鼻咽癌染色体12p12-11区段扩增基因的筛选及其功能研究
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摘要
鼻咽癌(nasopharyngeal carcinoma, NPC)是我国南方和东南亚各国发病率高、危害大的恶性肿瘤之一,具有明显的种族性和地域性,95%以上属于低分化癌和未分化癌类型,容易早期发生转移(转移是导致鼻咽癌患者死亡的主要原因),目前主要以放射治疗为主。虽然放疗设备及技术在不断进步,但鼻咽癌的五年生存率仍然维持在50%-60%左右,并未从根本上改善。因此探索其病因及寻找新的有效治疗方法尤为重要。
流行病学及病因学研究表明遗传因素(遗传易感性)、EB病毒感染和其它环境因素(主要是化学致癌物等)均与鼻咽癌的发生、发展有关。在这些因素的共同作用下,正常鼻咽上皮细胞发生遗传变异事件和表遗传学改变的不断积累,导致瘤基因的激活和抑瘤基因的失活,最终引起细胞的恶性转化。目前研究表明,鼻咽癌显著的种族易感性和家族聚集现象表明遗传基因的改变在鼻咽癌中起十分重要的作用,但与其他人类常见的恶性肿瘤相比,鼻咽癌发病的分子基础和相关的分子遗传学改变所知并不多,鼻咽癌发病的具体分子机制仍未阐明,因此寻找、鉴定与鼻咽癌发生发展密切相关的基因、研究其功能,是揭开鼻咽癌发病分子机制的必然途径,具有十分重要的意义。既往研究发现,一些原瘤基因(例如HRAS, cyclin D1、MDM2、STAT3、EGFR)和抑瘤基因(例如P53、P16P53、RASSF1A、DLC-1、LTF、BLU等)在鼻咽癌中异常表达,但尚未证实为鼻咽癌特异性瘤基因或抑瘤基因。因此需要我们从新的角度去重新认识和获得鼻咽癌关键的瘤基因和抑瘤基因信息。
我室尹志华和黄仲曦等利用香港、台湾、湖南和广东五个实验室发表的170例鼻咽癌比较基因组杂交数据,构建了鼻咽癌发生发展的树模型,以此来探讨鼻咽癌的发病机制。研究结果发现,鼻咽癌相关的重要染色体变异事件共11个,包括2个鼻咽癌发病的早期事件-3p26-13和+12p12,其中+12p12事件为首次报道。LOH及染色单体转移实验提示3p的丢失为鼻咽癌的早期事件以及在此区域存在鼻咽癌相关抑瘤基因。最近的研究进一步表明：3p21.3区域存在多个候选抑瘤基因(如RASSF1A1、BLU和LTF),其基因启动子区在鼻咽癌组织中常常存在高度甲基化导致其表达明显下调。这些证据均显示了树模型预测的准确性。因此,树模型首次发现的以扩增为主的+12p12-11为鼻咽癌的早期事件,为寻找鼻咽癌关键瘤基因提供了新的切入点。
[12p12-11扩增基因的筛选]
染色体扩增常导致瘤基因拷贝数增加和过表达,通过检测肿瘤组织中染色体扩增区域内候选瘤基因的过表达已经成为寻找特定肿瘤相关瘤基因的重要途径。染色体12p12-11长度为19Mb,包含123个已知基因和表达序列标签(expressed sequence tag, EST)。已有研究表明,+12p是大肠癌的早期事件,可能是生殖细胞肿瘤的晚期事件以及卵巢癌的中晚期事件。利用MEDLINE文献数据库及本实验室已完成的鼻咽癌cDNA微阵列数据资料,我们从12p12-11区初步确定了18个鼻咽癌相关候选瘤基因(DDX11、LDHB、CASC1、KRAS2、BCAT1、PTHLH、SOX5、PDE3A、CMAS、BHLHB3F、KCNJ8、PKP2、EKI1、PTX1、DNM1L、SSPN、RECQL、SLCO1A2),其中已明确的瘤基因及候选瘤基因有6个(DDX1、LDHB、CASC1、KRAS2、BCAT1、PTHLH),另外12个基因与细胞转化和增殖相关。采用RT-PCR和定量RY-PCR方法检测鼻咽癌和慢性鼻咽炎组织中这18个基因的表达水平,结果显示：其中9个基因(RECQL、PDE3A、SLCOo1A2、ASC1、DDX1、SSPN、PTHLH、BHLHB与PKP2)在慢性鼻咽炎与鼻咽癌组织中均无表达；5个基因(CMAS1、DNM1L、ETNK1、LDHB与SOy)表达水平在36例鼻咽癌及8例慢性鼻咽炎组织之间无显著差异(P>0.05)；而有4个基因(BACT1、KCNJ8、PTX1与KRAS2)在鼻咽癌组织中表达明显上调(P<0.05),提示其可能与鼻咽癌的发生发展相关。其中KCNJ8为ATP-敏感钾离子通道基因的一个亚类,与细胞的分泌和肌肉收缩有关,而与肿瘤发生及细胞的生长、增殖相关报道较少；PTX1主要参与内质网与高尔基体之间物质的膜运输,仅发现其在前列腺癌中表达下调；KRAS2在鼻咽癌中的研究颇多,其表达明显高于慢性鼻咽炎；而BCAT1(branched chain aminotransferase 1)基因编码支链氨基转移酶,在Burkitt's淋巴瘤、乳腺癌组织中明显上调,与细胞的增殖和凋亡有关,在鼻咽癌中尚未有相关报道。因此我们将其作为进一步研究的对象,探讨BXAT1与鼻咽癌的关系。
[BCAT1基因在鼻咽癌不同病理阶段中的表达]
从12p12-11筛选出的相关瘤基因回到癌前病变各阶段去考察能认识这些基因在鼻咽癌发病过程中的真正地位和意义,因此我们采用了免疫组化分析BCAT1蛋白在鼻咽正常上皮、增生活跃、轻中度、重度增生和鼻咽癌组织中的表达。结果表明,在正常假复层纤毛柱状上皮、复层鳞状上皮、轻中度增生、重度增生和鼻咽癌组织中均能检测到BCAT1蛋白不同程度的阳性表达,阳性信号定位于细胞胞浆中；BCAT1蛋白在正常上皮、轻中度增生、重度增生和鼻咽癌组织中阳性表达率分别为23.6%(17／72)、75%(18／24)、88.9%(8／9)和88.75(71／80)；结果提示在鼻咽部出现轻度增生早期病理改变时,BCAT1的表达就明显增高(P<0.05),这说明BCAT1基因对于鼻咽癌的发生可能起重要作用,这也为进一步分析该基因表达异常的分子机制提供了依据。
[探讨BCAT1基因在鼻咽癌中异常表达的可能机制]
探讨瘤基因表达上调的机制主要从遗传学改变和基因转录调控两方面进行。首先,通过PCR结合测序的方法分析20例鼻咽癌中BCAT1的11个外显子突变情况,共检测出1个外显子区SNP位点,提示基因突变对BCAT的表达无明显影响。其次,拷贝数增加被认为是瘤基因异常激活的一个重要机制,分析基因内部及其附近微卫星位点的扩增可以间接反映基因组中基因的扩增情况。采用Real-timePCR分析了BCAT1基因内部D12S1435、D12S1617和RH44650三个微卫星位点在鼻咽癌组织中扩增情况。结果表明,有42.4%(12／28)鼻咽癌组织中存在BCAT1基因微卫星位点的扩增。提示基因拷贝数的扩增可能对BCAT1在鼻咽癌组织中表达上调起一定作用。
真核基因的转录调控是一个非常复杂的过程,而转录因子参与的基因转录调控起重要作用。生物信息学分析显示在BCAT15’端非翻译区-155／-200bp区域包含有c-Myc结合位点。为了揭示c-Myc是否对BCATl的表达具有直接调控作用,我们进行了染色质免疫沉淀实验(ChIP)。结果证实,c-Myc转录因子可以直接结合于BCAT1基因启动子区的c-Myc结合位点。RT-PCR与免疫组化表明鼻咽癌中BCAT1和c-Myc的mRNA和蛋白的表达均上调,进一步利用RNA干扰技术建立了稳定干扰c-Myc表达的5-8F鼻咽癌细胞系(命名为5-8F/Si-c-myc),结果发现,封闭内源性c-Myc的表达可以下调BCAT1基因mRNA的表达水平。另外,我们将带有包含c-Myc结合位点的BCATl调控序列与荧光素酶报告基因连接构建重组报告基因载体,分别转染5-8F细胞、转染空白载体的5-8F细胞(即5-8F/si-control)以及5-8F/si-c-myc细胞中,经报告基因活性分析发现,封闭内源性c-Myc表达后,与对照组相比,重组报告基因的荧光素酶活性降低。而在转染c-Myc基因的COS7细胞系中具有较强的荧光素酶活性。上述实验结果说明c-Myc对BCAT1基因的表达具有直接的正调控作用。
[利用RNA干扰技术研究BCAT1基因在5-8F细胞的作用]
采用pSUPER.retro逆病毒载体系统,构建了针对BCAT1的RNAi载体(shBCAT1)。经测序结果证实后,利用脂质体转染技术将shBCAT1导入5-8F细胞,嘌呤霉素筛选获得抗性克隆,PCR证实抗性克隆中含有shBCAT1; RT-PCR结果表明,shBCAT1表达载体能特异性导致BCAT1 mRNA的表达下调了90%；Western Blotting分析表明细胞中的BCAT1蛋白表达下调了55%左右,这些说明我们已成功地建立了稳定干扰BCAT1基因的5-8F细胞系。
