TLR3在喉癌中的表达及siRNA沉默TLR3基因表达诱导喉癌细胞凋亡机制的研究

发布时间：2018-03-16 23:33

本文选题：喉肿瘤　切入点：小RNA干扰　出处：《广州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:喉癌是耳鼻咽喉科常见的恶性肿瘤之一,占头颈肿瘤的30%~50%,近年发病有上升趋势。美国癌症协会根据近30年的数据指出喉癌是所有恶性肿瘤五年生存率唯一没得到提高的肿瘤。随着分子生物学的发展,喉癌的发生发展己被证实是多基因、多步骤渐进发展的结果。寻找新的分子诊断标记物和治疗靶点,实现喉癌的个体化治疗已成为当前喉癌研究的重要方向之一。Toll样受体(Toll like receptors,TLRs)是一种模式识别受体(pattern recognition receptor,PRRs),能识别来源于宿主的病原体相关分子模式(athogen-associated molecular patterns,PAMPs)。其中Toll样受体3(Toll-like receptor 3,TLR3)是TLRs家族中重要成员之一,是一类最重要的PRRs[1],与其它TLRs信号转导不同的是,TLR3不依赖转接蛋白髓样分化因子88(myeloid differentiation foctor 88,MyD88),而是依赖TRIF(TIR-domain-containing molecule1,TICAM1)转接蛋白,即所谓TRIF途径。TLR3激活后,通过TRIF途径介导了细胞的凋亡,其凋亡过程是通过过受体相互作用(receptor-inferact-ing protein,RIP)介导的caspase-3激活诱导的途径。众多研究表明肿瘤的发生与凋亡受阻有关,诱导细胞凋亡已成为肿瘤治疗的重要思路。在多发性骨髓瘤细胞,乳腺癌细胞,黑色素瘤细胞中TLR3均参与于细胞凋亡过程。迄今为止,国内外针对喉癌中TLR3表达情况及其可能作用机制少见报道。本研究首先检测TLR3在喉鳞癌组织表达与喉癌临床特征相关情况;并利用小RNA干扰技术沉默TLR3在Hep-2细胞的表达进一步研究siTLR3诱导Hep-2细胞凋亡可能机制和siTLR3对Hep-2抑制迁移及侵袭能力生物学行为的影响;以期明确TLR3基因在喉癌中的地位及可能的作用机制,为喉癌的诊治提供新的筛选基因。本文旨在证实TLR3在喉鳞癌组织表达与喉癌临床特征相关情况;并初步探讨沉默TLR3基因后诱导喉癌hep-2细胞凋亡的可能调控机制。方法:(1)用免疫组化法检测TLR3蛋白在53例喉鳞癌组织和30例癌旁正常(对照组)组织中的表达,分析其与临床病理学特征的关系(2)设计并合成针对TLR3的小干扰RNA(small interference RNA,siRNA)3条序列及与TLR3无基因同源性的阴性对照(NC,negative control),在lipofectamine3000介导下转染Hep-2细胞,应用PCR及Western blot验证转染效率;(3)采用细胞划痕实验和transwell侵袭实验检测沉默TLR3对Hep-2细胞迁移和侵袭能力的影响。(4)通过细胞TUNEL检测法和流式细胞技术分别检测沉默TLR3后Hep-2细胞活力和细胞周期、细胞凋亡的变化。(5)采用Western blot检测沉默TLR3表达后caspase/bcl-2家族蛋白水平的表达。结论:(1)TLR3在喉癌肿瘤组织中的表达和68%(36/53)明显高于癌旁组织中的表达11%(6/53),差异具有统计学意义(P0.01)。TLR3的表达水平与患者年龄、性别及吸烟史无明显相关性,但与肿瘤T分期及淋巴结转移N分期显著相关,差异具有统计学意义(P0.01)。(2)TLR3siRNA-3组的TLR3表达最低(P0.05)。(3)细胞划痕实验和Transwell侵袭实验显示,siTLR3组细胞迁移和侵袭细胞数明显低于NC组(P0.05)。(4)与NC组相比,siTLR3组细胞的细胞周期被阻滞在G2/M期(P0.05),S期细胞明显减少(P0.05)。与NC组比,siTLR3实验组细胞凋亡率为较NC组的增加(P0.05)。siTLR3组的72小时细胞活力明显低于NC组(P0.05)(5)细胞TUNEL实验:siTLR3实验组每视野性中TUNEL检测阳性的细胞占20.1%,NC(阴性对照组)每视野性中TUNEL检测阳性的细胞占5%。两者相比具有统计学意义(P0.05)(6)Western blot结果显示,沉默TLR3基因后细胞cleaved-Caspase3、cleaved-Caspase8和cleaved-Caspase9均有不同程度的升高,抗凋亡蛋白Bcl-2的表达水平下降。
[Abstract]:Background: laryngeal cancer is one of the most common malignant tumor in otolaryngology head and neck cancer, accounting for 30%~50%, the incidence is rising in recent years. According to the American Cancer Society nearly 30 years of data suggest that laryngeal cancer is the five year survival rate of all malignant tumors not only improved the tumor. With the development of molecular biology, the occurrence and development of laryngeal carcinoma has proved to be multi gene, multi - step development. Looking for molecular diagnostic markers and novel therapeutic targets, implementation of individualized treatment of laryngeal cancer has become an important direction of current research of.Toll like receptors in laryngeal carcinoma (Toll like receptors, TLRs) is a kind of pattern recognition receptors (pattern, recognition receptor, PRRs) that can identify the source of pathogen associated molecular patterns from host (athogen-associated molecular patterns, PAMPs). The Toll like receptor 3 (Toll-like receptor 3, TLR3) is important in the TLRs family One of the most important, is a kind of PRRs[1], unlike other TLRs signal transduction is TLR3 independent adaptor protein myeloid differentiation factor 88 (myeloid differentiation 88 foctor, MyD88), but on TRIF (TIR-domain-containing molecule1, TICAM1) adaptor protein called TRIF to activate the.TLR3 pathway, mediated by TRIF pathway the cell apoptosis, the apoptosis process is through receptor interaction (receptor-inferact-ing protein RIP) mediated caspase-3 activation induced pathway. Many studies have shown that apoptosis and tumor blocked