【摘要】： 占人類基因1%的microRNA (miRNA)幾乎調(diào)節(jié)著至少30%的mRNA,它們通過(guò)調(diào)控相關(guān)靶基因mRNA的表達(dá)水平影響腫瘤細(xì)胞的表型。喉癌是最常見(jiàn)的頭頸部惡性腫瘤,也是僅次于肺癌的呼吸道第二高發(fā)癌。目前,尚少見(jiàn)有喉癌的miRNA特征性表達(dá)譜或與其生物學(xué)行為相關(guān)的差異表達(dá)miRNA的研究報(bào)道。 本項(xiàng)研究采用基于基因芯片技術(shù)的miRNA表達(dá)譜的分析方法,分別以福爾馬林固定石蠟包埋(formalin-fixed and paraffin-embedded, FFPE)組織和新鮮凍存組織為研究材料,以獲得喉鱗狀細(xì)胞癌特征性的miRNA表達(dá)譜型；進(jìn)而篩選出與喉鱗癌生物學(xué)特征密切相關(guān)的差異表達(dá)miRNA。同時(shí)比較從FFPE和新鮮凍存兩種組織材料中所獲得的喉鱗癌miRNA表達(dá)譜的差異,評(píng)價(jià)FFPE組織miRNA表達(dá)譜分析的有效性和可行性。這項(xiàng)研究的遠(yuǎn)期目標(biāo)為,從上述“分子變化譜型”中篩選出具有鑒別意義的miRNA,對(duì)喉癌發(fā)生與發(fā)展、淋巴結(jié)轉(zhuǎn)移或復(fù)發(fā)風(fēng)險(xiǎn)進(jìn)行評(píng)估預(yù)測(cè),以輔助診斷和指導(dǎo)治療。 首先,應(yīng)用Agilent公司的人類miRNA芯片,針對(duì)24例喉鱗癌FFPE組織樣品以及8例新鮮凍存的喉鱗癌患者配對(duì)癌組織和癌旁組織樣品的總RNA進(jìn)行了miRNA表達(dá)譜檢測(cè)。繼而,采用基因芯片分析軟件GeneSpring和統(tǒng)計(jì)學(xué)軟件R project分析了上述miRNA芯片的實(shí)驗(yàn)數(shù)據(jù)。結(jié)果顯示,從24例喉鱗癌FFPE織標(biāo)本中鑒定到319個(gè)miRNA,其中96個(gè)miRNA在全部24例中均有表達(dá)；從8例喉鱗癌新鮮凍存癌組織標(biāo)本中鑒定到342個(gè)miRNA,其中161個(gè)miRNA在全部8例中均有表達(dá)；從而初步獲得喉鱗癌的miRNA表達(dá)譜。在FFPE組織中檢測(cè)到的319個(gè)miRNA信號(hào)中有233個(gè)也能在新鮮凍存喉癌組織中檢測(cè)到；在FFPE組織中檢測(cè)到的79個(gè)與淋巴結(jié)轉(zhuǎn)移相關(guān)(p0.05)的miRNA中有16個(gè)也在新鮮凍存喉癌組織中發(fā)現(xiàn)的與淋巴結(jié)轉(zhuǎn)移相關(guān)的52個(gè)miRNA中出現(xiàn),這16個(gè)miRNA包括miR-17、miR-130b、miR-183、miR-30b、miR-20a、miR-96、miR-188-5p等；從而確認(rèn)了上述FFPE組織miRNA表達(dá)譜分析的有效性。進(jìn)而采用檢錯(cuò)率FDR0.05進(jìn)行校正,以篩選FFPE組織樣品中顯著差異表達(dá)的miRNA,獲得5個(gè)與喉鱗癌淋巴轉(zhuǎn)移密切相關(guān)的差異表達(dá)miRNA:miR-23a*、miR-28-5p、miR-15a、miR-16和miR-425。 本項(xiàng)研究表明,具有不同生物學(xué)行為的喉鱗癌之間存在miRNA的差異表達(dá),miR-23a*、miR-28-5p、miR-15a、miR-16和miR-425與喉鱗癌淋巴轉(zhuǎn)移密切相關(guān),有望通過(guò)進(jìn)一步擴(kuò)大樣本的深入研究建立起能夠起到鑒別或評(píng)估預(yù)測(cè)作用的分子模型。而醫(yī)院病理科存檔的FFPE組織可以提供符合基因芯片分析質(zhì)量要求的miRNA,是研究miRNA的重要樣品資源。
[Abstract]:MicroRNA (miRNA), which accounts for 1% of human genes, almost regulates at least 30% of mRNA,. It affects the phenotype of tumor cells by regulating the expression level of target gene mRNA. Laryngeal cancer is the most common malignant tumor of the head and neck and the second most common cancer of the respiratory tract after lung cancer. At present, there are few studies on miRNA characteristic expression profile or differential expression of miRNA related to biological behavior of laryngeal carcinoma. In this study, the miRNA expression profile of laryngeal squamous cell carcinoma was obtained by using formalin fixed paraffin embedded (formalin-fixed and paraffin-embedded, FFPE) tissue and fresh frozen tissue as materials. Furthermore, the differential expression of miRNA. closely related to the biological characteristics of laryngeal squamous cell carcinoma was screened out. At the same time, the differences of miRNA expression profiles of laryngeal squamous cell carcinoma (LSCC) obtained from FFPE and fresh frozen tissue materials were compared to