随后,我们进一步考察了BCAT1被干扰后对5-8F细胞生物学特性的影响。流式细胞仪分析结果表明：转染了shBCAT1的5-8F细胞(5-8F-shBCATl)阻滞于G1期,G1期细胞比例增加～16.66%(16.66+2.92,P=0.005),G2期细胞则减少了～6.91%(6.91+0.76.P=0.001),而对照组5-8F细胞与转染空白质粒的5-8F细胞(5-8F-blank vector)之间的细胞周期分布没有明显差异。MTT法检测发现,5-8F-shBCAT1细胞增殖速度明显低于对照组5-8F细胞与5-8F-blank vector细胞,统计学分析差异有显著性(P=-0.00)。平板克隆形成实验结果显示5-8F-shBCAT1细胞的克隆形成率明显低于转染载体组(7.2%vs 38.9%)。另外,迁移实验和侵袭实验证实,5-8F-shBCAT1细胞运动能力和侵袭能力明显降低。
IBCAT1与LARS2在鼻咽癌中的相互作用及LARS2在鼻咽癌中下调机制的研究]
生物信息学是后基因组时代的利器。STRING (http://string80. embl.de/)是目前已知的、揭示已知和预测蛋白-蛋白之间相互作用的、较广泛使用的数据库。该数据库揭示的蛋白-蛋白相互作用既包括直接相互作用(结合)也包括间接关联作用(功能相关),其率数据来源于基因组背景分析、高通量实验、共表达实验和已公布的数据库(如PUBMED, MIPS)。同时,利用STRING还可以比较不同物种间的数据。最新版STRING 8.1收录了630个物种、超过2,590,259种蛋白质数据。
在STRING的界面,输入BCAT1,可以找到直接和间接相关蛋白分子10个,它们分别是LRMP、hCG 26005、ENSP00000372、BCKDHA、BCKDHB、IARS2、IARS、LARS2、LARS和BCAT1。从这些基因的染色体定位数据中,我们发现LARS2基因位于鼻咽癌患者高频丢失区3p21。
LARS2 (leucyl aminoacyl-tRNA synthetase 2, LARS2)基因编码亮氨酰-tRNA合成酶。为了探讨LARS2基因在鼻咽癌中的可能作用,我们首先采用了RT-PCR、Real-time PCR方法检测LARS2在8例慢性鼻咽炎组织、36例鼻咽癌组织中的表达。结果发现,LARS2在78%(28／36)鼻咽癌组织中存在表达下调或缺失,提示LARS2可能是鼻咽癌相关候选抑瘤基因。接着,我们从遗传学(genetic)和表观遗传学(epigenetic)改变两方面探讨了LARS2基因失活的主要机制。1.通过PCR-SSCP并结合测序方法分析了25例鼻咽癌中启动子区和外显子1突变情况,结果未检测出突变和SNP,提示基因突变对于LARS2表达失活无明显影响。2.由于基因内部及其附近微卫星位点的丢失可以间接反映基因组中基因的缺失情况,我们采用了PCR-变性聚丙烯酰胺凝胶电泳-银染方法分析LARS2基因内部的RH25266和SHGC-12886二个微卫星位点在鼻咽癌组织中的丢失情况。结果表明,至少有28%(7／25)的鼻咽癌组织中存在LARS2基因微卫星位点的丢失,提示微卫星位点的LOH可能对LARS2在鼻咽癌细胞中表达下调起一定的作用。3.鉴于启动子区DNA异常甲基化是抑瘤基因在表观遗传学改变最为常见的方式,我们首先通过生物信息学软件对LARS2启动子区CpG岛分布情况进行了分析,发现LARS2启动子区存在一个227bp长的CpG岛。以此区域为模板,选择针对该区段的甲基化和非甲基化特异性引物,利用甲基化特异性PCR (methylation-specific PCR, MSPCR)技术,我们分析了慢性鼻咽炎和鼻咽癌组织中LARS2启动子甲基化情况。结果发现,64%(16／25)鼻咽癌组织和12.5%(1／8)慢性鼻咽炎组织存在LARS2基因启动子甲基化情况,但鼻咽癌组织中LARS2基因甲基化比率明显高于慢性鼻咽炎组织,两者存在明显的统计学差异(P=0.017),这提示启动子甲基化是导致LARS2基因表达下调的主要因素。
进一步比较发现,BCAT1蛋白和LARS2蛋白的相关性表现在这两个蛋白作用于同一条细胞代谢通路,即亮氨酸代谢。BCAT1在鼻咽癌中显著上调,而LARS2在鼻咽癌中表达下调,共同导致了中间产物L-亮氨酸的累积。作为一种支链氨基酸,L-亮氨酸通过改变mTOR (mammalian target of rapamycin)-raptor复合体的稳定性,激活：mTOR的信号传导通路。有研究表明,鼻咽癌组织常常存在mTOR的高表达。一方面,高表达的mTOR可以通过下游分子S6K1和4E-BP1调控编码细胞周期G1期和S期必需蛋白,如细胞周期素D1、Rb蛋白、低氧诱导因子-1(hypoxia inducible factor-1, HIF-1)、c-Myc和血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)、LIP-170等的mRNA翻译,最终导致细胞的恶性失控性增殖。另一方面,高表达的mTOR也可以通过诱导DNA转录调节因子Id2的表达影响上皮细胞的分化。基于以上实验和分析,我们认为BCAT1的表达增加和LARS2基因的表达下调两者之间存在间接的相互作用关系,它们共同参与推动鼻咽癌的发生和发展。
综上所述,通过本研究,我们首次从鼻咽癌发生早期事件+12p12-11区域内发现鼻咽癌相关瘤基因BCAT1,并系统、全面地考察了BCAT1蛋白在慢性鼻咽炎、鼻咽癌及鼻咽癌变不同阶段组织中的表达情况,探讨了导致其表达上调的可能机制,并进一步研究了BCAT1基因在鼻咽癌细胞中的功能。结果提示,BCAT1在慢性鼻咽上皮组织和正常鼻咽上皮中正常表达,而在鼻咽癌组织和鼻咽癌变不同阶段明显表达上调,而这种表达上调主要是由于基因扩增所致,而瘤基因c-Myc对其直接调控也发挥了一定作用。RNA干扰实验表明,干扰BCAT1的表达能够抑制鼻咽癌细胞的增殖,导致细胞周期的阻滞。此外,我们通过生物信息学发现了与BCAT1蛋白有间接相互作用的LARS2蛋白。LARS2基因在鼻咽癌组织中表达明显下调,其失活机制主要是基因启动子区甲基化。鼻咽癌中BCAT1显著上调和LARS2表达下调,共同导致了中间产物L-亮氨酸的累积,而L-亮氨酸则可以通过激活mTOR的信号传导通路最终导致细胞的恶性失控性增殖。当然,生物信息学预测结果还有待实验进一步验证。
Nasopharyngeal carcinoma (NPC) is one of the malignant tumors with a high incidence and causing a serious health problem in Southeast Asia and Southern China, which has distinct geographical and ethnic distribution. More than 95% of NPC is poorly differentiated and undifferentiated with high incidence of early metastasis (metastasis is the main cause of death in NPC). Radiation therapy is currently the first choice for NPC treatment. Although the radiotherapy equipment and technology were improved tremendously, but the five-year survival rate of NPC did not radically change and remains at about 50%-60%. Therefore, it is of importance to explore its causes and find a new and effective treatment to NPC.
