary glands(P<0.001), in combinationthe results of qMSP, it is presumed that the RUNX3 gene methylation is one of the reason inducing the down-regulation of RUNX3 in ACCs. Although there is norelationship between the promoter methylation of RUNX3 CpG island and theclinicopahtological parameters of ACCs, analysis the methylation status at thetranscription start site of the RUNX3 CpG island can be used in diagnosis and riskassessment of ACCs.
The promoter methylation is a common mechanism in the inactivation of tumorsuppressor genes in varieties of malignancies. In this study, we found the rate ofpromoter methylation of RUNX3 CpG island in salivary gland ACCs is significantlyhigher than it is in normal salivary gland, suggesting that the promoter methylation ofRUNX3 gene may be associated with the development of salivary gland ACCs. TheqMSP results reveal that the methylation ratio of RUNX3 CpG island differs in differentregion, hypermethylation initially occurs at the most 5' region of the RUNX3 CpGisland and spreads to the transcription site, which is supposed to be the critical site forgene silencing. Thus, the region spanning the transcription site, such as No. 6-8, shouldbe used to evaluate the methylation status of RUNX3 gene in tumors. Additionally, Norelationship between hypermethylation of RUNX3 gene and clinicopathologicalparameters in patients with salivary gland ACCs was found. A large clinical trial wasrequired to further evaluate the potential relationship.
Part three: Expression of RUNX3 in salivary gland adenoid cystic carcinomaand its association with tumor progress and prognosis
The long-term survival in patients with salivary gland adenoid cystic carcinoma isgloomy. Various parameters, including positive surgical margins, clinical stage,histopathological patterns, and perineural invasion, have been reported as relevantprognostic factor in patients with ACC. In addition to these chinicopathological factors,several investigators have explored the possible association between the proliferation orapoptosis-associated proteins and the survival in patients with salivary ACC. However,the precise mechanism responsible for its carcinogenesis has not been fully clarified.RUNX3 gene, supposed to be a new tumor suppressor, the silence expression of thisgene is highly correlated with the development and progress of gastric cancer, as well asthe therapy outcome and the survival in patients with gastric carcinomas. To detect theexpression of RUNX3 in a variety of malignant rumors might provide a better prognostic indicator. Moreover, the lack of RUNX3 expression is probably associatedwith distant metastasis in malignancies, such as gastric cancer. Although there is noreport on its expression in salivary ACC, which presents typically distant meatstasisresulting in a fatal outcome, on basis of the relationship between the RUNX3 expressionand distant meatastasis, RUNX3 expression might be conceivably associated with thetumorigenesis, progression, distant meatastasis, and survival of salivary ACC.
Experiment three: The quantitative RT-PCR (qRT-PCR), Western blot andimmunohistochemistry were applied for detecting the expression of RUNX3 in normalhuman salivary glands and adenoid cystic carcinomas in this part.
Nine normal adult salivary glands (including 3 parotid glands, 2 sublingual glands,and 2 submandibular glands) and seven adenoid cystic carcinomas frozen tissues wereobtained. The expressions of RUNX3 mRNA and protein were detected by usingqRT-PCR and western blot analysis, respectively. The relative RUNX3 mRNA levelwas measured by using the threshold cycle (Ct) method. In western blot analysis, therelative level of RUNX3 protein was decided by the ratio of RUNX3 gradation andGAPDH gradation with using Image-Pro Plus software. Seventy-three formalin-fixed,paraffin-embedded salivary ACC specimens were achieved. The immunohistochemistrywas applied to detect the subcellular location of RUNX3 protein in normal salivaryglands and ACC.
Results: The relative quantification of RUNX3 mRNA expression in ACC(0.09±0.05) was two-fold lower than that in normal salivary glands (0.19±0.08).Western blot analysis disclosed a RUNX3 protein of 45 kDa both in human normalsalivary glands and ACC.According to the results of gradation analysis, the relative ofRUNX3 protein expression in salivary gland ACC (0.79±0.03) was lower than that innormal salivary glands (0.88±0.03). However, no statistical difference between thesetwo groups. Immunohistochemistry results showed that RUNX3 protein located mainlyin the nuclear and cytoplasm of acinous cell and ductal cell in normal salivary gland;and only in the cytoplasm of tumor cell in ACC. In some cases of solid type ACC, noexpression RUNX3 protein was found.
Experiment four: The potential relationship between the expression of RUNX3 andthe clinicopathological factors and survival in patients with adenoid cystic carcinoma was analyzed in this part.
The semi-quantative RUNX3 level in 73 adenoid cystic carcinoma cases was detectedwith using Image-Pro Plus software. Continuous variables of RUNX3 labeling indexwere divided into three discrete exclusive variables according to the distribution of thesamples. The tertile cut-off values were as follows: High group (≥14.91%), intermediategroup (6.94%-14.91%), and low group (≤6.94%). Significant differences between theexpression of RUNX3 and clinicopathological parameters were compared. Theunivariate analysis with the Cox regression model was used to determine identifiedprognostic factors, and multivariate analysis with the Cox regression model was used toexplore combined effects. Survival analysis was computered by means of theKaplan-Meier method and significant levels were assessed by means of the log-rank test.Thereby, the application of RUNX3 expression in salivary gland ACC to predict thepatients' survival was assessed.
