【作者】 高伟；

【导师】 王斌全；

【作者基本信息】 山西医科大学 ， 耳鼻咽喉科学， 2013， 博士

【摘要】 【研究目的】1.获得喉鳞癌中差异性microRNA表达谱，从中挑选预研究的靶标分子；2.结合喉鳞癌临床病理参数评价Hsa-miR-145-5p及其靶基因FSCN1的临床意义，探讨它们与喉鳞癌预后的关系；3.验证Hsa-miR-145-5p及其靶基因FSCN1的调控关系，并明确他们在喉鳞癌细胞系Hep-2及TU-177中的体外、体内生物学功能；4.初步明确两者在喉鳞癌中调控紊乱的分子生物学机制。【研究方法】1.利用基因芯片技术获得喉鳞癌中差异性microRNA的表达谱；2.通过生物信息学预测并结合文献，定位与喉鳞癌侵袭转移相关的microRNA及其靶基因；3.利用qRT-PCR、Western blot及免疫组化技术回顾性研究它们与喉鳞癌患者临床病理参数及预后的关系；4.利用双荧光报告载体验证他们之前的靶向调控关系；5.利用基因转染技术，通过loss-of-function及gain-of-function体外观察他们对喉鳞癌细胞恶性表型的影响，通过裸鼠移植瘤模型观察抑制肿瘤效果；6.通过荧光共聚焦技术、电镜技术观察他们对喉鳞癌细胞骨架形成及细胞生物学结构的影响；7.利用生物信息学技术预测Hsa-miR-145-5p甲基化位点，结合甲基化测序进行定量验证；8.体外校正Hsa-miR-145-5p-FSCN1轴后，检测EMT关键分子的变化水平；数据全部录入SPSS21.0。率的比较用卡方检验，计量资料用t检验或方差检验，强度用秩和检验，生存分析用KM法及Cox模型。P<0.05认为差异有统计学意义。【研究结果】1.基因芯片筛查发现Hsa-miR-145-5p是喉鳞癌中显著下调的差异性microRNA之一；2.经生物信息学预测，同时利用双荧光报告载体验证了FSCN1是Hsa-miR-145-5p的直接靶基因；Hsa-miR-145-5p发挥对FSCN1转录后的调控作用；3. miR-145-5p（抑癌基因）在喉鳞癌组织中存在显著异常低表达，而其靶基因FSCN1（癌基因）则存在显著异常高表达；miR-145-5p与喉鳞癌患者T分期、颈淋巴结转移、临床分期、分化程度呈负相关，而FSCN1与喉鳞癌患者T分期、颈淋巴结转移、临床分期、分化程度呈正相关（P<0.05）；4. miR-145-5p低表达及FSCN1蛋白高表达提示喉鳞癌患者预后不良。其中FSCN1是喉鳞癌患者预后不良的单独影响因素；特别是同时伴有miR-145-5p低表达且FSCN1蛋白高表达这一分子特征是患者预后不良的独立风险因子。5.体外实验证实恢复miR-145-5p的表达或敲减FSCN1的表达，则可抑制喉鳞癌细胞增殖、平板克隆、迁移及侵袭的恶性表型，同时可将喉鳞癌细胞阻滞在G0/G1期，并促进其凋亡；化学修饰的miR-145-5p及si-FSCN1药物具有体内抑制肿瘤生长的效果；6. miR-145-5p启动子高甲基化引发其转录障碍，导致靶基因FSCN1调控紊乱，并在肿瘤细胞恶性间质转化（EMT）中发挥重要生物学效应。【研究结论】1.喉鳞癌中存在显著差异性microRNA表达谱。miR-145-5p在喉鳞癌中显著下调，扮演抑癌基因角色，而其靶基因FSCN1扮演癌基因角色。同时伴有miR-145-5p低表达且FSCN1高表达的个体预后不良，且这一分子特征是喉鳞癌患者预后的独立危险因素。2. miR-145-5p启动子高甲基化导致其功能失调，促进喉鳞癌细胞增殖、侵袭转移、间质转化等恶性表型，这一生物学效应是通过其靶基因FSCN1异常上调所介导。3.本研究以microRNA这一新视角，明确了miR-145-5p及靶基因FSCN1调控紊乱是喉癌中的重要分子事件。也为以microRNA为靶点对喉鳞癌行分子靶向治疗提供新思路及理论依据。更多还原

