發(fā)布時(shí)間：2018-10-13 17:54

【摘要】：【研究目的】 1.獲得喉鱗癌中差異性microRNA表達(dá)譜，從中挑選預(yù)研究的靶標(biāo)分子； 2.結(jié)合喉鱗癌臨床病理參數(shù)評(píng)價(jià)Hsa-miR-145-5p及其靶基因FSCN1的臨床意義，探討它們與喉鱗癌預(yù)后的關(guān)系； 3.驗(yàn)證Hsa-miR-145-5p及其靶基因FSCN1的調(diào)控關(guān)系，并明確他們?cè)诤眵[癌細(xì)胞系Hep-2及TU-177中的體外、體內(nèi)生物學(xué)功能； 4.初步明確兩者在喉鱗癌中調(diào)控紊亂的分子生物學(xué)機(jī)制。 【研究方法】 1.利用基因芯片技術(shù)獲得喉鱗癌中差異性microRNA的表達(dá)譜； 2.通過(guò)生物信息學(xué)預(yù)測(cè)并結(jié)合文獻(xiàn)，定位與喉鱗癌侵襲轉(zhuǎn)移相關(guān)的microRNA及其靶基因； 3.利用qRT-PCR、Western blot及免疫組化技術(shù)回顧性研究它們與喉鱗癌患者臨床病理參數(shù)及預(yù)后的關(guān)系； 4.利用雙熒光報(bào)告載體驗(yàn)證他們之前的靶向調(diào)控關(guān)系； 5.利用基因轉(zhuǎn)染技術(shù)，通過(guò)loss-of-function及gain-of-function體外觀察他們對(duì)喉鱗癌細(xì)胞惡性表型的影響，通過(guò)裸鼠移植瘤模型觀察抑制腫瘤效果； 6.通過(guò)熒光共聚焦技術(shù)、電鏡技術(shù)觀察他們對(duì)喉鱗癌細(xì)胞骨架形成及細(xì)胞生物學(xué)結(jié)構(gòu)的影響； 7.利用生物信息學(xué)技術(shù)預(yù)測(cè)Hsa-miR-145-5p甲基化位點(diǎn)，結(jié)合甲基化測(cè)序進(jìn)行定量驗(yàn)證； 8.體外校正Hsa-miR-145-5p-FSCN1軸后，檢測(cè)EMT關(guān)鍵分子的變化水平；數(shù)據(jù)全部錄入SPSS21.0。率的比較用卡方檢驗(yàn)，計(jì)量資料用t檢驗(yàn)或方差檢驗(yàn)，強(qiáng)度用秩和檢驗(yàn)，生存分析用KM法及Cox模型。P0.05認(rèn)為差異有統(tǒng)計(jì)學(xué)意義。 【研究結(jié)果】 1.基因芯片篩查發(fā)現(xiàn)Hsa-miR-145-5p是喉鱗癌中顯著下調(diào)的差異性microRNA之一； 2.經(jīng)生物信息學(xué)預(yù)測(cè)，，同時(shí)利用雙熒光報(bào)告載體驗(yàn)證了FSCN1是Hsa-miR-145-5p的直接靶基因；Hsa-miR-145-5p發(fā)揮對(duì)FSCN1轉(zhuǎn)錄后的調(diào)控作用； 3. miR-145-5p（抑癌基因）在喉鱗癌組織中存在顯著異常低表達(dá)，而其靶基因FSCN1（癌基因）則存在顯著異常高表達(dá)；miR-145-5p與喉鱗癌患者T分期、頸淋巴結(jié)轉(zhuǎn)移、臨床分期、分化程度呈負(fù)相關(guān)，而FSCN1與喉鱗癌患者T分期、頸淋巴結(jié)轉(zhuǎn)移、臨床分期、分化程度呈正相關(guān)（P0.05）； 4. miR-145-5p低表達(dá)及FSCN1蛋白高表達(dá)提示喉鱗癌患者預(yù)后不良。其中FSCN1是喉鱗癌患者預(yù)后不良的單獨(dú)影響因素；特別是同時(shí)伴有miR-145-5p低表達(dá)且FSCN1蛋白高表達(dá)這一分子特征是患者預(yù)后不良的獨(dú)立風(fēng)險(xiǎn)因子。 5.體外實(shí)驗(yàn)證實(shí)恢復(fù)miR-145-5p的表達(dá)或敲減FSCN1的表達(dá)，則可抑制喉鱗癌細(xì)胞增殖、平板克隆、遷移及侵襲的惡性表型，同時(shí)可將喉鱗癌細(xì)胞阻滯在G0/G1期，并促進(jìn)其凋亡；化學(xué)修飾的miR-145-5p及si-FSCN1藥物具有體內(nèi)抑制腫瘤生長(zhǎng)的效果； 6. miR-145-5p啟動(dòng)子高甲基化引發(fā)其轉(zhuǎn)錄障礙，導(dǎo)致靶基因FSCN1調(diào)控紊亂，并在腫瘤細(xì)胞惡性間質(zhì)轉(zhuǎn)化（EMT）中發(fā)揮重要生物學(xué)效應(yīng)。 【研究結(jié)論】 1.喉鱗癌中存在顯著差異性microRNA表達(dá)譜。miR-145-5p在喉鱗癌中顯著下調(diào)，扮演抑癌基因角色，而其靶基因FSCN1扮演癌基因角色。同時(shí)伴有miR-145-5p低表達(dá)且FSCN1高表達(dá)的個(gè)體預(yù)后不良，且這一分子特征是喉鱗癌患者預(yù)后的獨(dú)立危險(xiǎn)因素。 2. miR-145-5p啟動(dòng)子高甲基化導(dǎo)致其功能失調(diào)，促進(jìn)喉鱗癌細(xì)胞增殖、侵襲轉(zhuǎn)移、間質(zhì)轉(zhuǎn)化等惡性表型，這一生物學(xué)效應(yīng)是通過(guò)其靶基因FSCN1異常上調(diào)所介導(dǎo)。 3.本研究以microRNA這一新視角，明確了miR-145-5p及靶基因FSCN1調(diào)控紊亂是喉癌中的重要分子事件。也為以microRNA為靶點(diǎn)對(duì)喉鱗癌行分子靶向治療提供新思路及理論依據(jù)。
[Abstract]:[Study Purpose] 1. obtaining the differential microRNAs expression spectrum in the laryngeal squamous cell carcinoma, and selecting a target for pre-study therefrom; Molecular; 2. Evaluation of clinical significance of Hsa-miR-145-5p and its target gene FSCN1 in combination with clinical and pathological parameters of laryngeal squamous cell carcinoma 3. Verify the regulatory relationship between Hsa-miR-145-5p and its target gene FSCN1, and clarify their effects in laryngeal squamous cell carcinoma cell line Hep-2 and TU-177. in vivo biological function; 4. preliminary definition of both in laryngeal squamous cell carcinoma disturbance-controlled molecule Biological mechanism.[Study Method] 1. Use of gene chip technology to obtain laryngeal squamous cell the expression profile of differential microRNAs in cancer; 2. predicting and combining literature, positioning and laryngeal squamous cell carcinoma by bioinformatics Invasion and metastasis-related microRNAs and their target genes; 3. Review of the use of qRT-PCR, Western blot and immunohistochemistry The relationship between their clinical and pathological parameters and prognosis in patients with laryngeal squamous cell carcinoma were studied. 