Results: The semi-quantitative results of immunohistochemistry showed that the high,intermediate, and low RUNX3 expressions were 19.60±4.26%, 11.19±2.42%, and4.78±1.59%, respectively. And the RUNX3 expressions were 13.69±6.46% in thecribriform pattern, 11.78±4.40% in the tubular pattern, and 8.86±5.20% in the solidpattern. Low RUNX3 expression was significantly correlated with the histologicalpatterns of ACC, namely solid subtype (P=0.025), meanwhile, ACC with lower RUNX3expression significantly tended to be have more frequent distant metastasis (P=0.009),indicating the the association between down-regulation of RUNX3 expression andtumor progression. The RUNX3 expression percentage was 13.65±6.44% in tumorswith stage T1-3, and 10.24±5.19% in stage T4. There is only a weak tendency betweenthe low RUNX3 expression and stage T4 (P=0.061). No statistical differences betweenRUNX3 expression and other clinicopathologic factors, such as age, gender, tumor site,perineural invasion, and lymph nod involvement in ACCs, was detected. The 5-yearsurvival rate was 90% in the 18 patients with high levels of RUNX3 expression, 75.4%in the 37 patients with moderate level, and 48.27% in the 18 patients with lowlevel.Using univariate analysis of Cox regression model, the following variables werefound to be significantly associated with a worse prognosis, including low expression ofRUNX3 protein (P=0.012), lymph node involvement (P=0.007), and distant metastasis (P<0.001). Kaplan-Meier survival curves showed that the survival rates of patients withlow expression of RUNX3 was significantly worse than that of patients with moderateor high RUNX3 expression (P=0.022). Meanwhile, we observed that some otherclinicopathogical parameters, including stage T4 (P=0.003), lymph node involvement(P=0.004), and distant metastasis (P<0.001) were significantly related with decreasedsurvival. However, no significant association between overall survival and solidhistotype (P=0.771) or perineural invasion (P=0.554) was found. Multivariate survivalanalysis revealed that only distant metastasis was an independent significant prognosticfactor (P=0.043). Stage T4 only had a weak association with overall survival (P=0.063).RUNX3 gene, as a tumpr suppressor, is signigicantly associated with the survival rateand prognosis in patients with malignancies, and might be an independent predictor forthe prognosis. The 5-year survival rate is significant higher in tumor with highexpression of RUNX3 protein than this with low expression level. Meanwhile, theexpression of RUNX3 protein is correlated with cell differention, furthermore, the lowor lack expression of RUNX3 is potentially associated with distant metastasis. Theresults of this study indicate that the low expression of RUNX3 in salivary gland Aceis correlated with tumor development and metastasis. Together, the expression ofRUNX3 protein might be a candidate for diagnosis and prognostic marker in salivarygland ACCs. It has a pivotal role in the tumor proliferation and apoptosis.
Conclusions:
1. There is RUNX3 expression in adenoid cystic carcinoma cell lines ACC-2 andACC-3, and silence of RUNX3 in ACC-M. The DNA methyltransferase enzymeinhibitor, 5-Aza-dc, can elevate the RUNX3 level among these three cell lines.
2. The incidence of promoter methylation in RUNX3 gene CpG island in humansalivary gland adenoid cystic carcinoma is significanctly higher that it is innon-neoplastic salivary gland, indicating that the promoter methylation of RUNX3gene plays an critical role in the development of this malignancy.
3. The promoter methylation of RUNX3 CpG island spreads frorn the most 5' regionto the rranscriptiong start site in human salivary gland ACC, the incidence rate ofpromoter methylation in the transcriptiong start site is the lowest, suggesting this site might be a critical region for the methylation of RUNX3 gene.
4. There is no significant difference between the methylation status of RUNX3 geneand the clinicopathological parameters in patients with ACCs.
5. The expression of RUNX3 protein is down-regulated in salivary gland ACC. Thelower expression of this protein is significantly associated with rumor differentiation,distant metastasis, and prognosis. The expression of RUNX3 protein has a definitevalue in judging prognosis in salivary gland ACC.