Studies on epidemiology and etiology of NPC have shown that genetic factors (genetic susceptibility), EB virus infection and other environmental factors (mainly chemical carcinogens, etc.) are related to the occurrence and development of NPC. Under the combined effect of these factors, the genetic variation events affecting normal nasopharyngeal epithelial cells and the continuous accumulation of epigenetic changes led to activation of oncogene and inactivation of tumor suppressor genes, ultimately resulting in malignant cellular transformation. The current studies show that significant ethnic susceptibility to NPC and familial aggregation phenomenon indicate that changes of genetic genes play an important role in NPC, but compared to other human common malignant tumor, there is little known facts about the basis of molecular pathogenesis of NPC and the associated change in molecular genetics. The specific molecular mechanisms of NPC incidence have not been elucidated yet, so it is of great significance to look for and identify the genes closely related to the development of NPC and study their functions which is the inevitable way to reveal the molecular mechanism of NPC. Past research found that some proto-oncogenes(for example, HRAS, KRAS2,cyclin D1, MDM2, STAT3, EGFR, etc.) and tumor suppressor genes (P53,P16,RASSF1A, DLC-1, LTF, BLU, etc.) were expressed abnormal in NPC, but not yet confirmed as nasopharyngeal cancer-specific tumor gene or tumor suppressor gene. Therefore, we need to understand and access the information of the significant oncogenes and tumor suppressor genes related to NPC from a new perspective angle.
Zhi-Hua Yin and Zhong-Xi Huang in our Lab have constructed a tree model of NPC tumorigenesis to explore the pathogenetic mechanism of NPC by using comparative genomic hybridization data of 170 cases of NPC from five laboratories in Hong Kong, Taiwan, Hunan and Guangdong. The study showed 11 chromosomal aberrations in NPC, among which 2 early events in the pathogenesis of NPC were-3p26-13 and+12p12, the latter is the first reported. LOH and chromatid transfer experiments suggested the loss of 3p is an early event in NPC as well as some NPC tumor suppressor genes might exist at this region. Recent research further indicates that there are several candidate tumor suppressor genes (such as RASSF1A, BLU and LTF) located at 3p21.3, and also there are hypermethylation of gene promoter regions, leading to down regulation of gene expression. The evidences mentioned above suggest that the tree model prediction is accurate. Therefore, the amplification based+12p12-11 as the early event of NPC provides a new entry point to find the key oncogenes in the pathogenesis of NPC.
[The screening of amplified genes located at 12p12-11]
Chromosomal gain often leads to the amplification in copy number and overexpression of oncogene and it's important to search for specific tumor-related genes by detecting over-expression of candidate oncogenes in the amplified chromosome region. Chromosome 12p12-11,19Mb at length, contains 123 known genes and expressed sequence tag (expressed sequence tag, EST). Previous studies have shown that,+12p is an early event in colorectal cancer, and that may be the late events of germ cell tumors or late events of ovarian cancer. We preliminarily determined 18 candidate tumor-related genes, among which 6 real oncogenes were determined(DDX11, LDHB, CASC1, KRAS2, BCAT1, PTHLH) and other 12 genes (SOX5, PDE3A, CMAS, BHLHB3F, KCNJ8, PKP2, EKI1, PTX1, DNM1L, SSPN, RECQL, SLCO1A2) were associated with cell transformation and proliferation by using MEDLINE database and NPC cDNA microarray data from our laboratory.
We determined these 18 gene expression level between 36 cases of NPC and 8 cases of chronic nasopharyngitis tissues by RT-PCR and quantitative RT-PCR. The results showed 9 genes (RECQL, PDE3A, SLCO1A2, ASC1, DDX1, SSPN, PTHLH, BHLHB with PKP2) not expressed in NPC and in chronic nasopharyngitis tissues and the expression of 5 genes (CMAS1, DNM1L, ETNK1, LDHB and SOX) has no significant difference between 36 cases NPC and 8 cases chronic nasopharyngitis tissues (P> 0.05); While 4 genes (BACT1, KCNJ8, PTX1 and KRAS2) in NPC tissues were expressed significantly higher than that in controls (P<0.05), suggesting these 4 genes may be relevant to the development of NPC.
KCNJ8 is categorized into a sub-class of the ATP-sensitive potassium ion channel gene in relation to cell secretion and muscle contraction. At present there is no evidence that it is associated with the occurrence of cancer; PTX1 mainly involved in membrane transport of materials in endoplasmic reticulum and Golgi apparatus, down-expressed only found in prostate cancer; there are more studies of KRAS2 in NPC, its expression was significantly higher than that of the normal nasopharyngeal tissue and nasopharyngitis. BCAT1 (branched chain aminotransferase 1 gene) in Burkitt's lymphoma and breast cancer tissues were expressed significantly in relation to cell proliferation and apoptosis, but there is no relevant reports in NPC. Therefore, we select BCAT1 as a target for further study to explore its relationship with NPC development.
[the expression of BCAT1 protein in different NPC pathological stages]
To study tumor-related oncogenes selected from the 12p12-11 in various stages of precancerous lesions can be aware of the significance of these genes in NPC pathogenesis. Therefore, we used immunohistochemical method to analyze the expression of BCAT1 protein in the normal nasopharyngeal epithelium, low-moderate grade pre-invasive lesion, high grade pre-invasive lesion and NPC. The results showed that in the normal psudo-stratified ciliated epithelium, stratified epithelium, low-moderate grade pre-invasive lesion, high grade pre-invasive lesion and NPC, different levels of protein expression, positive signal located in the cytoplasm were able to be detected; The positive rate of the expression of BCAT1 protein in normal epithelium, low to moderate grade pre-invasive lesion, high grade pre-invasive and NPC were 23.6%(17/72),75%(18/24),88%(8/9) and 88.75 (71/80) respectively. The results suggest the expression of BCAT1 significantly increased (P<0.05) with the hyperplasia occurring in the nasopharynx of early pathological changes, which indicates the BCAT1 may play an important role in the occurrence of NPC and also provide a basis for further analysis of the molecular mechanism of gene expression abnormality.
[Explore possible mechanisms about abnormal expression of BCAT1 in NPC]
Genetic alterations and transcription des-regulation are two approaches to explore the mechanism of tumor gene up-regulation. Firstly, we analyzed the mutation status of 11 exons of BCAT1 from 20 cases of NPC by PCR-sequencing and one SNP site in exon1 was detected, suggesting mutation had no significant effect on the expression of BCAT1. Secondly, the copy number amplification is considered as a mechanism to activate an oncogene. Analyzing the amplification of microsatellite loci genes within and near the gene can indirectly reflect the gene amplification in the genome. We analyzed the amplification of three microsatellite loci (D12S1435, D12S1617 and RH44650) within BCAT1 by Real-time PCR in NPC. The results showed that 42.4%(12/28) of the NPC tissue have the amplification of microsatellite loci in BCATl. All above suggested that increase of the copy number may play a role in BCAT1 upregulation in NPC tissues to some degree.