4. Using the double fluorescence reporter vector to verify their pre-targeting regulatory relationship; 5. Using the gene transfection technique, observe them in vitro through the loss-of-function and the gain-of-function. The effect of malignant phenotype of squamous cell carcinoma was observed by nude mouse transplanted tumor model. Technology and electron microscopy to observe the effect of them on the formation and cellular biological structure of laryngeal squamous cell carcinoma; 7. Bioinformatics technology predicting the Hsa-miR-145-5p methylation site and carrying out quantitative verification in combination with the methylation sequencing; 8, after the Hsa-miR-145-5p-FSCN1 axis is corrected in vitro, detecting the change level of the critical molecules of the Hsa-miR-145-5p-FSCN1; Rank and Inspection of Strength The KM method and Cox model were used for the analysis of survival, and the difference was considered statistically significant.[Results] 1. Hsa-miR-145-5p is one of differentially expressed microRNAs in laryngeal squamous cell carcinoma due to chip screening; 2. Bioinformatics predicts that FSCN1 is Hsa-The direct target gene of miR-145-5p; Hsa-miR-145-5p plays a role in regulating the transcription of FSCN1; 3. The miR-145-5p (tumor suppressor gene) has a significant abnormal low expression in the laryngeal squamous cell carcinoma tissue, and the target gene FSCN1 (oncogene) has a significant abnormal high expression; and the miR-145-5p and the laryngeal squamous cell carcinoma patient T stage, cervical lymph node metastasis, clinical stage, differentiation, There was a negative correlation between FSCN1 and T stage, lymph node metastasis, clinical stage and degree of differentiation in patients with laryngeal squamous cell carcinoma (P <0.05); 4. Low expression of miR-145-5p and high expression of FSCN1 showed poor prognosis in patients with laryngeal squamous cell carcinoma. In vitro experiments confirm that the expression of miR-145-5p is restored or the expression of FSCN1 is knocked down, the malignant phenotype of laryngeal squamous carcinoma cell proliferation, tablet cloning, migration and invasion can be inhibited, Blocking in G0/ G1 phase and promoting apoptosis; chemically modified miR-145-5p and si-FSCN1 drugs have the effect of inhibiting tumor growth in vivo; 6. miR-145-5p promoter high Methylation caused its transcription disturbance, which led to the regulation disorder of target gene FSCN1 and played an important biological effect in malignant mesenchymal transition of tumor cells.[Study conclusion] 1. There was a significant difference in microRNAs expression in laryngeal squamous cell carcinoma. miR-145-5p was down-regulated in laryngeal squamous cell carcinoma, acting as tumor suppressor gene, and its target gene FSCN 1 plays an oncogenic role. At the same time, the low expression of miR-145-5p and high expression of FSCN1 is poor, and this molecular feature is an independent risk factor for the prognosis of laryngeal squamous cell carcinoma. 5p promoter hypermethylation leads to functional disorder, promotes the proliferation of laryngeal squamous carcinoma cells, invasion and metastasis, mesenchymal transition and other malignant phenotypes, and the biological effect is mediated by abnormal upregulation of the target gene FSCN1.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R739.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 榮舉,許麗艷,蔡唯佳,熊興東,李勁濤,方王楷,沈忠英,李恩民;Fascin 1基因在永生化食管上皮細(xì)胞癌變中的表達(dá)[J];癌癥;2004年03期