腺样囊性癌(adenoid cystic carcinoma，ACC)是涎腺中最为常见的恶性肿瘤之一，约占所有涎腺上皮性肿瘤的10％，其临床特点是生长缓慢，但侵袭性较强，易侵犯神经及血管，远处转移率高达40％。然而目前对该肿瘤的高侵袭性和远处转移的具体机制仍缺乏了解。人类Runt相关转录因子-3(human runt-related transcriptionfactor-3，RUNX3)是在胃癌中新发现的抑癌基因，由该基因甲基化引起的表达减少或缺失与胃癌的高侵袭性和远处转移密切相关，而在一些肿瘤细胞株中这种甲基化状态可以被DNA甲基化的逆转录酶5-氮唑-2'-脱氧胞苷(5-Aza-dc)在体外环境中逆转。基于RUNX3基因与肿瘤转移的相关性，因而对RUNX3基因及蛋白在涎腺腺样囊性癌细胞株中的表达情况作一探讨。
实验一分别用RT-PCR、免疫荧光化学和Western blot检测ACC-2、ACC-3和ACC-M细胞在5-Aza-dc处理前后的RUNX3的表达
无菌条件下取腺样囊性癌ACC-2和ACC-3细胞株以及肺高转移性腺样囊性癌ACC-M细胞株，将培养瓶置于二氧化碳孵箱中培养，培养条件为37℃、5％CO_2、饱和湿度。倒置相差显微镜下观察细胞活力，传代培养后加入300nmol／L 5-Aza-dc共培养72hrs后，分别提取总RNA和蛋白质，检测RUNX3 mRNA和蛋白质的表达。Western blot结果用Image-Pro Plus软件进行灰度分析，以GAPDH蛋白的灰度值进行标准校正，计算RUNX3蛋白产物的相对量。
实验结果：RT-PCR结果显示RUNX3 mRNA在ACC-2和ACC-3细胞株中存在弱表达，而在ACC-M中无表达。经过300nmol／L 5-Aza-dc处理后，RUNX3的表达在以上三株细胞株中的表达均提高。Western blot结果显示RUNX3蛋白分子量约为45kDa，通过灰度比值，RUNX3相对表达量在5-Aza-dc处理后比处理前有所增加，Western blot和RT-PCR的结果相似。激光共聚焦结果显示RUNX3蛋白主要定位在细胞浆中，经过300nmol／L 5-Aza-dc处理72hrs后，在ACC-2和ACC-3细胞核中出现表达，而在ACC-M细胞中仍然定位在细胞浆中。
这部分实验结果初步说明RUNX3基因甲基化可能是腺样囊性癌细胞株中RUNX3表达抑制的一个相关因素，而RUNX3在肺高转移性腺样囊性癌细胞株ACC-M中的表达缺失可能与该肿瘤的远处转移有关。由于RUNX3蛋白在细胞核内表达时才能激活相应的靶基因发挥作用，RUNX3蛋白在腺样囊性癌细胞株中的细胞浆中异位表达，可能与RUNX3蛋白的功能被抑制有关，其具体机制有待于进一步阐明。
第二章：涎腺腺样囊性癌中RUNX3基因启动子CpG岛甲基化的关键位点和演进规律
一些研究表明，RUNX3基因启动子CpG岛高甲基化是该基因表达沉默的主要方式之一。前一章结果表明腺样囊性癌细胞株ACC-2、ACC-3和ACC-M细胞株在DNA甲基化转移酶抑制剂5-Aza-dc共培养后RUNX3表达有所增加，提示在该肿瘤中RUNX3基因存在甲基化。RUNX3基因作为一个新的抑癌基因，在多种恶性肿瘤中由于启动子甲基化而导致表达抑制甚至缺失。但腺样囊性癌中RUNX3基因沉默的机制目前尚未阐明。因此在本章中对人涎腺腺样囊性癌及相应的正常涎腺组织标本中RUNX3基因启动子CpG岛多位点的甲基化状态与RUNX3基因甲基化和临床病理指标之间的相互关系作一比较分析，对腺样囊性癌中RUNX3基因甲基化的关键位点和演进规律作初步探讨，以更好地了解其分子机制。
实验二以定量甲基化特异性PCR(qMSP)法检测41例涎腺腺样囊性癌及其相应的正常涎腺组织中RUNX3基因的甲基化状态以及RUNX3基因启动子CpG岛第1-10位点中各位点的甲基化状态，用Logistic ANOVA模型分析RUNX3基因及各位点甲基化在涎腺腺样囊性癌中发生的危险度，并比较RUNX3基因启动子甲基化与腺样囊性癌临床病理因素之间的相关性。同时用Western blot法检测19例经过qMSP检测的腺样囊性癌及其匹配的正常涎腺组织中RUNX3蛋白的表达，以分析RUNX3基因启动子甲基化对蛋白表达的影响。
实验结果：实时定量qMSP结果显示腺样囊性癌及其相应正常涎腺组织中其RUNX3基因甲基化阳性率分别为42．69％-85．69％和22．17％-59．43％。RUNX3基因在第1、2位点(5’区)甲基化程度最高，随着向转录起始点方向演进，甲基化程度逐渐减弱，在转录起始点位置(第7-8位点)甲基化阳性率最低，向3’方向甲基化又略微升高。另外，在正常涎腺组织中RUNX3基因各位点甲基化定量值显著低于腺样囊性癌(P＜0．001)，同时在转录起始点位置甲基化阳性率最低。Logistic ANOVA模型分析结果进一步说明RUNX3基因启动子CpG岛的甲基化是人涎腺腺样囊性癌发生的一个重要因素，RUNX3基因CpG岛的甲基化从5’区向转录起始点方向演进，转录起始点区可能是RUNX3基因甲基化的关键位点。Western blot结果显示腺样囊性癌中RUNX3蛋白的表达量显著低于匹配的正常涎腺组织中的表达(P＜0．001)，而腺样囊性癌中RUNX3基因启动子CpG岛的甲基化阳性率显著高于正常涎腺标本(P＜0．001)，在一定程度上说明RUNX3基因甲基化是引起RUNX3蛋白表达下调的原因之一，也与肿瘤的发生相关。虽然RUNX3基因CpG岛各位点甲基化程度与各临床病理因素之间未发现有显著相关性，检测RUNX3基因转录起始点区域的甲基化状态在肿瘤的诊断和风险评估中仍有参考价值。
基因甲基化是引起肿瘤发生一个重要因素，本实验中人涎腺腺样囊性癌RUNX3基因启动子CpG岛甲基化的发生率显著高于正常涎腺组织，并且是导致RUNX3蛋白表达下调的原因之一，说明RUNX3基因甲基化与腺样囊性癌的发生存在密切联系。通过定量qMSP法检测各位点的甲基化状态说明各位点的甲基化程度存在差异，其甲基化现象是从5’区向转录起始点不断演进，而启动子区转录起始点可能是RUNX3基因甲基化的关键位点，在检测RUNX3基因甲基化状态时，应选择转录起始点周围的区域进行检测，如本实验中的第6-8位点。另外，本实验中未发现RUNX3基因的甲基化与肿瘤临床病理因素之间的相关性，这可能需要增加临床样本进行进一步评估。