Regulation of eukaryotic gene transcription is a very complicated process, and the participation of transcription factors play an important role in regulation of gene transcription. Bioinformatics analysis shows that BCAT1 5'untranslated region-155/-200bp area contains a c-Myc binding sites. In order to reveal whether c-Myc has a direct role in regulation of the expression of BCATl, we carried out chromatin immunoprecipitation experiments (ChIP). The results confirm c-Myc transcription factor can be directly binding the c-Myc binding site in BCAT1 gene promoter region. Further, we used RNAi to establish a stable 5-8F NPC cell line (named 5-8F/Si-c-Myc) with c-Myc expression interference and found that blocking of endogenous c-Myc expression could reduce the mRNA expression level of BCATl. In addition, we established a recombinant reporter gene vector by connecting BCAT1 regulatory sequences including c-Myc binding sites with the luciferase reporter gene, and then transfected 5-8F cell,5-8F cell with the blank vector (5-8F/si-control), as well as 5-8F/si-c-Myc cell. The reporter gene activity analysis showed that reporter gene activity of recombinant luciferase decreased after knockdown of endogenous c-Myc expression compared with the control group. The experimental results show that c-Myc has a direct positive regulatory role in the expression of BCAT1.
[Study the function of BCAT1 in 5-8F by RNA interference technology]
We constructed RNAi vectors targeting BCAT1 (shBCAT1) by pSUPER.retro retrovirus vector system. After the results confirmed by sequencing, we transfected shBCAT1 into 5-8F using the lipofection technique and puromycin resistant clones were selected, PCR confirmed that resistant clones contain shBCAT1; RT-PCR results showed that shBCAT1 expression vector could specifically lead to expression of BCAT1 reduced by 90%. Western Blotting analysis showed that the protein expression of BCAT1 decreased 55% or so in 5-8F. These results confirm that we have successfully established a stable interference system for BCAT1 gene in 5-8F.
Subsequently, we further examined the effects on 5-8F biological characteristics after the BCAT1 was blocked. Flow cytometric analysis showed that cell cycle distribution of 5-8F and 5-8F-blank vector did not change significantly; The 5-8F-shBCAT1 caws blocked in the G1 phase, G1 phase cells increased～16.66%(16.66±2.92,P=0.005), while the G2-phase cells decreased by～6.91%(6.91±0.76. P= 0.001). MTT assay found that the cell proliferation of 5-8F-shBCAT1 rate was significantly lower than control group,5-8F and 5-8F-blank vector, and the difference was statistically significant (P= 0.00). In addition, migration assay and invasion assay demonstrated that 5-8F-shBCAT1 could also significantly decrease the mobility and invasion ability of 5-8F cells.
[The interaction between BCAT1 and LARS2 and the inactivation Mechanism of LARS2 in NPC]
Bioinformatics is a tool for post-genomic era. STRING (http://string80.embl.de/) is a known and widely used database which reveals the protein-protein interaction between the known and predicted protein. The protein-protein interaction includes both the direct (binding) and the indirect interaction(association) and the background data come from genomic analysis, high-throughput experiments, co-expression experiments and published databases (such as the PUBMED, MIPS). At the same time, the data among different species can be compared by STRING. The latest version STRING 8.1 contains 630 species, more than 2,590,259 proteins. We found 10 protein molecules directly and indirectly interacted with BCAT1, which are LRMP, hCG_26005, ENSP00000372, BCKDHA, BCKDHB, IARS2, IARS, LARS2 and LARS. Moreover, we found that LARS2 gene is located in 3p21, which has a high-frequency loss in NPC. LARS2 (leucyl aminoacyl-tRNA synthetase 2) gene encodes leucyl-tRNA synthetase. In order to explore the possible role of LARS2 in NPC, we used RT-PCR and Real-time PCR to detect the expression of LARS2 in 8 cases of chronic nasopharyngitis and 36 cases of NPC. The results showed that, the mRNA expression of LARS2 was down-regulated or deleted in 78%(28/36) of the NPC tissue, suggesting that LARS2 may be a candidate tumor suppressor gene in NPC. Then, we studied the primary mechanism for LARS2 inactivation from the aspects of genetics and epigenetics. Firstly, we studied mutation in the promoter region and exon 1 in 25 cases of NPC by PCR-SSCP combined with sequencing analysis. The results did not detect the mutation and SNP, indicated that mutation had no effect on inactivation of LARS2 expression. Secondly, as the loss of microsatellite loci within and near the gene can indirectly reflect the absence of genes in the genome, we used PCR-denaturing polyacrylamide gel electrophoresis to analyze the loss of the two microsatellite loci (RH25266 and SHGC-12886) within LARS2 in NPC. The results showed that at least 28%(7/25) of NPC have loss of microsatellite loci in LARS2, suggesting that LOH of microsatellite loci may downregulate the expression of LARS2 in NPC in a certain degree. Thirdly, hypermethylation of gene promoter is the most common epigenetic change of tumor suppressor gene. Therefore, whether there exist CpG islands in the promoter region of LARS2 was analyzed and a 227 bp long CpG island in the promoter region was found. Then, we designed the methylation and the non-methylation-specific primers of this region. Finally, we analyzed methylation status of LARS2 promoter in the chronic nasopharyngitis and NPC by methylation-specific PCR (methylation-specific PCR, MSPCR) technology; The results showed that 64%(16/25) of NPC tissue and 12.5%(1/8) of Chronic nasopharyngitis tissue have the LARS2 gene promoter methylation, but the methylation ratio of LARS2 in NPC is significantly higher than that in chronic nasopharyngitis tissue. Statistical significant difference exists between the two groups (P=0.017), suggesting that promoter methylation is the main factor leading to down-regulation of LARS2.
Further comparison showed that both BCAT1 and LARS2 proteins participated in the same cell metabolism pathway, namely, leucine metabolism. Up-regulation of BCAT1 and down-regulation of LARS2 in NPC together led to accumulation of the intermediate product-L-leucine. As a branched-chain amino acid, L-leucine activates mTOR signaling pathways by changing the mTOR (mammalian target of rapamycin)-raptor complex stability. Studies have shown that mTOR is often highly expressed in NPC. On one hand, high expression of mTOR lead to uncontrolled proliferation of malignant cells eventually by the downstream S6K1 and 4E-BP1 molecules regulating mRNA translation of cell cycle required protein such as G1 phase and S phase protein [such as cyclin D1, Rb protein, hypoxia-inducible factor-1 (hypoxia inducible factor-1, HIF-1), c-Myc and vascular endothelial growth factor (vascular endothelial growth factor, VEGF), CLIP-170, etc.]. On the other hand, high expression of mTOR can also affect epithelial cell differentiation by inducing the expression of DNA transcription factor Id2. Based on the above experiments and analysis, we believe that there is an indirect interaction relationship between the up-expression of BCAT1 and down-expression of LARS2, which together involved in promoting the occurrence and development of NPC.
In summary, through this study, we found a NPC-related oncogene-BCAT1 from the early event of NPC located at+12p12-11 for the first time, and systematically and comprehensively examined the expression level of BCAT1 protein in chronic nasopharyngitis, NPC and nasopharyngeal cancer tissue at different stages and studied the possible mechanisms of upregulation of it in NPC. The results suggest BCAT1 protein has normal expression in chronic nasopharyngeal epithelial tissue while the expression significantly increased in NPC and different stages of nasopharyngeal carcinogenesis. The upregulation was mainly due to gene amplification, while the c-Myc also played a role for its direct regulation of BCAT1. RNAi showed that the BCAT1 knockdown could inhibit the proliferation of 5-8F, leading to cell cycle arrest. In addition, we discovered that LARS2 protein could interact with BCAT1 protein indirectly by bioinformatics studies. LARS2 is significantly down-regulated in NPC and promoter hypermethylation is the main mechanism of its inactivation. Up-regulation of BCAT1 and down-regulation of LARS2 in NPC together led to accumulation of the intermediate product-L-leucine, which could lead to uncontrolled proliferation of malignant cell by activating mTOR signaling pathway. The bioinformatics predicting results remain to be elucidated by experimental validation.
