喉鳞癌中Hsa-miR-145-5p及靶基因FSCN1调控紊乱的新机制及临床意义

发布时间：2018-10-13 17:54

【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。P0.05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（P0.05）； 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。
[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.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.65

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