喉鳞癌microRNA表达谱变化及miR-125b对喉癌Hep-2细胞增殖影响的研究

发布时间：2018-11-13 14:16

【摘要】：第一部分喉鳞癌microRNA表达谱变化及候选靶基因分析背景：microRNA(微小RNA,简称miRNA)是一种非编码RNA,在恶性肿瘤的基因表达转录后调节中发挥作用。本部分研究miRNA在喉鳞癌组织中表达谱变化,为LSCC研究提供了新的方向。方法：通过激光显微切割技术(LCM)分离6例喉癌组织肿瘤细胞,采用miRNA芯片筛查喉癌组织与癌旁正常组织中miRNA表达谱变化,在48对喉癌组织及癌旁正常组织中,采用实时定量PCR技术验证miRNA芯片结果,通过生物信息学软件预测这些差异表达miRNAs的靶基因,在24对喉癌组织中检测候选基因的表达水平。结果：通过miRNA芯片检测,筛查得到29个差异表达的miRNAs,经过实时定量PCR验证后,有6个被证实包括表达上调的miR-21、miR-93、miR-205和miR-708,表达下调的miR-125b和miR-145。使用软件预测共有25个候选靶基因,经检测后有10个候选基因在LSCC中表达水平有差异。结论：这些差异表达的miRNA在喉癌的发生和演进过程中可能发挥了重要作用,并且为miRNA的功能研究提供了方向。第二部分miR-125b通过EIF2C2抑制喉癌Hep-2细胞增殖背景：失控的自主性增殖是恶性肿瘤细胞具有的主要特征之一,细胞周期控制紊乱导致肿瘤细胞增殖调控异常。本部分在体外和体内水平研究上调miR-125b对喉癌Hep-2细胞增殖的影响及机制。方法：采用慢病毒载体构建miR-125b表达载体,感染Hep-2细胞后建立能够稳定高表达miR-125b的稳转细胞株Hep-2-miR-125b和对照细胞株Hep-2-vector,WST-1实验检测细胞增殖能力,流式细胞术检测细胞周期和凋亡变化。并将Hep-2-miR-125b和Hep-2-vector细胞接种NOD/SCID小鼠,观察小鼠移植瘤生长情况。采用双荧光素酶报告基因检测系统初步验证EIF2C2(eukaryotic translation initiation factor2C-2,真核转录起始因子2C-2)是miR-125b的靶基因,在两株细胞中检测候选基因EIF2C2mRNA表达和蛋白水平变化。结果：Hep-2-miR-125b能够稳定、高水平表达miR-125b,miR-125b过表达后能够抑制Hep-2细胞的增殖能力,抑制NOD/SCID小鼠体内成瘤能力,使Hep-2细胞停滞于G0-G1期,凋亡没有变化。双荧光素酶报告基因检测系统显示miR-125b能够抑制EIF2C2载体40%的荧光素酶活性,验证EIF2C2是miR-125b的靶基因,并在mRNA水平和蛋白水平上进一步证实miR-125b可以抑制EIF2C2的表达。结论：上调miR-125b通过靶向EIF2C2抑制喉癌Hep-2细胞增殖,本研究为以miR-125b作为靶点治疗喉癌提供了一定的理论依据。
[Abstract]:Part I changes of microRNA expression profile and candidate target gene analysis in laryngeal squamous cell carcinoma background: microRNA (minimal RNA, (miRNA) is a non-coding RNA, that plays a role in the posttranscriptional regulation of gene expression in malignant tumors. In this part, we study the changes of miRNA expression profile in laryngeal squamous cell carcinoma (LSCC), and provide a new direction for the study of LSCC. Methods: six cases of laryngeal carcinoma tumor cells were isolated by laser microdissection technique (LCM). The miRNA expression patterns were screened by miRNA microarray in 48 pairs of laryngeal cancer tissues and adjacent normal tissues. Real-time quantitative PCR was used to verify the results of miRNA microarray. The target genes of these differentially expressed miRNAs were predicted by bioinformatics software. The expression level of candidate genes was detected in 24 pairs of laryngeal carcinoma tissues. Results: by miRNA chip analysis, 29 differentially expressed miRNAs, were screened after real-time quantitative PCR verification, and 6 of them were confirmed to include up-regulated miR-21,miR-93,miR-205 and down-regulated miR-125b and miR-145.. A total of 25 candidate target genes were predicted by software, and 10 candidate genes were detected with different expression levels in LSCC. Conclusion: these differentially expressed miRNA may play an important role in the development and progression of laryngeal carcinoma and provide a direction for the study of the function of miRNA. The second part: miR-125b inhibits the proliferation of laryngeal carcinoma Hep-2 cells through EIF2C2: uncontrolled autonomous proliferation is one of the main characteristics of malignant tumor cells. The disorder of cell cycle control leads to abnormal regulation of tumor cell proliferation. The effect and mechanism of upregulation of miR-125b on the proliferation of laryngeal carcinoma Hep-2 cells were studied in vitro and in vivo. Methods: miR-125b expression vector was constructed by using lentivirus vector. After infection with Hep-2 cells, stable and highly expressed miR-125b cell line Hep-2-miR-125b and control cell line Hep-2-vector, were established. WST-1 assay was used to detect cell proliferation and flow cytometry was used to detect cell cycle and apoptosis. Hep-2-miR-125b and Hep-2-vector cells were inoculated into NOD/SCID mice to observe the growth of transplanted tumor. Double luciferase reporter gene detection system was used to preliminarily verify that EIF2C2 (eukaryotic translation initiation factor2C-2, eukaryotic transcription initiation factor (2C-2) is the target gene of miR-125b. The expression of candidate gene EIF2C2mRNA and the change of protein level were detected in two cell lines. Results: Hep-2-miR-125b could be stable, and high level expression of miR-125b,miR-125b could inhibit the proliferation of Hep-2 cells, inhibit the tumorigenic ability of NOD/SCID mice, and make Hep-2 cells stop at G0-G1 phase. Apoptosis did not change. Double luciferase reporter gene detection system showed that miR-125b could inhibit 40% luciferase activity of EIF2C2 vector, which confirmed that EIF2C2 was the target gene of miR-125b. It was further confirmed that miR-125b could inhibit the expression of EIF2C2 at mRNA level and protein level. Conclusion: upregulation of miR-125b inhibits the proliferation of laryngeal carcinoma Hep-2 cells through targeted EIF2C2. This study provides a theoretical basis for the treatment of laryngeal carcinoma with miR-125b as the target.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.65

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