本文選題：喉癌 + S100A4　； 參考：《中國醫(yī)科大學(xué)》2010年博士論文

【摘要】： 前言 喉鱗狀細(xì)胞癌(laryngeal squamous cell carcinoma, LSCC)是頭頸部常見的惡性腫瘤之一。在我國,東北地區(qū)是喉癌的高發(fā)區(qū)。過去的10年中,我國喉癌的發(fā)病率呈現(xiàn)上升的趨勢。喉癌對放療及化療均不敏感,手術(shù)是目前治療的主要手段,但手術(shù)會給患者造成不同程度的損傷。此外,在診斷和治療過程中普遍存在的一個問題是：除聲門型喉癌外,其他類型喉癌的發(fā)病比較隱蔽,一旦確診,患者往往多已進(jìn)入中、晚期。因此對喉癌進(jìn)行早期診斷,并在基因水平尋求新的治療手段成為當(dāng)前急需解決的問題。 惡性腫瘤被認(rèn)為是一種遺傳和表觀遺傳性疾病�；蛟诩膊“l(fā)生和發(fā)展的分子機(jī)制中扮演非常重要的作用,基因序列改變可以導(dǎo)致相應(yīng)基因的表達(dá)異常進(jìn)而導(dǎo)致表型的變化。不僅如此,基因的表達(dá)還受表觀遺傳的控制。近年來,表觀遺傳學(xué)研究已成為基因表達(dá)調(diào)控的研究熱點(diǎn)之一。表觀遺傳調(diào)控機(jī)制包括DNA甲基化、組蛋白修飾和RNA干擾等,而DNA甲基化是表觀遺傳學(xué)研究最深入、最重要的一種機(jī)制,與許多疾病如腫瘤的發(fā)生和發(fā)展密切相關(guān)。由于DNA甲基化是一個可逆的修飾過程,DNA甲基轉(zhuǎn)移酶抑制劑如氮雜脫氧胞苷(5-Aza-CdR)通過抑制DNA甲基化轉(zhuǎn)移酶活性,改變腫瘤基因的甲基化狀態(tài),從而使基因的功能得到恢復(fù)或增強(qiáng),進(jìn)而達(dá)到腫瘤治療的目的。前期我們應(yīng)用二維電泳和質(zhì)譜技術(shù)以及豐富的生物信息資源研究喉癌5-Aza-CdR相關(guān)的蛋白表達(dá)譜,對5-Aza-CdR相關(guān)的差異蛋白點(diǎn)進(jìn)行質(zhì)譜鑒定和分析,結(jié)果發(fā)現(xiàn)S100A4是差異表達(dá)顯著的蛋白質(zhì)之一,提示S100A4基因可能是喉癌發(fā)生過程中的一個重要基因。人類S100A4基因定位于1q21,編碼由101個氨基酸組成的多肽,分子量約為11.7kDa。S100A4具有廣泛的細(xì)胞內(nèi)外功能,如影響細(xì)胞骨架形成、改變細(xì)胞形狀、參與信號傳導(dǎo)等。S100A4表達(dá)升高與食管癌、胃癌、結(jié)腸癌及黑色素瘤等的發(fā)生有關(guān),但S100A4參與腫瘤發(fā)生的機(jī)制仍不十分清楚,迄今未見其與喉癌發(fā)生的相關(guān)研究報道。本研究旨在探討S100A4基因在喉癌發(fā)生中的作用及可能的分子機(jī)制。 材料與方法 以臨床喉癌標(biāo)本和喉癌Hep2細(xì)胞系為實(shí)驗(yàn)材料,應(yīng)用RT-PCR和Western Blot檢測喉癌組織中S100A4基因的表達(dá)情況。應(yīng)用MSP方法檢測喉癌組織中S100A4基因DNA甲基化情況。針對S100A4基因設(shè)計siRNA,體外轉(zhuǎn)錄合成S100A4-siRNA,利用TransMessenger轉(zhuǎn)染試劑將其轉(zhuǎn)染喉癌細(xì)胞系Hep2,通過RT-PCR和Western Blot檢測轉(zhuǎn)染前后喉癌Hep2細(xì)胞中S100A4基因表達(dá)水平以評價轉(zhuǎn)染效率；應(yīng)用MTT、流式細(xì)胞術(shù)、Transwell和RT-PCR以及Western Blot分別檢測干擾S100A4基因表達(dá)對Hep2細(xì)胞生物學(xué)特性的影響；針對啟動子區(qū)甲基化位點(diǎn),構(gòu)建S100A4野生型及突變型熒光報告素酶表達(dá)載體,轉(zhuǎn)染Hep2細(xì)胞,檢測這些甲基化位點(diǎn)對S100A4基因轉(zhuǎn)錄調(diào)節(jié)的作用。