流行病学及病因学研究表明遗传因素(遗传易感性)、EB病毒感染和其它环境因素(主要是化学致癌物等)均与鼻咽癌的发生、发展有关。在这些因素的共同作用下,正常鼻咽上皮细胞发生遗传变异事件和表遗传学改变的不断积累,导致瘤基因的激活和抑瘤基因的失活,最终引起细胞的恶性转化。目前研究表明,鼻咽癌显著的种族易感性和家族聚集现象表明遗传基因的改变在鼻咽癌中起十分重要的作用,但与其他人类常见的恶性肿瘤相比,鼻咽癌发病的分子基础和相关的分子遗传学改变所知并不多,鼻咽癌发病的具体分子机制仍未阐明,因此寻找、鉴定与鼻咽癌发生发展密切相关的基因、研究其功能,是揭开鼻咽癌发病分子机制的必然途径,具有十分重要的意义。既往研究发现,一些原瘤基因(例如HRAS, cyclin D1、MDM2、STAT3、EGFR)和抑瘤基因(例如P53、P16P53、RASSF1A、DLC-1、LTF、BLU等)在鼻咽癌中异常表达,但尚未证实为鼻咽癌特异性瘤基因或抑瘤基因。因此需要我们从新的角度去重新认识和获得鼻咽癌关键的瘤基因和抑瘤基因信息。
我室尹志华和黄仲曦等利用香港、台湾、湖南和广东五个实验室发表的170例鼻咽癌比较基因组杂交数据,构建了鼻咽癌发生发展的树模型,以此来探讨鼻咽癌的发病机制。研究结果发现,鼻咽癌相关的重要染色体变异事件共11个,包括2个鼻咽癌发病的早期事件-3p26-13和+12p12,其中+12p12事件为首次报道。LOH及染色单体转移实验提示3p的丢失为鼻咽癌的早期事件以及在此区域存在鼻咽癌相关抑瘤基因。最近的研究进一步表明：3p21.3区域存在多个候选抑瘤基因(如RASSF1A1、BLU和LTF),其基因启动子区在鼻咽癌组织中常常存在高度甲基化导致其表达明显下调。这些证据均显示了树模型预测的准确性。因此,树模型首次发现的以扩增为主的+12p12-11为鼻咽癌的早期事件,为寻找鼻咽癌关键瘤基因提供了新的切入点。
[12p12-11扩增基因的筛选]
染色体扩增常导致瘤基因拷贝数增加和过表达,通过检测肿瘤组织中染色体扩增区域内候选瘤基因的过表达已经成为寻找特定肿瘤相关瘤基因的重要途径。染色体12p12-11长度为19Mb,包含123个已知基因和表达序列标签(expressed sequence tag, EST)。已有研究表明,+12p是大肠癌的早期事件,可能是生殖细胞肿瘤的晚期事件以及卵巢癌的中晚期事件。利用MEDLINE文献数据库及本实验室已完成的鼻咽癌cDNA微阵列数据资料,我们从12p12-11区初步确定了18个鼻咽癌相关候选瘤基因(DDX11、LDHB、CASC1、KRAS2、BCAT1、PTHLH、SOX5、PDE3A、CMAS、BHLHB3F、KCNJ8、PKP2、EKI1、PTX1、DNM1L、SSPN、RECQL、SLCO1A2),其中已明确的瘤基因及候选瘤基因有6个(DDX1、LDHB、CASC1、KRAS2、BCAT1、PTHLH),另外12个基因与细胞转化和增殖相关。采用RT-PCR和定量RY-PCR方法检测鼻咽癌和慢性鼻咽炎组织中这18个基因的表达水平,结果显示：其中9个基因(RECQL、PDE3A、SLCOo1A2、ASC1、DDX1、SSPN、PTHLH、BHLHB与PKP2)在慢性鼻咽炎与鼻咽癌组织中均无表达；5个基因(CMAS1、DNM1L、ETNK1、LDHB与SOy)表达水平在36例鼻咽癌及8例慢性鼻咽炎组织之间无显著差异(P>0.05)；而有4个基因(BACT1、KCNJ8、PTX1与KRAS2)在鼻咽癌组织中表达明显上调(P<0.05),提示其可能与鼻咽癌的发生发展相关。其中KCNJ8为ATP-敏感钾离子通道基因的一个亚类,与细胞的分泌和肌肉收缩有关,而与肿瘤发生及细胞的生长、增殖相关报道较少；PTX1主要参与内质网与高尔基体之间物质的膜运输,仅发现其在前列腺癌中表达下调；KRAS2在鼻咽癌中的研究颇多,其表达明显高于慢性鼻咽炎；而BCAT1(branched chain aminotransferase 1)基因编码支链氨基转移酶,在Burkitt's淋巴瘤、乳腺癌组织中明显上调,与细胞的增殖和凋亡有关,在鼻咽癌中尚未有相关报道。因此我们将其作为进一步研究的对象,探讨BXAT1与鼻咽癌的关系。
[BCAT1基因在鼻咽癌不同病理阶段中的表达]
从12p12-11筛选出的相关瘤基因回到癌前病变各阶段去考察能认识这些基因在鼻咽癌发病过程中的真正地位和意义,因此我们采用了免疫组化分析BCAT1蛋白在鼻咽正常上皮、增生活跃、轻中度、重度增生和鼻咽癌组织中的表达。结果表明,在正常假复层纤毛柱状上皮、复层鳞状上皮、轻中度增生、重度增生和鼻咽癌组织中均能检测到BCAT1蛋白不同程度的阳性表达,阳性信号定位于细胞胞浆中；BCAT1蛋白在正常上皮、轻中度增生、重度增生和鼻咽癌组织中阳性表达率分别为23.6%(17／72)、75%(18／24)、88.9%(8／9)和88.75(71／80)；结果提示在鼻咽部出现轻度增生早期病理改变时,BCAT1的表达就明显增高(P<0.05),这说明BCAT1基因对于鼻咽癌的发生可能起重要作用,这也为进一步分析该基因表达异常的分子机制提供了依据。
[探讨BCAT1基因在鼻咽癌中异常表达的可能机制]
探讨瘤基因表达上调的机制主要从遗传学改变和基因转录调控两方面进行。首先,通过PCR结合测序的方法分析20例鼻咽癌中BCAT1的11个外显子突变情况,共检测出1个外显子区SNP位点,提示基因突变对BCAT的表达无明显影响。其次,拷贝数增加被认为是瘤基因异常激活的一个重要机制,分析基因内部及其附近微卫星位点的扩增可以间接反映基因组中基因的扩增情况。采用Real-timePCR分析了BCAT1基因内部D12S1435、D12S1617和RH44650三个微卫星位点在鼻咽癌组织中扩增情况。结果表明,有42.4%(12／28)鼻咽癌组织中存在BCAT1基因微卫星位点的扩增。提示基因拷贝数的扩增可能对BCAT1在鼻咽癌组织中表达上调起一定作用。
真核基因的转录调控是一个非常复杂的过程,而转录因子参与的基因转录调控起重要作用。生物信息学分析显示在BCAT15’端非翻译区-155／-200bp区域包含有c-Myc结合位点。为了揭示c-Myc是否对BCATl的表达具有直接调控作用,我们进行了染色质免疫沉淀实验(ChIP)。结果证实,c-Myc转录因子可以直接结合于BCAT1基因启动子区的c-Myc结合位点。RT-PCR与免疫组化表明鼻咽癌中BCAT1和c-Myc的mRNA和蛋白的表达均上调,进一步利用RNA干扰技术建立了稳定干扰c-Myc表达的5-8F鼻咽癌细胞系(命名为5-8F/Si-c-myc),结果发现,封闭内源性c-Myc的表达可以下调BCAT1基因mRNA的表达水平。另外,我们将带有包含c-Myc结合位点的BCATl调控序列与荧光素酶报告基因连接构建重组报告基因载体,分别转染5-8F细胞、转染空白载体的5-8F细胞(即5-8F/si-control)以及5-8F/si-c-myc细胞中,经报告基因活性分析发现,封闭内源性c-Myc表达后,与对照组相比,重组报告基因的荧光素酶活性降低。而在转染c-Myc基因的COS7细胞系中具有较强的荧光素酶活性。上述实验结果说明c-Myc对BCAT1基因的表达具有直接的正调控作用。
[利用RNA干扰技术研究BCAT1基因在5-8F细胞的作用]
采用pSUPER.retro逆病毒载体系统,构建了针对BCAT1的RNAi载体(shBCAT1)。经测序结果证实后,利用脂质体转染技术将shBCAT1导入5-8F细胞,嘌呤霉素筛选获得抗性克隆,PCR证实抗性克隆中含有shBCAT1; RT-PCR结果表明,shBCAT1表达载体能特异性导致BCAT1 mRNA的表达下调了90%；Western Blotting分析表明细胞中的BCAT1蛋白表达下调了55%左右,这些说明我们已成功地建立了稳定干扰BCAT1基因的5-8F细胞系。
