【作者】 刘佳；

【导师】 富伟能；

【作者基本信息】 中国医科大学 ， 遗传学， 2010， 博士

【摘要】 前言喉鳞状细胞癌(laryngeal squamous cell carcinoma, LSCC)是头颈部常见的恶性肿瘤之一。在我国,东北地区是喉癌的高发区。过去的10年中,我国喉癌的发病率呈现上升的趋势。喉癌对放疗及化疗均不敏感,手术是目前治疗的主要手段,但手术会给患者造成不同程度的损伤。此外,在诊断和治疗过程中普遍存在的一个问题是：除声门型喉癌外,其他类型喉癌的发病比较隐蔽,一旦确诊,患者往往多已进入中、晚期。因此对喉癌进行早期诊断,并在基因水平寻求新的治疗手段成为当前急需解决的问题。恶性肿瘤被认为是一种遗传和表观遗传性疾病。基因在疾病发生和发展的分子机制中扮演非常重要的作用,基因序列改变可以导致相应基因的表达异常进而导致表型的变化。不仅如此,基因的表达还受表观遗传的控制。近年来,表观遗传学研究已成为基因表达调控的研究热点之一。表观遗传调控机制包括DNA甲基化、组蛋白修饰和RNA干扰等,而DNA甲基化是表观遗传学研究最深入、最重要的一种机制,与许多疾病如肿瘤的发生和发展密切相关。由于DNA甲基化是一个可逆的修饰过程,DNA甲基转移酶抑制剂如氮杂脱氧胞苷(5-Aza-CdR)通过抑制DNA甲基化转移酶活性,改变肿瘤基因的甲基化状态,从而使基因的功能得到恢复或增强,进而达到肿瘤治疗的目的。前期我们应用二维电泳和质谱技术以及丰富的生物信息资源研究喉癌5-Aza-CdR相关的蛋白表达谱,对5-Aza-CdR相关的差异蛋白点进行质谱鉴定和分析,结果发现S100A4是差异表达显著的蛋白质之一,提示S100A4基因可能是喉癌发生过程中的一个重要基因。人类S100A4基因定位于1q21,编码由101个氨基酸组成的多肽,分子量约为11.7kDa。S100A4具有广泛的细胞内外功能,如影响细胞骨架形成、改变细胞形状、参与信号传导等。S100A4表达升高与食管癌、胃癌、结肠癌及黑色素瘤等的发生有关,但S100A4参与肿瘤发生的机制仍不十分清楚,迄今未见其与喉癌发生的相关研究报道。本研究旨在探讨S100A4基因在喉癌发生中的作用及可能的分子机制。材料与方法以临床喉癌标本和喉癌Hep2细胞系为实验材料,应用RT-PCR和Western Blot检测喉癌组织中S100A4基因的表达情况。应用MSP方法检测喉癌组织中S100A4基因DNA甲基化情况。针对S100A4基因设计siRNA,体外转录合成S100A4-siRNA,利用TransMessenger转染试剂将其转染喉癌细胞系Hep2,通过RT-PCR和Western Blot检测转染前后喉癌Hep2细胞中S100A4基因表达水平以评价转染效率；应用MTT、流式细胞术、Transwell和RT-PCR以及Western Blot分别检测干扰S100A4基因表达对Hep2细胞生物学特性的影响；针对启动子区甲基化位点,构建S100A4野生型及突变型荧光报告素酶表达载体,转染Hep2细胞,检测这些甲基化位点对S100A4基因转录调节的作用。利用生物信息学转录因子预测软件预测可能与S100A4基因上游启动子区特异序列结合的转录因子,并用EMSA、染色质免疫沉淀技术结合PCR技术对预测结果进行验证以评价S100A4启动子区特异甲基化序列与相关反式作用元件的结合能力。实验结果1.RT-PCR和Western blot结果显示,人喉癌组织以及转移淋巴结中S100A4mRNA和蛋白表达水平均高于癌旁对照组织；2. MSP结果表明,在人喉癌组织中S100A4基因启动子以及第一内含子存在低甲基化,且与S100A4基因表达上调相关；3. S100A4-siRNA对喉癌细胞中S100A4 mRNA和蛋白表达的影响：S100A4-siRNA转染Hep2细胞第5天,S100A4 mRNA表达水平在S100A4-siRNA组较对照组显著降低。S100A4-siRNA转染Hep2细胞第7天,S100A4蛋白表达水平在S100A4-siRNA组较对照组显著降低,表明S100A4表达受到抑制；4.S100A4-siRNA对人喉癌Hep2细胞生物学行为的影响：与对照组相比,在S100A4-siRNA组,喉癌Hep2细胞的增殖能力和侵袭力显著降低、凋亡率显著升高；5. S100A4调控区转录因子结合位点的预测结果：经P-MATCH软件预测发现在S100A4存在转录因子c-Myb、c／Fbp、AP2和MSX-1的结合位点；6.荧光素酶结果显示S100A4调控区甲基化变化对基因活性起调控作用；7. EMSA结果显示在体外c-Myb和c／Ebp可以S100A4特异性甲基化序列结合,而MSX-1和AP2不能与S100A4特异性结合；8.染色质免疫沉淀结合PCR技术检测结果证明在体内c-Myb和c／Ebp与S100A4调控区特异性结合。结论1.S100A4基因在喉癌组织和淋巴结转移组织中表达上调,其参与喉癌的发生和发展；2.喉鳞癌中S100A4表达上调原因之一为S100A4基因启动子和第一内含子区低甲基化；3.S100A4基因具有促进喉癌细胞的增殖和侵袭、抑制细胞凋亡的能力；4.c-Myb和c／Ebp可以与S100A4基因特异性结合,其中c-Myb结合位点的甲基化状态影响S100A4基因的表达。更多还原

