【作者】 王铠；

【导师】 凌天牖；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 口腔鳞状细胞癌(Oral Squamous Cell Carcinoma, OSCC)是头颈部最常见的恶性肿瘤。尽管传统的手术、放疗、化疗及其综合治疗对早期OSCC可获得较好的治疗效果,但对于晚期或复发的病人难以达到满意的疗效。因此,研究OSCC发病的分子机制,寻找其早期诊断和治疗的分子标志物,将有助于发现新的治疗方法和提高口腔肿瘤病人的生存率和生存质量。越来越多的研究提示,恶性肿瘤实质上是一种基因疾病,其发生、发展涉及到多种基因的改变。抑瘤基因(tumor suppressor gene, TSG)在人体内往往发挥非常重要的生理功能,其失活、缺失与肿瘤的发生发展密切相关。许多抑瘤基因的发现都是通过研究等位基因杂合性丢失或纯合性丢失(loss of heterozygosity/homozygosity, LOH)而实现的。研究表明,染色体3p21.3区域在肺癌、鼻咽癌等多种肿瘤组织中表现为高频缺失,并存在多个与肿瘤发生密切相关的候选抑瘤基因。多个研究小组发现,该区域在OSCC中同样存在不同程度高频缺失(23%-50%)。目前,该区域内候选抑瘤基因在OSCC发生发展中的作用还鲜少有人研究,大量的信息还有待进一步挖掘。基于以上原因,本研究拟在文献复习及生物信息学方法分析的基础上,选择该区域内鲜有研究的候选抑瘤基因作为研究对象。筛选在OSCC中表达下调的候选基因并进一步分析其表达失活的可能原因及其在OSCC中的生物学功能。[3p21.3区候选抑瘤基因在口腔鳞状细胞癌中的筛选]首先,通过文献复习,生物信息学的方法分析基因的可能功能,确定11个基因(AUXD1、BAP1、FUS1、GNAT1、LARS2、LTF, NPRL2、RASSF1A、SEMA3F、SEMA3B和ZNF35)为初步研究对象。采用RT-PCR方法分析11个基因在12例OSCC癌组织及其旁侧正常对照组织中的表达情况。结果发现,LTF, GNAT1、SEMA3B、AUXD1基因分别在50%(6／12),58.3%(7／12),41.7%(5／12),41.7%(5／12)的口腔鳞癌组织中表达下调或缺失,与在对应正常口腔组织中的表达存在明显差异(P<0.05)。SEMA3F、RASSFIA基因在口腔鳞癌组织中均表现为25%(3／12)的表达下调或缺失。而ZNF35和LARS2基因在口腔鳞癌组织中表现为33.3%(4／12)和25%(3／12)上调表达。其它3个基因(NPRL2、BAP1、FUS1)在口腔鳞癌组织及其对侧正常对照组织中未见明显表达差异(P>0.05)。初步筛选的结果提示,LTF、GNAT1、EMA3B、AUXD14个基因在OSCC组织中呈现较高频率的下调表达,可能与OSCC发生发展密切相关。为此,我们选择这4个基因作为进一步研究的对象。[启动区异常甲基化在基因表达中的作用]为能较为准确地判断4个基因在OSCC组织中的表达情况,我们采用RT-PCR方法,分析了4个基因在舌癌细胞系TCA8113细胞和36对OSCC组织标本中的mRNA表达水平。结果发现,除AUXD1基因在TCA8113细胞中表达外,LTF、GNAT1，SEMA3B均表达下调或缺失。与癌旁对照口腔组织相比,LTF, GNAT1、SEMA3B、AUXD1基因分别在61.1%(22／36),44.4%(16／36),50%(18／36),47.2%(17／36)的口腔鳞癌组织中表达下调或缺失,统计学分析,4个基因在两组间的表达具有显著差异(P<0.05)。为考察4个基因的异常表达与临床相关因素的关系。采用χ2检验对数据进行统计分析,结果提示,LTF, AXUD1、GNAT1和SEMA3B的表达均与患者性别无相关性；而仅LTF基因的表达下调与OSCC发生转移有关,有转移的患者,其LTF的表达明显低于没有转移的患者(P<0.05),其它三个基因均与患者肿瘤转移无明显相关性(P>0.05)。为进一步探讨基因启动子区甲基化对于基因在OSCC组织或细胞中表达异常的影响,课题采用甲基化特异性PCR (methylation specific PCR, MSP)分析了LTF, GNAT1、SEMA3B在其表达下调的OSCC组织中各自启动子区甲基化的情况。结果发现,LTF, GNATl和SEMA3B在OSCC组织中启动子区均有高甲基化,甲基化率分别为72.7%(16／22)、75%(12／16)、77.8%(14／18),而在癌旁对照组织中三个基因也存在不同程度的的甲基化,其频率也分别为22.7%(5／22)、56.25%(9／16)和38.9%(8／18)。χ2分析表明,LTF和SEMA3B在两种组织中存在启动子区甲基化差异(P=-0.000<0.01),但GNAT1基因启动子区的甲基化在两组间不具有显著差异(P=0.264>0.01)。甲基转移酶抑制剂5-杂氮-2-脱氧胞苷(5-aza-2’-deoxycytidine, 5-Aza-Cdc)处理舌癌细胞株TCA8113,通过RT-PCR和甲基化特异性PCR分别检测了处理前和用不同浓度的药物处理细胞后基因mRNA表达水平和其启动子区甲基化的情况。结果提示：高甲基化能明显抑制舌癌细胞株TCA8113中LTF基因和SEMA3B基因的mRNA表达,而去甲基化后能部分逆转细胞中两个基因的启动子甲基化状态,重新激活或上调该基因的表达。这些结果均提示启动子区高甲基化对于LTF基因和SEMA3B基因表达失活具有重要的作用。[LTF基因在舌癌细胞中的功能研究]相对RT-PCR方法,Real-time PCR能更为准确地检测基因mRNA表达水平,为此,我们在前期分析的基础上,采用Real-time PCR方法进一步验证了LTF基因在OSCC及其癌旁正常对照组织中的表达情况。结果LTF基因表达在OSCC组织中的平均Ct值(9.490±1.675)明显大于癌旁对照组(6.212±4.00),说明LTF基因在OSCC组织中的表达明显下调(P<0.01),其下调频率为63.3%(19／30),并与RT-PCR初筛结果相似。蛋白质是基因功能的执行者,蛋白质的表达水平与基因功能的发挥密切相关。因此,在检测了LTF mRNA表达水平之后,我们进一步采用免疫组化方法考察了30例OSCC患者石蜡切片中LTF蛋白表达分布的情况。结果显示,LTF蛋白主要表达在细胞胞浆中,并主要分布在鳞状上皮中。30例OSCC石蜡切片中,除4例表现较弱信号外,其余26例癌旁组织上皮均可检测到较强的阳性信号(++)或(+++),而在癌组织中,除12例检测到阳性信号外,其余18例表现为阴性信号(一),或较弱信号(+),其下调频率为60%(18／30),两组间存在明显差异(P<0.01)。为了进一步探讨LTF在舌癌细胞中的作用,我们从购买的pCMV6-XL5-LTF载体中,采用酶切方法分离获得包含LTF开放阅读框序列在内的2.8kb cDNA片段,经测序证实LTF cDNA存在三个插入或错义SNP位点(23R, A29T, K47R),并经生物信息学分析推测这些位点的多态性可能与LTF的生物学功能无关。将LTF基因的开放阅读框序列克隆到pcDNA3.1(-)载体中,构建了LTF基因的真核表达载体LTF/pcDNA3.1(-),并重新命名为pcLTFo利用脂质体转染技术将pcLTF导入TCA8113细胞,G418筛选获得抗性克隆,进一步采用RT-PCR检测LTF基因的mRNA表达,采用Western Blotting检测LTF蛋白的表达,结果表明我们已成功地建立稳定转染pcLTF的TCA8113细胞系TCA-LTF。免疫细胞化学实验显示表达的LTF蛋白主要定位于胞浆。