随后,我们进一步考察了BCAT1被干扰后对5-8F细胞生物学特性的影响。流式细胞仪分析结果表明：转染了shBCAT1的5-8F细胞(5-8F-shBCATl)阻滞于G1期,G1期细胞比例增加～16.66%(16.66+2.92,P=0.005),G2期细胞则减少了～6.91%(6.91+0.76.P=0.001),而对照组5-8F细胞与转染空白质粒的5-8F细胞(5-8F-blank vector)之间的细胞周期分布没有明显差异。MTT法检测发现,5-8F-shBCAT1细胞增殖速度明显低于对照组5-8F细胞与5-8F-blank vector细胞,统计学分析差异有显著性(P=-0.00)。平板克隆形成实验结果显示5-8F-shBCAT1细胞的克隆形成率明显低于转染载体组(7.2%vs 38.9%)。另外,迁移实验和侵袭实验证实,5-8F-shBCAT1细胞运动能力和侵袭能力明显降低。
IBCAT1与LARS2在鼻咽癌中的相互作用及LARS2在鼻咽癌中下调机制的研究]
生物信息学是后基因组时代的利器。STRING (http://string80. embl.de/)是目前已知的、揭示已知和预测蛋白-蛋白之间相互作用的、较广泛使用的数据库。该数据库揭示的蛋白-蛋白相互作用既包括直接相互作用(结合)也包括间接关联作用(功能相关),其率数据来源于基因组背景分析、高通量实验、共表达实验和已公布的数据库(如PUBMED, MIPS)。同时,利用STRING还可以比较不同物种间的数据。最新版STRING 8.1收录了630个物种、超过2,590,259种蛋白质数据。
在STRING的界面,输入BCAT1,可以找到直接和间接相关蛋白分子10个,它们分别是LRMP、hCG 26005、ENSP00000372、BCKDHA、BCKDHB、IARS2、IARS、LARS2、LARS和BCAT1。从这些基因的染色体定位数据中,我们发现LARS2基因位于鼻咽癌患者高频丢失区3p21。
LARS2 (leucyl aminoacyl-tRNA synthetase 2, LARS2)基因编码亮氨酰-tRNA合成酶。为了探讨LARS2基因在鼻咽癌中的可能作用,我们首先采用了RT-PCR、Real-time PCR方法检测LARS2在8例慢性鼻咽炎组织、36例鼻咽癌组织中的表达。结果发现,LARS2在78%(28／36)鼻咽癌组织中存在表达下调或缺失,提示LARS2可能是鼻咽癌相关候选抑瘤基因。接着,我们从遗传学(genetic)和表观遗传学(epigenetic)改变两方面探讨了LARS2基因失活的主要机制。1.通过PCR-SSCP并结合测序方法分析了25例鼻咽癌中启动子区和外显子1突变情况,结果未检测出突变和SNP,提示基因突变对于LARS2表达失活无明显影响。2.由于基因内部及其附近微卫星位点的丢失可以间接反映基因组中基因的缺失情况,我们采用了PCR-变性聚丙烯酰胺凝胶电泳-银染方法分析LARS2基因内部的RH25266和SHGC-12886二个微卫星位点在鼻咽癌组织中的丢失情况。结果表明,至少有28%(7／25)的鼻咽癌组织中存在LARS2基因微卫星位点的丢失,提示微卫星位点的LOH可能对LARS2在鼻咽癌细胞中表达下调起一定的作用。3.鉴于启动子区DNA异常甲基化是抑瘤基因在表观遗传学改变最为常见的方式,我们首先通过生物信息学软件对LARS2启动子区CpG岛分布情况进行了分析,发现LARS2启动子区存在一个227bp长的CpG岛。以此区域为模板,选择针对该区段的甲基化和非甲基化特异性引物,利用甲基化特异性PCR (methylation-specific PCR, MSPCR)技术,我们分析了慢性鼻咽炎和鼻咽癌组织中LARS2启动子甲基化情况。结果发现,64%(16／25)鼻咽癌组织和12.5%(1／8)慢性鼻咽炎组织存在LARS2基因启动子甲基化情况,但鼻咽癌组织中LARS2基因甲基化比率明显高于慢性鼻咽炎组织,两者存在明显的统计学差异(P=0.017),这提示启动子甲基化是导致LARS2基因表达下调的主要因素。
进一步比较发现,BCAT1蛋白和LARS2蛋白的相关性表现在这两个蛋白作用于同一条细胞代谢通路,即亮氨酸代谢。BCAT1在鼻咽癌中显著上调,而LARS2在鼻咽癌中表达下调,共同导致了中间产物L-亮氨酸的累积。作为一种支链氨基酸,L-亮氨酸通过改变mTOR (mammalian target of rapamycin)-raptor复合体的稳定性,激活：mTOR的信号传导通路。有研究表明,鼻咽癌组织常常存在mTOR的高表达。一方面,高表达的mTOR可以通过下游分子S6K1和4E-BP1调控编码细胞周期G1期和S期必需蛋白,如细胞周期素D1、Rb蛋白、低氧诱导因子-1(hypoxia inducible factor-1, HIF-1)、c-Myc和血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)、LIP-170等的mRNA翻译,最终导致细胞的恶性失控性增殖。另一方面,高表达的mTOR也可以通过诱导DNA转录调节因子Id2的表达影响上皮细胞的分化。基于以上实验和分析,我们认为BCAT1的表达增加和LARS2基因的表达下调两者之间存在间接的相互作用关系,它们共同参与推动鼻咽癌的发生和发展。
综上所述,通过本研究,我们首次从鼻咽癌发生早期事件+12p12-11区域内发现鼻咽癌相关瘤基因BCAT1,并系统、全面地考察了BCAT1蛋白在慢性鼻咽炎、鼻咽癌及鼻咽癌变不同阶段组织中的表达情况,探讨了导致其表达上调的可能机制,并进一步研究了BCAT1基因在鼻咽癌细胞中的功能。结果提示,BCAT1在慢性鼻咽上皮组织和正常鼻咽上皮中正常表达,而在鼻咽癌组织和鼻咽癌变不同阶段明显表达上调,而这种表达上调主要是由于基因扩增所致,而瘤基因c-Myc对其直接调控也发挥了一定作用。RNA干扰实验表明,干扰BCAT1的表达能够抑制鼻咽癌细胞的增殖,导致细胞周期的阻滞。此外,我们通过生物信息学发现了与BCAT1蛋白有间接相互作用的LARS2蛋白。LARS2基因在鼻咽癌组织中表达明显下调,其失活机制主要是基因启动子区甲基化。鼻咽癌中BCAT1显著上调和LARS2表达下调,共同导致了中间产物L-亮氨酸的累积,而L-亮氨酸则可以通过激活mTOR的信号传导通路最终导致细胞的恶性失控性增殖。当然,生物信息学预测结果还有待实验进一步验证。
Nasopharyngeal carcinoma (NPC) is one of the malignant tumors with a high incidence and causing a serious health problem in Southeast Asia and Southern China, which has distinct geographical and ethnic distribution. More than 95% of NPC is poorly differentiated and undifferentiated with high incidence of early metastasis (metastasis is the main cause of death in NPC). Radiation therapy is currently the first choice for NPC treatment. Although the radiotherapy equipment and technology were improved tremendously, but the five-year survival rate of NPC did not radically change and remains at about 50%-60%. Therefore, it is of importance to explore its causes and find a new and effective treatment to NPC.