【Abstract】 Laryngeal squamous cell carcinoma (LSCC) is one of the most common head and neck cancers. In China, the northeast part is the high incidence region of laryngeal carcinoma. The incidence of LSCCs has been rising in China over past decades. Laryngeal cancer cells are not sensitive to radiotherapy and chemotherapy, so surgery is the primary method in treating LSCC patients. However, surgery could bring different degrees of damage to LSCC patients. At presence, a common question in the diagnosis and treatment process is the hidden incidence of laryngeal carcinoma, which means patients diagnosed are often found in the middle or late stage. Therefore, it is urgent requirement to find a new way to detect or diagnose LSCC in early stage.Malignant tumors are considered as genetic and epigenetic diseases. Genes play an important role in tumorignesis. The alteration of gene seqence could resullt in the abnormal expressions of the related gene usually leading to the abnormal phenotype. However, gene expression is also controlled by epigenetic events. In recent years, gene expressions regulated by epigenetic mechanisms have become a hotspot in studying human diseases. DNA methylation, histone modifications and RNA interference are involved in epigenetic processes, among which DNA methylation is highly concerned. Epigenetic mechanisms are closely related to the occurrence and development of many diseases including cancers. DNA methylation is a reversible process of modification. DNA methylation inhibitors, such as 5’-aza-deoxycytidine (5’-Aza-CdR) which can inhibit DNA methyltransferase activity and change the methylation status of tumor-related genes so that gene function is restored or enhanced, become prospective reagents in treating tumor cells. By using 2-DE (two dimensional gel electrophoresis) and mass spectrometry (MS) technologies combining bioinformatics resource, we previously obtained 5-AZA-CdR-associated protein profiles and some important genes such as S100A4 in laryngeal carcinoma. Human S100A4 gene, which is located on 1q21, codes a 11.7kDa peptide with.101 amino acids. S100A4 participates in biological processes such as cytoskeleton formation, cell shape and signal transduction. The abnormal expression levels of S100A4 were found in esophageal cancer, stomach cancer, colon cancer and melanoma, but the mechanism of S100A4 involved in tumorigenesis is still unclear, and the associated data of S100A4 with laryngeal cancer have not been reported. This study aims to discover the function as well as the possible mechanism of S100A4 gene in the genesis and development of LSCC.Materials and MethodsClinical laryngeal carcinoma specimens and laryngeal squamous cell line Hep2 were used in this study. S100A4 gene expression was detected by using RT-PCR and Western blot. The methylation status of S100A4 gene was detected by using mathylation-specific PCR (MSP). Specific siRNA for S100A4 gene (S100A4-siRNA) designed and synthesized in vitro was transfected into Hep2 cells by TransMessenger Transfection reagent. The inhibitory effect of S100A4-siRNA on S100A4 gene in Hep2 cell line was evaluated by RT-PCR and Western Blot. Biological changes of Hep2 cells tranfected by S100A4-siRNA were detected by MTT, flow cytometry, transwell, RT-PCR and Western Blot. The wild-type and mutant-type fluorescent report luciferase expression vectors including the specifically methylated sites of S100A4 promoter were constructed to detect and analyse the transcriptional regulation role of S100A4 gene. The bioinformatics prediction software was used to predict the transcription factors which may bind S100A4. The predicted transcription factors were tested by using EMSA and CHIP. 1. RT-PCR and Western blot results showed that both S100A4 mRNA and protein levels were significantlly higher in human laryngeal carcinoma and metastatic lymph nodes than those in the adjacent control tissues.2. MSP results showed that hypomethylation within S100A4 promoter and first intron was found and related to the overexpression of S100A4 in human laryngeal carcinoma.3. Influence of S100A4-siRNA on the expression of S100A4 mRNA and protein:In day 5 of S100A4-siRNA transfection to Hep2 cells, S100A4 mRNA level was significantly lower than that in the control group and in day 7 of S100A4-siRNA transfection to Hep2 cells, S100A4 protein level was significantly lower than that in the control group, which indicated that S100A4 expression was inhibited by S100A4-siRNA.4. Influence of S100A4-siRNA on biological characteristics of laryngeal carcinoma cells Hep2:Compared to the control group, the abilities of proliferation and invasiveness were significantly lower, and apoptotic cells were significantly higher in Hep2 cells tranfected by S100A4-siRNA.5. Predictions of S100A4 regulatory site binding to transcription factors:The searching results from P-MATCH software displayed that transcription factor of c-Myb, c/Ebp, AP2, and MSX-1 might bind the corresponding sites of S100A4 regulatory region.6. Luciferase results showed that the methylation status of S100A4 regulatory region could change the promoter activities of S100A4.7. EMS A results showed that c-Myb and c/Ebp except AP2 and MSX-1 specifically bound S100A4 in vitro.8. ChIP results proved the specific binding of c-Myb and c/Ebp to S100A4 in vivo.Conclusion1. Overexpression of S100A4 in human laryngeal carcinoma and metastatic lymph nodes participates in the genesis and development of LSCC. 2. The methylation status of S100A4 promoter and first intron contributes to the overexpression of S100A4 in LSCC.3. S100A4 can promote the growth and invasiveness and inhibit the apoptosis of laryngeal cancer cells, which indicates S100A4 is an important oncogene associated with LSCC.4. C-Myb and c/Ebp could bind S100A4 gene specifically, and the C-Myb methylation status in binding sites of S100A4 gene affects the expression of S100A4 gene更多还原