第三章：RUNX3在涎腺腺样囊性癌和正常涎腺中的表达及与患者预后的相关性
涎腺腺样囊性癌患者的远期预后较差。目前的一些研究认为外科切缘阳性、临床分期、病理类型、神经侵犯等是腺样囊性癌患者的一个预后因素。同时，在分子水平一些研究者认为一些增殖或凋亡相关基因与腺样囊性癌患者的预后相关。RUNX3作为在胃癌中新发现的一种抑癌基因，其表达的沉默与胃癌的发生和发展及远处转移有着高度的相关性，而且也影响着胃癌的治疗效果和预后，因此检测恶性肿瘤中RUNX3的表达可能提供一个比较好的预后指标。由于腺样囊性癌患者出现远处转移是导致其预后差的一个主要因素，虽然在涎腺腺样囊性癌中没有关于RUNX3表达的报道，基于RUNX3与肿瘤远处转移的相关性，探讨RUNX3在ACC中的表达对判断该肿瘤的发生进展和预后都将有一个参考作用。
塞验三分别用荧光定量qRT-PCR、Western blot和免疫组织化学检测RUNX3在正常涎腺和腺样囊性癌中的表达
从液氮中取出9例正常涎腺(包括3例腮腺、2例舌下腺及2例颌下腺)和7例腺样囊性癌组织，分别以荧光定量RT-PCR和Western blot检测RUNX3 mRNA和蛋白的表达情况。qRT-PCR结果以标准曲线法测定RUNX3 mRNA的相对表达量。Western blot结果用Image-Pro Plus软件进行灰度分析，以GAPDH蛋白的灰度值进行标准校正，计算RUNX3蛋白产物的相对量。同时用免疫组织化学检测73例石蜡包埋的腺样囊性癌组织中RUNX3蛋白在肿瘤细胞中的定位表达情况。
实验结果：荧光定量qRT-PCR结果显示，与正常涎腺组织中RUNX3 mRNA的表达量(0．19±0．08)相比，RUNX3 mRNA在腺样囊性癌中的表达水平(0．09±0．05)下调了2．2倍。Western blot结果显示在人正常涎腺和腺样囊性癌中均存在分子量约为45kDa的RUNX3蛋白的表达。通过灰度值显示：腺样囊性癌组织中RUNX3蛋白的相对表达量(0．79±0．03)低于正常涎腺组织中的相对表达量(0．88±0．03)。免疫组化显示在正常涎腺组织中，RUNX3蛋白主要表达在腺泡细胞和导管细胞的细胞浆和细胞核中；在腺样囊性癌中则均表达在细胞浆中，在一些实体型ACC中则无表达。
实验四分析RUNX3表达与腺样囊性癌临床病理指标和预后的相关性
以Image-Pro Plus图像分析软件检测RUNX3蛋白在73例腺样囊性癌中的表达水平，并以Tertile法将其分为高表达组(≥14．91％)、中表达组(6．94％-14．91％)和低表达组(≤6．94％)，并分析RUNX3的表达水平与73例腺样囊性癌的临床病理指标的相关性。同时以Cox回归模型评价各临床病理指标与预后的相关性，以Kaplan-Meier生存曲线检测RUNX3表达水平对患者预后的影响，从而评估RUNX3在涎腺腺样囊性癌中的表达对评价患者的预后的临床价值。
实验结果：免疫组化半定量结果显示RUNX3高、中和低表达组中RUNX3的阳性表达率分别为19．60±4,26％、11．19±2．42％和4．78±1．59％。而筛孔型、导管型和实性型腺样囊性癌中RUNX3的表达率分别为：13．69±6．46％、11．78±4．40％和8．864±5．20％，RUNX3低表达与腺样囊性癌的组织病理类型(实性型)密切相关(P=0．025)，同时RUNX3低表达的腺样囊性癌具有高度的远处转移可能(P=0．009)，这些结果提示RUNX3的表达抑制与肿瘤的分化及进展存在密切联系。在T1-3期腺样囊性癌中RUNX3的表达率为13．65±6．44％，在T4期中其表达率为10．24±5．19％。统计学结果显示RUNX3低表达与肿瘤临床分期存在弱相关(P=0．061)。RUNX3的低表达与患者的年龄、性别、肿瘤部位、神经浸润和淋巴转移无统计学差异。预后分析显示18例RUNX3高表达、37例RUNX3中表达及18例RUNX3低表达腺样囊性癌患者的5年生存率分别为90％、75．4％和48．27％。单因素Cox回归模型分析显示RUNX3低表达(P=0．012)，淋巴结转移(P=0．007)，远处转移(P＜0．001)与患者预后显著相关。Kaplan-Meier生存曲线结果显示RUNX3高表达组的生存率显著高于RUNX3低表达组(P=0．022)。同时临床分期(T4期)(P=0．003)，淋巴转移(P=0．004)和远处转移(P＜0．001)与患者的预后显著相关。然而腺样囊性癌的病理类型(实体型)(P=0．771)和神经浸润(P=0．554)与患者的预后无统计学差异。而多因素Cox回归模型显示只有远处转移是患者预后的独立指标(P=0．043)，而T4分期与患者的预后存在弱相关(P=0．063)。
目前的研究认为RUNX3作为一个抑癌基因，其表达水平与患者预后和生存期显著相关，可能是影响恶性肿瘤预后的独立指标。RUNX3高表达的恶性肿瘤中，其5年生存率显著高于RUNX3低表达者，并且与细胞分化程度有关，高分化的恶性肿瘤中RUNX3表达水平高于低分化者，且RUNX3低表达与肿瘤的远处转移能力显著相关。本实验的结果表明RUNX3蛋白的表达可能与人涎腺腺样囊性癌的病理分化和转移相关，提示RUNX3蛋白的表达下调可能对ACC的发生发展具有重要作用，检测该肿瘤中RUNX3蛋白的表达情况可能有助于腺样囊性癌的筛查、临床诊断和预后判断。
结论：
1．RUNX3在腺样囊性癌细胞株ACC-2和ACC-3中存在表达，而在肺高转移腺样囊性癌细胞株ACC-M中则无表达。经过300nmol／L的5-Aza-dc处理72小时后能提高RUNX3在这些细胞株中的表达。
2．人涎腺腺样囊性癌中RUNX3基因甲基化的概率显著高于正常涎腺组织，提示RUNX3基因启动子CpG岛甲基化是人涎腺腺样囊性癌发生的一个重要因素。
3．RUNX3基因CpG岛的甲基化的规律是从5’区向转录起始点方向演进，转录起始点的甲基化程度最低，提示该位点为RUNX3基因甲基化的关键位点。
4．RUNX3基因CpG岛的甲基化状态与肿瘤临床病理指标之间无显著相关性。
5．RUNX3在人涎腺腺样囊性癌中存在表达。RUNX3蛋白低表达与肿瘤的分化和远处转移及患者的预后存在显著关系，对临床预后的判断具有诊断价值。
Part one: Effect of 5-Aza-dc on RUNX3 expression in salivary gland adenoidcystic carcinoma cell lines ACC-2, ACC-3 and ACC-M
