S100A4基因DNA甲基化在喉癌中的作用及调控机制的研究

发布时间：2018-06-26 19:15

本文选题：喉癌 + S100A4　；参考：《中国医科大学》2010年博士论文

【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。
[Abstract]:Preface
Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors in the head and neck. In China, the northeastern region is a high incidence area of larynx cancer. In the past 10 years, the incidence of larynx cancer in our country is on the rise. Larynx cancer is not sensitive to radiotherapy and chemotherapy. Surgery is the main means of treatment at present, but the operation will be given to patients. In addition, in the process of diagnosis and treatment, a common problem in the diagnosis and treatment process is that the incidence of other types of larynx is more concealed than glottic carcinoma. Once the diagnosis is confirmed, the patients often enter the middle and late stages. Therefore, the early diagnosis of larynx cancer and the search for new treatment methods at the gene level have become urgent. The problem that needs to be solved.
Malignant tumors are considered to be a genetic and epigenetic disease. Genes play a very important role in the molecular mechanisms of the occurrence and development of disease. Gene sequence changes can lead to abnormal expression of the corresponding genes and lead to phenotypic changes. Research has become one of the hotspots in the regulation of gene expression. Epigenetic regulation includes DNA methylation, histone modification and RNA interference. DNA methylation is the most important and most important mechanism in epigenetic study, which is closely related to the occurrence and development of many diseases, such as tumor. Because DNA methylation is a reversible process. DNA methyltransferase inhibitors, such as nitrogen heterodeoxycytidine (5-Aza-CdR), change the methylation status of the tumor gene by inhibiting the activity of DNA methyltransferase, thereby restoring or enhancing the function of the gene, thus achieving the purpose of cancer treatment. 5-Aza-CdR related protein expression profiles of larynx cancer were studied by information resources, and 5-Aza-CdR related protein points were identified and analyzed by mass spectrometry. The results showed that S100A4 was one of the significant differentially expressed proteins, suggesting that S100A4 gene might be an important gene in the process of larynx carcinogenesis. Human S100A4 gene was located in 1q21 and encoded by 101 Amino acid polypeptide, with a molecular weight about 11.7kDa.S100A4, has a wide range of intracellular and extracellular functions, such as the influence of cytoskeleton formation, changes in cell shape, and involvement of.S100A4 in signal transduction, such as esophageal cancer, gastric cancer, colon cancer and melanoma, but the mechanism of S100A4 involvement in the carcinogenesis is still not very clear. It is not reported that it is related to laryngeal cancer. This study aims to explore the role of S100A4 gene in laryngeal carcinogenesis and its possible molecular mechanism.
Materials and methods
The expression of S100A4 gene in larynx cancer tissues was detected by RT-PCR and Western Blot in the specimens of larynx cancer and the Hep2 cell line of larynx cancer. MSP method was used to detect the DNA methylation of the S100A4 gene in the laryngeal carcinoma tissue. The S100A4 gene was designed for siRNA, in vitro transcriptional synthesis, and the TransMessenger transfection reagent would be used. The transfection of the larynx cell line Hep2, the expression level of S100A4 gene in the Hep2 cells of larynx cancer before and after transfection was detected by RT-PCR and Western Blot to evaluate the transfection efficiency. MTT, flow cytometry, Transwell and RT-PCR, and Western Blot were used to detect the effects of the interfering S100A4 gene expression on the biological characteristics of the cell, and the promoter region. Methylation sites, construct S100A4 wild type and mutant fluorescent reporter protein expression vector, transfect Hep2 cells, detect the role of these methylation sites on the regulation of S100A4 gene transcription. Using bioinformatics transcription factor prediction software to predict the transcription factors that may be associated with the specific sequence of the upstream promoter region of the S100A4 gene, and use EMSA to dye the transcription factors. The color chromatin immunoprecipitation technique combined with the PCR technique to verify the prediction results to evaluate the binding ability of the S100A4 promoter region specific methylation sequence with the related trans acting elements.
experimental result
1.RT-PCR and Western blot results showed that the expression levels of S100A4mRNA and protein in human laryngeal carcinoma tissues and metastatic lymph nodes were higher than those in adjacent tissues.
2. MSP results showed that there was hypomethylation of S100A4 gene promoter and intron 1 in human laryngeal carcinoma tissues, and was associated with up regulation of S100A4 gene expression.
The effect of 3. S100A4-siRNA on the expression of S100A4 mRNA and protein in larynx cancer cells: S100A4-siRNA transfected Hep2 cells for fifth days, the expression level of S100A4 mRNA significantly reduced.S100A4-siRNA transfected Hep2 cells in S100A4-siRNA group for seventh days, and the expression level of S100A4 protein decreased significantly in the S100A4-siRNA group than that in the control group. To inhibit;
The effect of 4.S100A4-siRNA on the biological behavior of human larynx cancer Hep2 cells: compared with the control group, the proliferation and invasive ability of the Hep2 cells in the S100A4-siRNA group were significantly decreased, and the apoptosis rate was significantly increased.
5. S100A4 regulatory region transcriptional factor binding site prediction results: P-MATCH software prediction found that the existence of S100A4 transcription factor c-Myb, C / Fbp, AP2 and MSX-1 binding site;
6. luciferase results showed that the methylation of S100A4 regulatory region played a regulatory role in gene activity.
7. EMSA results showed that c-Myb and C / Ebp could bind to S100A4 specific methylation sequence in vitro, while MSX-1 and AP2 could not be specifically combined with S100A4.
8. chromatin immunoprecipitation combined with PCR technology showed that c-Myb and C / Ebp were specifically bound to S100A4 regulatory region in vivo.
conclusion
The expression of 1.S100A4 gene is up-regulated in laryngeal carcinoma and lymph node metastasis, and is involved in the occurrence and development of laryngeal carcinoma.
2. one of the reasons for the up regulation of S100A4 in laryngeal squamous cell carcinoma is S100A4 gene promoter and low intromethylation in the first intron.
3.S100A4 gene has the ability to promote the proliferation and invasion of laryngeal carcinoma cells and inhibit cell apoptosis.
4.c-Myb and C / Ebp can bind specifically to S100A4 gene, and the methylation status of c-Myb binding sites affects S100A4 gene expression.
【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.65

【参考文献】