利用生物信息學(xué)轉(zhuǎn)錄因子預(yù)測軟件預(yù)測可能與S100A4基因上游啟動子區(qū)特異序列結(jié)合的轉(zhuǎn)錄因子,并用EMSA、染色質(zhì)免疫沉淀技術(shù)結(jié)合PCR技術(shù)對預(yù)測結(jié)果進(jìn)行驗(yàn)證以評價S100A4啟動子區(qū)特異甲基化序列與相關(guān)反式作用元件的結(jié)合能力。 實(shí)驗(yàn)結(jié)果 1.RT-PCR和Western blot結(jié)果顯示,人喉癌組織以及轉(zhuǎn)移淋巴結(jié)中S100A4mRNA和蛋白表達(dá)水平均高于癌旁對照組織； 2. MSP結(jié)果表明,在人喉癌組織中S100A4基因啟動子以及第一內(nèi)含子存在低甲基化,且與S100A4基因表達(dá)上調(diào)相關(guān)； 3. S100A4-siRNA對喉癌細(xì)胞中S100A4 mRNA和蛋白表達(dá)的影響：S100A4-siRNA轉(zhuǎn)染Hep2細(xì)胞第5天,S100A4 mRNA表達(dá)水平在S100A4-siRNA組較對照組顯著降低。S100A4-siRNA轉(zhuǎn)染Hep2細(xì)胞第7天,S100A4蛋白表達(dá)水平在S100A4-siRNA組較對照組顯著降低,表明S100A4表達(dá)受到抑制； 4.S100A4-siRNA對人喉癌Hep2細(xì)胞生物學(xué)行為的影響：與對照組相比,在S100A4-siRNA組,喉癌Hep2細(xì)胞的增殖能力和侵襲力顯著降低、凋亡率顯著升高； 5. S100A4調(diào)控區(qū)轉(zhuǎn)錄因子結(jié)合位點(diǎn)的預(yù)測結(jié)果：經(jīng)P-MATCH軟件預(yù)測發(fā)現(xiàn)在S100A4存在轉(zhuǎn)錄因子c-Myb、c／Fbp、AP2和MSX-1的結(jié)合位點(diǎn)； 6.熒光素酶結(jié)果顯示S100A4調(diào)控區(qū)甲基化變化對基因活性起調(diào)控作用； 7. EMSA結(jié)果顯示在體外c-Myb和c／Ebp可以S100A4特異性甲基化序列結(jié)合,而MSX-1和AP2不能與S100A4特異性結(jié)合； 8.染色質(zhì)免疫沉淀結(jié)合PCR技術(shù)檢測結(jié)果證明在體內(nèi)c-Myb和c／Ebp與S100A4調(diào)控區(qū)特異性結(jié)合。 結(jié)論 1.S100A4基因在喉癌組織和淋巴結(jié)轉(zhuǎn)移組織中表達(dá)上調(diào),其參與喉癌的發(fā)生和發(fā)展； 2.喉鱗癌中S100A4表達(dá)上調(diào)原因之一為S100A4基因啟動子和第一內(nèi)含子區(qū)低甲基化； 3.S100A4基因具有促進(jìn)喉癌細(xì)胞的增殖和侵襲、抑制細(xì)胞凋亡的能力； 4.c-Myb和c／Ebp可以與S100A4基因特異性結(jié)合,其中c-Myb結(jié)合位點(diǎn)的甲基化狀態(tài)影響S100A4基因的表達(dá)。
[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.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R739.65

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本文編號：2071119