Adenoid cystic carcinomas (ACC)are one of the most common malignancies insalivary gland, and comprise approximately 10% of all epithelial salivary neoplasms. Itis characterized by slow growth, followed by high neural and blood vessal invasion. Thedistant metastasis rate is appropriately 40% which results in a poor long term survival.Surgery followed by postoperative radiation therapy is the treatment of choice. It isreported that alternation of the antioncogen and cell adhesion molecules has beenproposed as factors associated with the aggressive behavior in salivary gland adenoidcystic carcinoma. However the underlying mechanism for the aggressive nature and thefatal course of this tumor are pooly investigated. The human runt-related transcriptionfactor-3 (RUNX3) is a tumor suppressor gene in gastric cancer. The inhibition orsilence of this gene expression is supposed to be highly associated with invasion anddistant metastasis of a variety of malignant tumor cell lines, whilst, the promotermethylation of RUNX3 gene is the main cause. However, after using the DNAmethyltransferase enzyme inhibitor, 5-Aza-dc, the expression of this gene can berestored in vitro. Promoter methylation is a common mechanism to inactivate tumorsuppressor genes in tumorigenesis, which plays an important role in the development ofsalivary gland tumors; however, the expression of RUNX3 in salivary tumor cell lines israre. Thus, it is necessary to investigate the expression of RUNX3 gene and protein insalivary gland adnoid cystic carcinoma cell lines, namely ACC-2, ACC-3, and ACC-M.
Experiment one: in this part, RT-PCR, laser scanning confocal microscope (LSCM),and western blot were used to detect the expression of RUNX3 gene and protein insalivary gland adenoic cystic carcinoma cell lines, ACC-2, ACC-3, and ACC-M,before/after treated with 5-Aza-dc, restropectively.
Three adenoid cystic carcinoma cell lines (ACC-2, ACC-3, and ACC-M) were usedin this vitro experiment. Those cell lines were cultured in RPMI-1640 with 10% fetalbovine serum, and incubated them at 37℃in a humidified atmosphere containing5%CO_2. 5-Aza-dc (300nmol/L) was added into the medium, and the cell lines were cultured together with 5-Aza-dc for 72 hours. After that the total RNA and proteinswere isolated from the cultured cells with or without treated with 5-Aza-dc. RT-PCRand western blot were used to detect the RUNX3 mRNA and protein expression in thesethree cells lines, respectively. In western blot analysis, the relative level of RUNX3protein was decided by the ratio between RUNX3 gradation and GAPDH gradationwith using Image-Pro Plus software.