【Abstract】 Objective:MicroRNAs (miRNAs) are endogenous short non-coding RNA molecules thatregulate gene expression by repressing translation or cleaving RNA transcripts in asequence-specific manner. We aim to get the expression profiles of microRNAs inlaryngeal squamous cell carcinoma (LSCC). We investigated the association ofmiR-145-5p and its target gene FSCN1expression with clinicopathologic factors andtheir prognostic value in LSCC. Finaly, we identify the main function of miR-145-5pand FSCN1in Hep-2and TU-177.Methods:We used microRNA chip to identify the expression profiles in LSCC. Thebioinformatics was used in prediction of regulation between miR-145-5p and FSCN1which was validated by fluorescent report vector. Quantitative RT-PCR and westernblot analyses were used to examine mRNA and protein levels in10fresh LSCCspecimens and10corresponding adjacent normal margin (ANM) tissues frompatients undergoing surgery in2012. We used immunohistochemistry toretrospectively study188paraffin blocks of LSCC samples from patients who hadundergone surgery between2000and2006and had not received special treatmentbefore the diagnosis. Univariate analysis of patient survival involved the Kaplan–Meier method. Multivariate analyses involved the Cox proportional hazards model.The gene transfection and siRNA technology was used in order to explore theirfunctions on the malignant phenotype of laryngeal squamous carcinoma cells in vitro. The biological information technology was used in forecasting methylation sites ofHsa-miR-145-5p which was validated by quantitative methylation sequencing.Finally, we detected the changes of key molecules of epithelial-mesenchymaltransition (EMT) after Hsa-miR-145-5p-FSCN1axis was rescued in vitro.Results:Hsa-miR-145-5p, one of the multifarious microRNA, was significantlydown-regulated in laryngeal squamous cell carcinoma. FSCN1was the direct targetgene of miR-145-5p. It could inhibit the proliferation, clone, migration, invasion oflaryngeal squamous carcinoma cell which were arrested in G0/G1phase, andpromoted into apoptosis by recovery of miR-145-5p or siRNA of FSCN1.Low expression of miR-145-5p and high expression pattern of FSCN1weresignificantly observed in laryngeal squamous cell carcinoma. The low expression ofmiR-145-5p was positively correlated with poor tumor differentiation, cervical lymphnode metastasis (N+), and advanced clinical stage (III+IV), but not sex, denger ormetastasis. In addition, a high expression of FSCN1was associated with advancedtumor stage (T3+T4). The expression of fascin-1was higher in smokers thannon-smokers. A high expression of FSCN1or low expression of miR-145-5p wasassociated with poor prognosis. Low expression of miR-145-5p with high expressionof FSCN1is an independent risk factor for LSCC patients with poor prognosis.MiR-145-5p promoter hypermethylation caused the imbalance ofhsa-miR-145-5p-FSCN1axis, resulting in regulation disorder of FSCN1, whichplayed an important biological effect in tumor cell malignant mesenchymal transition(EMT).Conclusions:The regulation disorder between miR-145-5p and FSCN1is important molecularevent in the process of malignant progression and metastasis in laryngeal squamous cell carcinoma. miR-145-5p is an actor as tumor suppressor gene but proto-oncogeneof FSCN1which might be prognostic of poor outcome with LSCC after surgery. Ourstudy may lead to establishing new molecular therapeutic targets and/or prognosticbiomarkers in LSCC.更多还原