evaluate the validity and feasibility of miRNA expression profile analysis in FFPE tissues. The long-term objective of this study is to screen out the differential miRNA, from the above "molecular change patterns" to evaluate and predict the risk of laryngeal cancer occurrence and development, lymph node metastasis or recurrence, in order to assist in diagnosis and guidance of treatment. Firstly, the total RNA expression profiles of 24 laryngeal squamous cell carcinoma (LSCC) FFPE tissues and 8 fresh frozen laryngeal squamous cell carcinoma (LSCC) samples were detected by Agilent human miRNA microarray in matched tissues and adjacent tissues of laryngeal squamous cell carcinoma (LSCC). Then, GeneSpring and R project were used to analyze the experimental data of these miRNA chips. The results showed that 319 miRNA, samples were identified from 24 cases of laryngeal squamous cell carcinoma (LSCC), 96 miRNA were detected in all 24 cases, 342 miRNA, were identified from 8 samples of fresh frozen tissue of laryngeal squamous cell carcinoma (LSCC), and 161 miRNA were detected in all 8 cases. Thus the miRNA expression profile of laryngeal squamous cell carcinoma was preliminarily obtained. 233 of the 319 miRNA signals detected in FFPE tissues could also be detected in fresh frozen laryngeal cancer tissues. Sixteen of 79 miRNA associated with lymph node metastasis (p0.05) detected in FFPE tissues were also found in 52 miRNA related to lymph node metastasis in fresh frozen laryngeal carcinoma tissues, including miR-17,miR-130b,miR-183,miR-30b,miR-20a,miR-96,miR-188-5p. Therefore, the validity of miRNA expression profile analysis of FFPE tissue was confirmed. Then the error detection rate (FDR0.05) was used to correct for the screening of significantly differentially expressed miRNA, in FFPE tissue samples. Five differentially expressed miRNA:miR-23a*,miR-28-5p,miR-15a,miR-16 and miR-425. were obtained which were closely related to lymphatic metastasis of laryngeal squamous cell carcinoma (LSCC). This study shows that the differential expression of miRNA in laryngeal squamous cell carcinoma with different biological behaviors is closely related to lymphatic metastasis of laryngeal squamous cell carcinoma. It is expected that a molecular model which can identify or evaluate the prediction can be established by further expanding the study of the sample. The FFPE tissue archived in hospital pathology department can provide miRNA, which meets the quality requirement of gene chip analysis. It is an important sample resource to study miRNA.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.6

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 陳愛(ài)榮;1、葉酸代謝酶相關(guān)亞甲基四氫葉酸還原酶基因多態(tài)性與2型糖尿病及代謝綜合征易感性的關(guān)聯(lián)研究 2、膀胱癌石蠟包埋組織microRNA檢測(cè)及臨床意義的初步探討[D];蘭州大學(xué);2010年

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