Results: RT-PCR and western blot analysis showed weak expression of RUNX3mRNA and protein in ACC-2 and ACC-3, and no expression of RUNX3 in ACC-3 cellline. The western blot analysis disclosed a RUNX3 protein of 45kDa in the three ACCcell lines. After treated by 300 nmol/L 5-Aza-dc for 72 hours, the expression level ofRUNX3 in ACC-2 and ACC-3 was improved, and in ACC-M was restored by analyzingthe gradation ratio of RUNX3 protein and GAPDH protein. A similar result was foundbetween RT-PCR and western blot analysis. The results of LSCM showed that theRUNX3 protein located mainly in the cytoplasm of the ACC cell lines. After treatedwith 300nmol/L 5-Aza-dc for 72 hours, both nuclear and cytoplasmic location ofRUNX3 positive signals were found in the ACC-2 and ACC-3 cells. However, the weakpositive signal was still only found in the cytoplasm of ACC-M cells
In this part, the expression of RUNX3 gene and protein in salivary gland ACC celllines, ACC-2, ACC-3, and ACC-M, before/after treated with 5-Aza-dc, was exploredrestropectively. These results indicate that the promoter methylation of RUNX3 mightplay an important role in the inhibition of RUNX3 expression in ACC cell lines. Thesilence expression of RUNX3 in high lung metastasis salivary adenoid cystic carcinomacell line, ACC-M, provide a clew that the RUNX3 inhibition is associated with distantmetastasis of ACC. Because RUNX3 can fuction as a tumor suppressor when locates inthe nuclear of cells, the cytoplasmic mislocalization of RUNX3 in ACC cells might becorrelated with the development and progression of ACC.
Part two: Promoter methylation and protein expression of the RUNX3 gene inthe clinicopathologic assessment of salivary gland adenoid cystic carcinoma
The methylation of CpG island in promoter region is the main mechanism of theRUNX3 gene silence. In part one, the results showed that the RUNX3 expression waselevated in adenoid cystic carcinoma cell lines ACC-2, ACC-3, and ACC-M after being treated by 5-Aza-dc, which indicates the association between promoter methylation inRUNX3 gene and the progression of salivary gland adnoid cystic carcinoma. As a tumorsuppressor, the RUNX3 expression was inhibited due to the promoter methylation in avariety of malignant tumors. However the mechanism of inhibition or sitence ofRUNX3 gene in salivary gland adenoid cystic carcinoma is pooly investigated. Thus, inthis chapter, the methylation status in successive ten regions ranging from the 5' regionto the transcription start site within the RUNX3 CpG island and the expression levels ofRUNX3 protein in ACC samples and compared non-neoplastic salivary glands wereexplored, and the relationship between the methylation status of RUNX3 gene andRUNX3 protein expression or the clinicopathological parameters was also analyzed, aswell as the mechanism of the critical regions and spreading of the RUNX3 CpG islandmethylation in ACC.
Experiment two: The quantitive MSP method was used to detect the methylationstatus of CpG island in various regions (No. 1-10) of RUNX3 promoter region, and theWestern blot method was used for detecting the expression of RUNX3 protein in 41salivary gland ACC samples and the corresponding non-neoplastic salivary glands. ALogistic ANOVA model is used to analysis the risk ratio between the methylation statusof CpG island in RUNX3 gene and development of salivary ACC, meanwhile, thepossible association among the methylation of RUNX3 gene, the clinicopathologicalparameters of ACCs, and RUNX3 protein expression was compared.
Results: The results of qMSP show that the hypermethylation initially occurs atthe most 5' region of the RUNX3 CpG island and spreads to the transcription start site.And the methylation rate is highest in region No. 1 and No. 2 among the successive tenregions ranging from the 5' region to the transcription start site within the RUNX3 CpGisland, and lowest in the transcription start site both in ACCs and normal salivary glands.Furthermore, there is no methylation in the transcription start site in nomal salivaryglands tissues. Together with the results of Logistic ANOVA model analysis, thoseresult indicats that the transcription start site within the RUNX3 promoter CpG island iscritical for gene silencing. Western blot results reveal that the RUNX3 protein level inACC was significantly lower than in normal salivary glands(P<0.001), in combinationthe results of qMSP, it is presumed that the RUNX3 gene methylation is one of the reason inducing the down-regulation of RUNX3 in ACCs. Although there is norelationship between the promoter methylation of RUNX3 CpG island and theclinicopahtological parameters of ACCs, analysis the methylation status at thetranscription start site of the RUNX3 CpG island can be used in diagnosis and riskassessment of ACCs.
The promoter methylation is a common mechanism in the inactivation of tumorsuppressor genes in varieties of malignancies. In this study, we found the rate ofpromoter methylation of RUNX3 CpG island in salivary gland ACCs is significantlyhigher than it is in normal salivary gland, suggesting that the promoter methylation ofRUNX3 gene may be associated with the development of salivary gland ACCs. TheqMSP results reveal that the methylation ratio of RUNX3 CpG island differs in differentregion, hypermethylation initially occurs at the most 5' region of the RUNX3 CpGisland and spreads to the transcription site, which is supposed to be the critical site forgene silencing. Thus, the region spanning the transcription site, such as No. 6-8, shouldbe used to evaluate the methylation status of RUNX3 gene in tumors. Additionally, Norelationship between hypermethylation of RUNX3 gene and clinicopathologicalparameters in patients with salivary gland ACCs was found. A large clinical trial wasrequired to further evaluate the potential relationship.