随后我们检测了LTF基因稳定表达后对TCA8113细胞的生物学特性的影响。利用MTT法和平板克隆形成实验观察LTF基因稳定表达后对LTF细胞增殖能力和克隆形成能力的影响。MTT的结果表明TCA-LTF细胞的增殖速度明显低于对照组(P<0.05)；平板克隆形成实验结果显示TCA-LTF细胞的克隆形成率明显低于对照组(27.7%vs 51.1%／47.7%)。流式细胞分析结果表明,与对照组相比,转染pcLTF后可以使TCA8113细胞停滞于G0-G1期,G0-G1期细胞比例增加(66.37%vs 53.7%),S期(24.6%vs 32.83%)和G2-M期细胞比例减少(9.05%vs 13.47%)。提示LTF基因稳定表达后,阻止细胞周期进程,导致TCA8113细胞的增殖能力和体外克隆形成能力降低。总之,本课题以染色体3p21.3缺失区域为切入点,在oscc组织及其癌旁对照组织中分析了该区域内11个候选抑瘤基因的表达情况。结果提示,在oscc组织中,LTF、GNAT1、SEMA3B和AUXD14个基因的mRNA表达呈较高水平下调或缺失,并且启动子甲基化可能是影响LTF和SEMA3B表达下调的重要机制之一,但与GNAT1基因表达下调无关。恢复LTF基因的表达,改善了舌癌细胞TCA8113的恶性生物学特性。这些研究结果将有助于了解口腔黏膜癌变的分子机制,也为寻找新的治疗方法和用于早期诊断、治疗的靶标分子提供一定的理论和实验依据。更多还原

【Abstract】 Oral squamous cell carcinoma (OSCC) remains to be the major malignant tumor in head and neck tumor. Although the traditional surgery, radiotherapy, chemotherapy and combined therapy have a great therapeutic efficacy to early oral cancer, a satisfied effect can not be achieved to those patients who were in advanced stage or recurrence stage. Therefore, studying the molecular mechanism of carcinogenesis of oral mucous membrane and finding the molecular marker of early diagnosis and treatment can attribute to acquire new therapy and raise survival rate and quality.Increasing study suggested that malignant tumor is actually a gene disease and the occurrence and development involved the change of several genes. Tumor suppressor gene (TSG) always plays an important role in physiological function and its inactivation and deletion are closely related to the occurrence and development of tumor. Many tumor suppressor genes are found by studying the loss of heterozygosity or homozygosity of allele. Study showed high frequency deletion of chromosome 3p21.3 was existed in several tumors such as lung cancer, nasopharyngeal carcinoma (NPC), and so on. Several groups find that the region of chromosome 3p21.3 also exist different degree of high frequency deletion in OSCC. Currently, little study has done to the role of TSGs at this region in the occurrence and development of OSCC and amount of information remain to further be mined.Based on the reasons above, in this study, we prepare to choose the candidate TSGs little reported as the research object. Screening the candidate genes down-expression in OSCC and further analyze the possible reason of expression inactivation and the biological function in OSCC.[The screening of candidate TSGs located at 3p21.3 in OSCC]We preliminarily determined 11 genes as the research object, such as AUXD1、BAP1、FUS1、GNAT1、LARS2、LTF、NPRL2、RASSF1A、SEMA3B、SEMA3F and ZNF1, by literature review and analyzing the possible function of genes with bioinformatics. RT-PCR was performed to analyze the expression of 11 genes at the transcription level in primary OSCC biopsies and matched tumor-adjacent normal tissues. The results showed that LTF, GNAT1, SEMA3B, AUXD1 were down-regulation or deletion in 50%(6/12),58.3%(7/12),41.7%(5/12),41.7%(5/12) respectively in OSCC and there were significant difference compared to matched tumor-adjacent normal tissues (P<0.05). SEMA3F, RASSF1A were down-regulated or deletion in 25%(3/12), while ZNF35, LARS2 were up-regulated in 33.3%(4/12) and 25%(3/12) in OSCC. No significant difference in transcriptional levels of other 3 genes (NPRL2、BAP1、FUS1) was found between OSCC and matched tumor-adjacent normal tissues (P>0.05). Results of preliminary screening suggested LTF, GNAT1, SEMA3B, AUXD1 were highly down-regulated in OSCC. Therefore, we choose these 4 gens as the object for further research.