Studies on epidemiology and etiology of NPC have shown that genetic factors (genetic susceptibility), EB virus infection and other environmental factors (mainly chemical carcinogens, etc.) are related to the occurrence and development of NPC. Under the combined effect of these factors, the genetic variation events affecting normal nasopharyngeal epithelial cells and the continuous accumulation of epigenetic changes led to activation of oncogene and inactivation of tumor suppressor genes, ultimately resulting in malignant cellular transformation. The current studies show that significant ethnic susceptibility to NPC and familial aggregation phenomenon indicate that changes of genetic genes play an important role in NPC, but compared to other human common malignant tumor, there is little known facts about the basis of molecular pathogenesis of NPC and the associated change in molecular genetics. The specific molecular mechanisms of NPC incidence have not been elucidated yet, so it is of great significance to look for and identify the genes closely related to the development of NPC and study their functions which is the inevitable way to reveal the molecular mechanism of NPC. Past research found that some proto-oncogenes(for example, HRAS, KRAS2,cyclin D1, MDM2, STAT3, EGFR, etc.) and tumor suppressor genes (P53,P16,RASSF1A, DLC-1, LTF, BLU, etc.) were expressed abnormal in NPC, but not yet confirmed as nasopharyngeal cancer-specific tumor gene or tumor suppressor gene. Therefore, we need to understand and access the information of the significant oncogenes and tumor suppressor genes related to NPC from a new perspective angle.
Zhi-Hua Yin and Zhong-Xi Huang in our Lab have constructed a tree model of NPC tumorigenesis to explore the pathogenetic mechanism of NPC by using comparative genomic hybridization data of 170 cases of NPC from five laboratories in Hong Kong, Taiwan, Hunan and Guangdong. The study showed 11 chromosomal aberrations in NPC, among which 2 early events in the pathogenesis of NPC were-3p26-13 and+12p12, the latter is the first reported. LOH and chromatid transfer experiments suggested the loss of 3p is an early event in NPC as well as some NPC tumor suppressor genes might exist at this region. Recent research further indicates that there are several candidate tumor suppressor genes (such as RASSF1A, BLU and LTF) located at 3p21.3, and also there are hypermethylation of gene promoter regions, leading to down regulation of gene expression. The evidences mentioned above suggest that the tree model prediction is accurate. Therefore, the amplification based+12p12-11 as the early event of NPC provides a new entry point to find the key oncogenes in the pathogenesis of NPC.
[The screening of amplified genes located at 12p12-11]
Chromosomal gain often leads to the amplification in copy number and overexpression of oncogene and it's important to search for specific tumor-related genes by detecting over-expression of candidate oncogenes in the amplified chromosome region. Chromosome 12p12-11,19Mb at length, contains 123 known genes and expressed sequence tag (expressed sequence tag, EST). Previous studies have shown that,+12p is an early event in colorectal cancer, and that may be the late events of germ cell tumors or late events of ovarian cancer. We preliminarily determined 18 candidate tumor-related genes, among which 6 real oncogenes were determined(DDX11, LDHB, CASC1, KRAS2, BCAT1, PTHLH) and other 12 genes (SOX5, PDE3A, CMAS, BHLHB3F, KCNJ8, PKP2, EKI1, PTX1, DNM1L, SSPN, RECQL, SLCO1A2) were associated with cell transformation and proliferation by using MEDLINE database and NPC cDNA microarray data from our laboratory.
We determined these 18 gene expression level between 36 cases of NPC and 8 cases of chronic nasopharyngitis tissues by RT-PCR and quantitative RT-PCR. The results showed 9 genes (RECQL, PDE3A, SLCO1A2, ASC1, DDX1, SSPN, PTHLH, BHLHB with PKP2) not expressed in NPC and in chronic nasopharyngitis tissues and the expression of 5 genes (CMAS1, DNM1L, ETNK1, LDHB and SOX) has no significant difference between 36 cases NPC and 8 cases chronic nasopharyngitis tissues (P> 0.05); While 4 genes (BACT1, KCNJ8, PTX1 and KRAS2) in NPC tissues were expressed significantly higher than that in controls (P<0.05), suggesting these 4 genes may be relevant to the development of NPC.
KCNJ8 is categorized into a sub-class of the ATP-sensitive potassium ion channel gene in relation to cell secretion and muscle contraction. At present there is no evidence that it is associated with the occurrence of cancer; PTX1 mainly involved in membrane transport of materials in endoplasmic reticulum and Golgi apparatus, down-expressed only found in prostate cancer; there are more studies of KRAS2 in NPC, its expression was significantly higher than that of the normal nasopharyngeal tissue and nasopharyngitis. BCAT1 (branched chain aminotransferase 1 gene) in Burkitt's lymphoma and breast cancer tissues were expressed significantly in relation to cell proliferation and apoptosis, but there is no relevant reports in NPC. Therefore, we select BCAT1 as a target for further study to explore its relationship with NPC development.
[the expression of BCAT1 protein in different NPC pathological stages]
To study tumor-related oncogenes selected from the 12p12-11 in various stages of precancerous lesions can be aware of the significance of these genes in NPC pathogenesis. Therefore, we used immunohistochemical method to analyze the expression of BCAT1 protein in the normal nasopharyngeal epithelium, low-moderate grade pre-invasive lesion, high grade pre-invasive lesion and NPC. The results showed that in the normal psudo-stratified ciliated epithelium, stratified epithelium, low-moderate grade pre-invasive lesion, high grade pre-invasive lesion and NPC, different levels of protein expression, positive signal located in the cytoplasm were able to be detected; The positive rate of the expression of BCAT1 protein in normal epithelium, low to moderate grade pre-invasive lesion, high grade pre-invasive and NPC were 23.6%(17/72),75%(18/24),88%(8/9) and 88.75 (71/80) respectively. The results suggest the expression of BCAT1 significantly increased (P<0.05) with the hyperplasia occurring in the nasopharynx of early pathological changes, which indicates the BCAT1 may play an important role in the occurrence of NPC and also provide a basis for further analysis of the molecular mechanism of gene expression abnormality.
[Explore possible mechanisms about abnormal expression of BCAT1 in NPC]
Genetic alterations and transcription des-regulation are two approaches to explore the mechanism of tumor gene up-regulation. Firstly, we analyzed the mutation status of 11 exons of BCAT1 from 20 cases of NPC by PCR-sequencing and one SNP site in exon1 was detected, suggesting mutation had no significant effect on the expression of BCAT1. Secondly, the copy number amplification is considered as a mechanism to activate an oncogene. Analyzing the amplification of microsatellite loci genes within and near the gene can indirectly reflect the gene amplification in the genome. We analyzed the amplification of three microsatellite loci (D12S1435, D12S1617 and RH44650) within BCAT1 by Real-time PCR in NPC. The results showed that 42.4%(12/28) of the NPC tissue have the amplification of microsatellite loci in BCATl. All above suggested that increase of the copy number may play a role in BCAT1 upregulation in NPC tissues to some degree.