Part three: Expression of RUNX3 in salivary gland adenoid cystic carcinomaand its association with tumor progress and prognosis
The long-term survival in patients with salivary gland adenoid cystic carcinoma isgloomy. Various parameters, including positive surgical margins, clinical stage,histopathological patterns, and perineural invasion, have been reported as relevantprognostic factor in patients with ACC. In addition to these chinicopathological factors,several investigators have explored the possible association between the proliferation orapoptosis-associated proteins and the survival in patients with salivary ACC. However,the precise mechanism responsible for its carcinogenesis has not been fully clarified.RUNX3 gene, supposed to be a new tumor suppressor, the silence expression of thisgene is highly correlated with the development and progress of gastric cancer, as well asthe therapy outcome and the survival in patients with gastric carcinomas. To detect theexpression of RUNX3 in a variety of malignant rumors might provide a better prognostic indicator. Moreover, the lack of RUNX3 expression is probably associatedwith distant metastasis in malignancies, such as gastric cancer. Although there is noreport on its expression in salivary ACC, which presents typically distant meatstasisresulting in a fatal outcome, on basis of the relationship between the RUNX3 expressionand distant meatastasis, RUNX3 expression might be conceivably associated with thetumorigenesis, progression, distant meatastasis, and survival of salivary ACC.
Experiment three: The quantitative RT-PCR (qRT-PCR), Western blot andimmunohistochemistry were applied for detecting the expression of RUNX3 in normalhuman salivary glands and adenoid cystic carcinomas in this part.
Nine normal adult salivary glands (including 3 parotid glands, 2 sublingual glands,and 2 submandibular glands) and seven adenoid cystic carcinomas frozen tissues wereobtained. The expressions of RUNX3 mRNA and protein were detected by usingqRT-PCR and western blot analysis, respectively. The relative RUNX3 mRNA levelwas measured by using the threshold cycle (Ct) method. In western blot analysis, therelative level of RUNX3 protein was decided by the ratio of RUNX3 gradation andGAPDH gradation with using Image-Pro Plus software. Seventy-three formalin-fixed,paraffin-embedded salivary ACC specimens were achieved. The immunohistochemistrywas applied to detect the subcellular location of RUNX3 protein in normal salivaryglands and ACC.
Results: The relative quantification of RUNX3 mRNA expression in ACC(0.09±0.05) was two-fold lower than that in normal salivary glands (0.19±0.08).Western blot analysis disclosed a RUNX3 protein of 45 kDa both in human normalsalivary glands and ACC.According to the results of gradation analysis, the relative ofRUNX3 protein expression in salivary gland ACC (0.79±0.03) was lower than that innormal salivary glands (0.88±0.03). However, no statistical difference between thesetwo groups. Immunohistochemistry results showed that RUNX3 protein located mainlyin the nuclear and cytoplasm of acinous cell and ductal cell in normal salivary gland;and only in the cytoplasm of tumor cell in ACC. In some cases of solid type ACC, noexpression RUNX3 protein was found.
Experiment four: The potential relationship between the expression of RUNX3 andthe clinicopathological factors and survival in patients with adenoid cystic carcinoma was analyzed in this part.
The semi-quantative RUNX3 level in 73 adenoid cystic carcinoma cases was detectedwith using Image-Pro Plus software. Continuous variables of RUNX3 labeling indexwere divided into three discrete exclusive variables according to the distribution of thesamples. The tertile cut-off values were as follows: High group (≥14.91%), intermediategroup (6.94%-14.91%), and low group (≤6.94%). Significant differences between theexpression of RUNX3 and clinicopathological parameters were compared. Theunivariate analysis with the Cox regression model was used to determine identifiedprognostic factors, and multivariate analysis with the Cox regression model was used toexplore combined effects. Survival analysis was computered by means of theKaplan-Meier method and significant levels were assessed by means of the log-rank test.Thereby, the application of RUNX3 expression in salivary gland ACC to predict thepatients' survival was assessed.