[The role of aberrant promoter methylation in gene expression]To understand exactly the expression statues of 4 genes (LTF, GNAT1, SEMA3B, AUXD1) in OSCC, we analyzed the expression levels of 4 genes in tongue cancer cell line-TCA8113 and 36 OSCC and matched tumor-adjacent normal tissues by RT-PCR. Results revealed LTF, GNAT1, SEMA3B were all down-regulated or deleted except AUXD1 expressed in TCA8113. Compared to matched tumor-adjacent normal tissues, LTF, GNAT1, SEMA3B, AUXD1 were down-regulated or deleted in 61.1%(22/36),44.4%(16/36),50%(18/36),47.2%(17/36) in OSCC biopsies. Statistical analysis showed that the 4 genes have statistical significance between the two groups (P<0.05).These data were further analyzed to determine the relationship between the aberrant expression of 4 genes and clinical related factor withχ2 test.Statistical analysis indicated that no any significant correlation was found between gender and expression of LTF、AXUD1、GNAT1 and SEMA3B (P>0.05); Only the under-expression of LTF but not other three genes was significantly correlated with metastasis. The expression level of LTF in patients with metastasis was significantly lower than those with no metastasis (P<0.05).To further explore the role of promoter methylation in abnormal expression of genes in OSCC biopsies or cell lines, we analyzed the promoter methylation statues of LTF, GNAT and SEMA3B in OSCC patients with down-regulated expression of each 3 genes. MSPCR analysis revealed LTF, GNAT, SEMA3B promoter hypermethylation in 72.7%(16/22),75%(12/16),77.8%(14/18) primary OSCCs respectively, and in 22.7%(5/22)、56.25%(9/16) and 38.9%(8/18) matched tumor-adjacent normal tissues respectively.χ2 analysis indicated that there was a significant difference in methylation frequency of LTF (P=0.000<0.01) between OSCCs and tumor-adjacent normal tissues, while no difference in that of GNAT1 between this two groups (P=0.264>0.01). In the other hand, restoration of LTF or SEMA3B expression along with decrease in methylated allele of LTF or SEMA3B could be achieved in TCA8113 cell line after the treatment with 5-aza-2’-deoxycytidine (Aza), a DNA methyltransferase inhibitor. It suggested that promoter hypermethylation should be the important mechanism responsible for inactivation of LTF or SEMA3B in OSCC.[Investigation of LTF functions in tongue cancer cells]Real-time PCR can detect more exactly than RT-PCR in mRNA expression levels. Based on previous analysis, we further determined the expression levels of LTF in OSCC biopsies and matched tumor-adjacent normal tissues using real-time PCR method. Results showed the average of Ct value (9.490±1.675) denoting the expression level of LTF in OSCC biopsies was higher than that in matched tumor-adjacent normal tissues (6.212±4.00). It is similar to previous results of RT-PCR that LTF expression was obviously down-regulation (P<0.01) and the ratio of down-regulation was 63.3%(19/30).To better understand the role of LTF protein in OSCC, immunohistochemical method was