2 劉天潤(rùn);楊安奎;陳福進(jìn);曾木圣;宋明;郭朱明;陳文寬;歐陽(yáng)電;李秋梨;陳艷峰;張?jiān)?曾宗淵;;221例晚期喉鱗癌患者術(shù)后的生存和預(yù)后分析[J];癌癥;2009年03期

3 劉莉;丁彥青;;結(jié)直腸癌組織Fascin-1蛋白表達(dá)的意義[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2007年02期

4 王蘋;付濤;王緒銳;祝威;;應(yīng)用微陣列芯片分析喉鱗狀細(xì)胞癌miRNA與正常黏膜表達(dá)差異的初步研究[J];臨床耳鼻咽喉頭頸外科雜志;2010年12期

5 畢麗青;王翠玲;;山西某醫(yī)院2008-2010年7335例腫瘤病例調(diào)查分析[J];山西醫(yī)科大學(xué)學(xué)報(bào);2012年03期

6 武永春;張燕萍;馬利剛;;太原市2009年惡性腫瘤發(fā)病資料分析[J];山西醫(yī)藥雜志;2012年10期

7 季文樾,杜強(qiáng),關(guān)超,王殿閣;1115例喉癌患者的生存分析[J];中華耳鼻咽喉科雜志;2004年01期

8 劉鳴，肖占榮，申玉山，董春梅;聲門上型喉癌與頸淋巴結(jié)轉(zhuǎn)移[J];中華耳鼻咽喉科雜志;1995年01期

9 于靖寰,季文樾,關(guān)超,于剛;聲門上型喉癌頸淋巴結(jié)轉(zhuǎn)移的臨床病理研究[J];中華耳鼻咽喉科雜志;1997年06期

10 趙舒薇;;喉癌治療進(jìn)展[J];腫瘤學(xué)雜志;2012年09期

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