Results: The semi-quantitative results of immunohistochemistry showed that the high,intermediate, and low RUNX3 expressions were 19.60±4.26%, 11.19±2.42%, and4.78±1.59%, respectively. And the RUNX3 expressions were 13.69±6.46% in thecribriform pattern, 11.78±4.40% in the tubular pattern, and 8.86±5.20% in the solidpattern. Low RUNX3 expression was significantly correlated with the histologicalpatterns of ACC, namely solid subtype (P=0.025), meanwhile, ACC with lower RUNX3expression significantly tended to be have more frequent distant metastasis (P=0.009),indicating the the association between down-regulation of RUNX3 expression andtumor progression. The RUNX3 expression percentage was 13.65±6.44% in tumorswith stage T1-3, and 10.24±5.19% in stage T4. There is only a weak tendency betweenthe low RUNX3 expression and stage T4 (P=0.061). No statistical differences betweenRUNX3 expression and other clinicopathologic factors, such as age, gender, tumor site,perineural invasion, and lymph nod involvement in ACCs, was detected. The 5-yearsurvival rate was 90% in the 18 patients with high levels of RUNX3 expression, 75.4%in the 37 patients with moderate level, and 48.27% in the 18 patients with lowlevel.Using univariate analysis of Cox regression model, the following variables werefound to be significantly associated with a worse prognosis, including low expression ofRUNX3 protein (P=0.012), lymph node involvement (P=0.007), and distant metastasis (P<0.001). Kaplan-Meier survival curves showed that the survival rates of patients withlow expression of RUNX3 was significantly worse than that of patients with moderateor high RUNX3 expression (P=0.022). Meanwhile, we observed that some otherclinicopathogical parameters, including stage T4 (P=0.003), lymph node involvement(P=0.004), and distant metastasis (P<0.001) were significantly related with decreasedsurvival. However, no significant association between overall survival and solidhistotype (P=0.771) or perineural invasion (P=0.554) was found. Multivariate survivalanalysis revealed that only distant metastasis was an independent significant prognosticfactor (P=0.043). Stage T4 only had a weak association with overall survival (P=0.063).RUNX3 gene, as a tumpr suppressor, is signigicantly associated with the survival rateand prognosis in patients with malignancies, and might be an independent predictor forthe prognosis. The 5-year survival rate is significant higher in tumor with highexpression of RUNX3 protein than this with low expression level. Meanwhile, theexpression of RUNX3 protein is correlated with cell differention, furthermore, the lowor lack expression of RUNX3 is potentially associated with distant metastasis. Theresults of this study indicate that the low expression of RUNX3 in salivary gland Aceis correlated with tumor development and metastasis. Together, the expression ofRUNX3 protein might be a candidate for diagnosis and prognostic marker in salivarygland ACCs. It has a pivotal role in the tumor proliferation and apoptosis.
Conclusions:
1. There is RUNX3 expression in adenoid cystic carcinoma cell lines ACC-2 andACC-3, and silence of RUNX3 in ACC-M. The DNA methyltransferase enzymeinhibitor, 5-Aza-dc, can elevate the RUNX3 level among these three cell lines.
2. The incidence of promoter methylation in RUNX3 gene CpG island in humansalivary gland adenoid cystic carcinoma is significanctly higher that it is innon-neoplastic salivary gland, indicating that the promoter methylation of RUNX3gene plays an critical role in the development of this malignancy.
3. The promoter methylation of RUNX3 CpG island spreads frorn the most 5' regionto the rranscriptiong start site in human salivary gland ACC, the incidence rate ofpromoter methylation in the transcriptiong start site is the lowest, suggesting this site might be a critical region for the methylation of RUNX3 gene.
4. There is no significant difference between the methylation status of RUNX3 geneand the clinicopathological parameters in patients with ACCs.
5. The expression of RUNX3 protein is down-regulated in salivary gland ACC. Thelower expression of this protein is significantly associated with rumor differentiation,distant metastasis, and prognosis. The expression of RUNX3 protein has a definitevalue in judging prognosis in salivary gland ACC.
引文
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[5]Nakamura S,Senzaki,Yoshikawa M,et al.Dynamic regulation of the expression of neurotrophin receptor by Runx3.Development 2008;135:1703-1711.
[6]Ito K,Liu Q,Salto-Tellez,et al.RUNX3,A Novel Tumor Suppressor,Is Frequently Inactivated in Gastric Cancer by Protein Mislocalization.Cancer Res 2005;65:7743-7750.
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[15]Salto-Tellez M,Peh BK,Ito K,et al.RUNX3 protein is overexpressed in human basal cell carcinomas.Oncogene 2006;25:7646-7649.
[16]Hiramatsu T,Osaki M,Ito Y,et al.Expression of RUNX3 protein in human esophageal mucosa and squamous cell carcinoma.Pathobiology 2005;72:316-324.
[17]Araki K,Osaki M,Nagahama Y,et al.Expression of RUNX3 protein in human lung adenocarcinoma:implications for tumor progression and prognosis.Cancer Sci 2005;96:227-231.
[18]Wei D,Gong W,Oh SC,et al.Loss of RUNX3 expression significantly affects the clinical outcome of gastric cancer patients and its restoration causes drastic suppression of tumor growth and metastasis.Cancer Res 2005;65:4809-4816.
[19]Sakakura C,Hasegawa K,Miyagawa K,et al.Possible involvement of RUNX3 silencing in the peritoneal metastases of gastric cancers.Clin Cancer Res 2005;11:6479-6488.
[20]Lau QC,Raja E,Salto-Tellez M,et al.RUNX3 is frequently inactivated by dual mechanisms of protein mislocalization and promoter hypermethylation in breast cancer.Cancer Res 2006;66:6512-6520.
[21]Subramaniam MM,Chan JY,Soong R,et al.RUNX3 inactivation by frequent promoter hypermethytlation and protein mislocalzation constitute an early event in breast cancer progression.Brest Cancer Res Treat.2009;113:113-121.
[22]Sakakura C,Miyagawa K,Fukuda KJ,et al.Frequent silencing of RUNX3 in esophageal squamous cell carcinomas is associated with radioresistance and poor prognosis.Oncogene 2007;26:5927-5938.
[23]He JF,Ge MH,Zhu X,et al.Expression of Runx3 in salivary adenoid cystic carcinoma:Implication for tumor progression and prognosis.Cancer Sci 2008;37:1334-1340.
[24]Ito K,Lim AC,Salto-Tellez M,Motoda L,et al.RUNX3 Attenuates beta-Catenin/T Cell Factors in Intestinal Tumorigenesis.Cancer Cell 2008;14:226-237.
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