also used to detect the protein levels and location of LTF in 30 OSCC specimens. The results showed LTF protein was mainly located in the cytoplasm and stratified squamous epithelial cell. Additionally, positive strong signal (++)or(+++) can be detected in 26 tumor-adjacent normal tissues besides 4 expressed weak signal, while 60%(18/30) of the OSCC tissues showed negative (-) or very low expression level (+) of LTF protein.In order to investigate the functions of LTF in tongue cancer cells, using enzyme digestion methods, we obtained-2.8kb cDNA containing the open reading frame (ORF) sequence of LTF gene from bought pCMV6-XL5-LTF. Sequencing results showed that LTF cDNA sequence contained three missense SNP site (23R, T29A, R47K), but these had no influence on LTF function according to bioinformatic analysis. Subsequently, the eukaryotic expression vector of LTF (named pcLTF) was constructed by inserting the LTF ORF into the pcDNA3.1(-) vector. Then the pcLTF vector was transferred into TCA8113 cells by Lipofectamine transfection technology. After G418 selection, several G418-resistant cell clones were obtained. It was demonstrated that LTF expressed stably both at mRNA and protein level in the G418-resistant cell clones detected by RT-PCR and western blotting, respectively, showing that the TCA8113 cell line stably expressing LTF gene (named TCA-LTF) was successfully established. The recombinant LTF protein was detected mainly in the cytoplasm of TCA-LTF by immunocytochemistry.Subsequently, we examined the changes in biological characteristics TCA-LTF cells. The proliferative activity and clonality ability of TCA-LTF were measured by MTT analysis and colony formation assay, respectively. Compared with TCA8113 cells transfected with blank vector (named TCA-pc3.1) or untransfected TCA8113 cells, TCA-LTF cells showed especial proliferation inhibition (P<0.05) and reduction in colony formation efficiency (27.7% vs 51.1%/47.7%). Flow cytometry (FCM) analysis showed that TCA-LTF could block the cell cycle progression of TCA8113 cells in G0-G1 phase, as the pencentage of G0-G1 phase cells were increased (66.37% vs 53.7%) while S phase (24.6% vs 32.83%) and G2-M phase cells (9.05%vs 13.47%) were reduced. These results indicated that LTF overexpression blocked TCA8113 cells at G0-G1 phase resulting in attenuated proliferation and clony forming ability in vitro.In summary, we elected the deletion region of chromosome 3p21.3 as a breakthrough point to find other TSG candidates closely related OSCC, expression of 11 genes from which in OSCC biopsies and matched tumor-adjacent normal tissues have been done. Our results suggest that 4 genes, such as LTF, GNAT1, SEMA3B, AUXD1, expresses is significantly down-regulated or absent in OSCC tissues. Promoter hypermethylation plays a key role in inactivating LTF and SEMA3B in OSCC, but not in down-regulation expression of GNAT1. Re-expression of LTF in TCA8113 cells can confer partial reversion of the malignant phenotype of tongue cance cells. These results will help us comprehensively understand molecular mechanism underlying OSCC carcinogenesis, and provide the theoretical and experimental evidence for finding new therapy method and biomarker used in early diagnosis or therapy.更多还原