Regulation of eukaryotic gene transcription is a very complicated process, and the participation of transcription factors play an important role in regulation of gene transcription. Bioinformatics analysis shows that BCAT1 5'untranslated region-155/-200bp area contains a c-Myc binding sites. In order to reveal whether c-Myc has a direct role in regulation of the expression of BCATl, we carried out chromatin immunoprecipitation experiments (ChIP). The results confirm c-Myc transcription factor can be directly binding the c-Myc binding site in BCAT1 gene promoter region. Further, we used RNAi to establish a stable 5-8F NPC cell line (named 5-8F/Si-c-Myc) with c-Myc expression interference and found that blocking of endogenous c-Myc expression could reduce the mRNA expression level of BCATl. In addition, we established a recombinant reporter gene vector by connecting BCAT1 regulatory sequences including c-Myc binding sites with the luciferase reporter gene, and then transfected 5-8F cell,5-8F cell with the blank vector (5-8F/si-control), as well as 5-8F/si-c-Myc cell. The reporter gene activity analysis showed that reporter gene activity of recombinant luciferase decreased after knockdown of endogenous c-Myc expression compared with the control group. The experimental results show that c-Myc has a direct positive regulatory role in the expression of BCAT1.
[Study the function of BCAT1 in 5-8F by RNA interference technology]
We constructed RNAi vectors targeting BCAT1 (shBCAT1) by pSUPER.retro retrovirus vector system. After the results confirmed by sequencing, we transfected shBCAT1 into 5-8F using the lipofection technique and puromycin resistant clones were selected, PCR confirmed that resistant clones contain shBCAT1; RT-PCR results showed that shBCAT1 expression vector could specifically lead to expression of BCAT1 reduced by 90%. Western Blotting analysis showed that the protein expression of BCAT1 decreased 55% or so in 5-8F. These results confirm that we have successfully established a stable interference system for BCAT1 gene in 5-8F.
Subsequently, we further examined the effects on 5-8F biological characteristics after the BCAT1 was blocked. Flow cytometric analysis showed that cell cycle distribution of 5-8F and 5-8F-blank vector did not change significantly; The 5-8F-shBCAT1 caws blocked in the G1 phase, G1 phase cells increased～16.66%(16.66±2.92,P=0.005), while the G2-phase cells decreased by～6.91%(6.91±0.76. P= 0.001). MTT assay found that the cell proliferation of 5-8F-shBCAT1 rate was significantly lower than control group,5-8F and 5-8F-blank vector, and the difference was statistically significant (P= 0.00). In addition, migration assay and invasion assay demonstrated that 5-8F-shBCAT1 could also significantly decrease the mobility and invasion ability of 5-8F cells.
[The interaction between BCAT1 and LARS2 and the inactivation Mechanism of LARS2 in NPC]
Bioinformatics is a tool for post-genomic era. STRING (http://string80.embl.de/) is a known and widely used database which reveals the protein-protein interaction between the known and predicted protein. The protein-protein interaction includes both the direct (binding) and the indirect interaction(association) and the background data come from genomic analysis, high-throughput experiments, co-expression experiments and published databases (such as the PUBMED, MIPS). At the same time, the data among different species can be compared by STRING. The latest version STRING 8.1 contains 630 species, more than 2,590,259 proteins. We found 10 protein molecules directly and indirectly interacted with BCAT1, which are LRMP, hCG_26005, ENSP00000372, BCKDHA, BCKDHB, IARS2, IARS, LARS2 and LARS. Moreover, we found that LARS2 gene is located in 3p21, which has a high-frequency loss in NPC. LARS2 (leucyl aminoacyl-tRNA synthetase 2) gene encodes leucyl-tRNA synthetase. In order to explore the possible role of LARS2 in NPC, we used RT-PCR and Real-time PCR to detect the expression of LARS2 in 8 cases of chronic nasopharyngitis and 36 cases of NPC. The results showed that, the mRNA expression of LARS2 was down-regulated or deleted in 78%(28/36) of the NPC tissue, suggesting that LARS2 may be a candidate tumor suppressor gene in NPC. Then, we studied the primary mechanism for LARS2 inactivation from the aspects of genetics and epigenetics. Firstly, we studied mutation in the promoter region and exon 1 in 25 cases of NPC by PCR-SSCP combined with sequencing analysis. The results did not detect the mutation and SNP, indicated that mutation had no effect on inactivation of LARS2 expression. Secondly, as the loss of microsatellite loci within and near the gene can indirectly reflect the absence of genes in the genome, we used PCR-denaturing polyacrylamide gel electrophoresis to analyze the loss of the two microsatellite loci (RH25266 and SHGC-12886) within LARS2 in NPC. The results showed that at least 28%(7/25) of NPC have loss of microsatellite loci in LARS2, suggesting that LOH of microsatellite loci may downregulate the expression of LARS2 in NPC in a certain degree. Thirdly, hypermethylation of gene promoter is the most common epigenetic change of tumor suppressor gene. Therefore, whether there exist CpG islands in the promoter region of LARS2 was analyzed and a 227 bp long CpG island in the promoter region was found. Then, we designed the methylation and the non-methylation-specific primers of this region. Finally, we analyzed methylation status of LARS2 promoter in the chronic nasopharyngitis and NPC by methylation-specific PCR (methylation-specific PCR, MSPCR) technology; The results showed that 64%(16/25) of NPC tissue and 12.5%(1/8) of Chronic nasopharyngitis tissue have the LARS2 gene promoter methylation, but the methylation ratio of LARS2 in NPC is significantly higher than that in chronic nasopharyngitis tissue. Statistical significant difference exists between the two groups (P=0.017), suggesting that promoter methylation is the main factor leading to down-regulation of LARS2.
Further comparison showed that both BCAT1 and LARS2 proteins participated in the same cell metabolism pathway, namely, leucine metabolism. Up-regulation of BCAT1 and down-regulation of LARS2 in NPC together led to accumulation of the intermediate product-L-leucine. As a branched-chain amino acid, L-leucine activates mTOR signaling pathways by changing the mTOR (mammalian target of rapamycin)-raptor complex stability. Studies have shown that mTOR is often highly expressed in NPC. On one hand, high expression of mTOR lead to uncontrolled proliferation of malignant cells eventually by the downstream S6K1 and 4E-BP1 molecules regulating mRNA translation of cell cycle required protein such as G1 phase and S phase protein [such as cyclin D1, Rb protein, hypoxia-inducible factor-1 (hypoxia inducible factor-1, HIF-1), c-Myc and vascular endothelial growth factor (vascular endothelial growth factor, VEGF), CLIP-170, etc.]. On the other hand, high expression of mTOR can also affect epithelial cell differentiation by inducing the expression of DNA transcription factor Id2. Based on the above experiments and analysis, we believe that there is an indirect interaction relationship between the up-expression of BCAT1 and down-expression of LARS2, which together involved in promoting the occurrence and development of NPC.
In summary, through this study, we found a NPC-related oncogene-BCAT1 from the early event of NPC located at+12p12-11 for the first time, and systematically and comprehensively examined the expression level of BCAT1 protein in chronic nasopharyngitis, NPC and nasopharyngeal cancer tissue at different stages and studied the possible mechanisms of upregulation of it in NPC. The results suggest BCAT1 protein has normal expression in chronic nasopharyngeal epithelial tissue while the expression significantly increased in NPC and different stages of nasopharyngeal carcinogenesis. The upregulation was mainly due to gene amplification, while the c-Myc also played a role for its direct regulation of BCAT1. RNAi showed that the BCAT1 knockdown could inhibit the proliferation of 5-8F, leading to cell cycle arrest. In addition, we discovered that LARS2 protein could interact with BCAT1 protein indirectly by bioinformatics studies. LARS2 is significantly down-regulated in NPC and promoter hypermethylation is the main mechanism of its inactivation. Up-regulation of BCAT1 and down-regulation of LARS2 in NPC together led to accumulation of the intermediate product-L-leucine, which could lead to uncontrolled proliferation of malignant cell by activating mTOR signaling pathway. The bioinformatics predicting results remain to be elucidated by experimental validation.
引文
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