related apoptosis has become an important way for tumor therapy. In multiple myeloma cells, breast cancer cells, TLR3 melanoma cells were involved in the apoptotic process. So far, both at home and abroad for the expression of TLR3 in laryngeal carcinoma and its possible mechanism is rarely reported. This study firstly detected The expression and clinical features of laryngeal carcinoma TLR3 in laryngeal squamous cell carcinoma; and the use of small RNA further study on siTLR3 induced apoptosis in Hep-2 cells may influence the biological behavior of inhibition of migration and invasion mechanism and siTLR3 on the expression of Hep-2 silencing TLR3 in Hep-2 cells of interference; in order to clear the status of TLR3 gene in laryngeal carcinoma and its possible mechanism provide new genes for screening, diagnosis and treatment of laryngeal carcinoma. The purpose of this paper is to confirm the expression and clinical features of laryngeal carcinoma TLR3 in laryngeal squamous cell carcinoma; and to explore the possible regulatory mechanism of TLR3 gene silencing induced apoptosis of HEp-2 cells. Methods: (1) immunohistochemistry was used to detect the expression of TLR3 in 53 cases of laryngeal squamous cell carcinoma and 30 cases of adjacent normal (control group) in tissues, and analyze its relationship with clinical pathological feature (2) design and synthesis of small interfering RNA targeting TLR3 (small interference RNA, SiRNA) 3 sequences and TLR3 gene homologous negative control (NC, negative, control) in lipofectamine3000 mediated transfection of Hep-2 cells, the transfection efficiency of PCR Western blot and application verification; (3) the effect of silencing TLR3 by cell scratch assay and Transwell invasion assay on migration and invasion of Hep-2 cells. (4) Hep-2 after silencing TLR3 cell viability and cell cycle were detected by cell TUNEL assay and flow cytometry, changes of cell apoptosis. (5) using Western blot detection after silencing TLR3 expression of caspase/bcl-2 family protein expression. Conclusion: (1) the expression of TLR3 in laryngeal carcinoma and 68% tumor tissues (36/53 11%) was significantly higher than that in the adjacent tissues (6/53), the difference was statistically significant (P0.01) the expression level of.TLR3 with age, sex and smoking were significantly associated with the tumor, but T staging and lymph node metastases Shift N stage were significantly correlated, the difference was statistically significant (P0.01). (2) TLR3siRNA-3 group TLR3 was the lowest (P0.05). (3) showed cell scratch assay and Transwell invasion assay, cell migration and invasion of siTLR3 cells was significantly lower than that in NC group (P0.05). (4) compared with the NC group, cells group siTLR3 cell cycle arrest in G2/M phase (P0.05), S phase cells decreased significantly (P0.05). Compared with group NC, the apoptosis rate of siTLR3 cells in experimental group increased than those of group NC (P0.05).SiTLR3 group of 72 hours the cell viability was significantly lower than in group NC (P0.05) (5) TUNEL cell experiment siTLR3: each experimental group as the wild TUNEL detection in positive cells accounted for 20.1%, NC (negative control group) as per TUNEL compared to wild positive cells accounted for 5%. of the two has statistical significance (P0.05) (6) Western blot showed that TLR3 gene silencing cells after cleaved-Caspase3, cleaved-Caspase8 and cleaved-Caspase9 The expression of anti apoptotic protein Bcl-2 decreased in varying degrees.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.65

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