长链非编码RNA在局部晚期鼻咽癌中的功能及作用机制研究

发布时间：2018-05-05 22:10

本文选题：鼻咽癌 + lncRNA　；参考：《广州医科大学》2017年硕士论文

【摘要】：【背景】长链非编码RNA(long non-coding RNA,lnc RNA)是一类长度超过200nt的RNA,它们本身并不编码蛋白,而是以RNA的形式在多层面上(表观遗传调控,转录调控以及转录后调控等)调节基因的表达【1】。近年来的研究表明,Lnc RNA广泛参与各种生物学的过程,Lnc RNA的异常表达与包括肿瘤在内的多种疾病密切相关。已有证据证明lnc RNA参与肝癌、胰腺癌、胶质瘤等多种恶性肿瘤的发生发展【2-5】,但lnc RNA在鼻咽癌中的功能机制研究鲜有报道。鼻咽癌的发病率有明显的地域差异性,在我国南方、北非及部分地中海地区是高发区域,特别在我国南方地区鼻咽癌明显高发。鼻咽癌预后与疾病分期密切相关,由于鼻咽肿瘤生长部位隐蔽,早期没有任何症状和体征,确诊时已有70%的患者是Ⅲ或ⅣA期的局部晚期鼻咽癌【6】。近年来,尽管鼻咽癌的诊治水平有了较大的提高,但其具体的发生机制仍不十分明确,临床治疗仍缺乏有效手段,5年生存率仅70%左右【7】,局部治疗失败和远处转是治疗失败的重要原因,因此,提高局部控制率和预防远处转移是局部晚期鼻咽癌患者治疗上面临的巨大挑战。近年来,lnc RNA越来越多的成为人们研究的热点,但lnc RNA与鼻咽癌的作用关系尚有待研究,尤其lnc RNA与局部晚期鼻咽癌的关系更值得我们去探讨。LncRNA基因芯片是目前广泛应用与研究1nc RNA表达谱的一种理想有效的方法。通过Lnc RNA基因芯片,能够快速高通量的获得与特定生物学过程或疾病相关的Lnc RNA的表达变化,从而为后续的Lnc RNA功能研究或生物标志物筛选提供极大便利。【目的】本课题旨在研究与局部晚期鼻咽癌相关的lnc RNA及其相关生物学功能,并初步探讨其发生机制,为后续鼻咽癌的研究提供理论和科学依据。【方法】1、采用Human Lnc RNA Array V4.0检测局部晚期鼻咽癌组织及其对照的癌旁组织,得到两种组织之间lnc RNA和m RNA差异表达谱,综合NCBI Refseq,UCSC,RNAdb及Lnc RNAs等数据库资源,对芯片结果进行聚类分析、GO分析及Pathway分析等初步生物信息学分析。2、体外培养鼻咽癌细胞CNE1和HONE1以及鼻咽正常上皮细胞NP69,运用Real-time PCR技术验证目标lnc RNA在CNE1和HONE1与NP69的表达水平。3、采用si RNA干扰技术干扰目标lnc RNA表达后,运用Real-time PCR方法检测si RNA的转染效率,干扰目标lnc RNA后检测目标lnc RNA对鼻咽癌细胞生物学特性的影响。4、应用CCK8法、Boyden侵袭实验、Transwell迁移实验和细胞划痕实验以及细胞凋亡实验,分别检测干扰目标lnc RNA后,对鼻咽癌细胞株CNE1和HONE1增殖、侵袭、迁移以及凋亡能力的影响。【结果】第一部分:1、通过基因芯片技术成功筛选差异表达的lnc RNA和m RNA,设定筛选标准:P值≤0.05,Foldchange≥1.5,筛选得到324个差异表达lnc RNAs,上调226个,下调98个;同时有188个差异表达m RNAs,上调98个,下调90个。2、Pathway结果显示:lnc RNA潜在的靶基因m RNA可能参与的信号通路共有22条,其中表达上调的信号通路6条,下调的13条。GO分析表明,上调表达的m RNA生物过程、细胞组成、分子功能富集程度最高的条目分别是cell chemotaxis,collagen trimer和chemokine activity;下调表达的分别是protein localization to cytoskeleton,cell cortex和protein homodimerization activity。第二部分:1、实时荧光定量PCR实验结果显示,AW450413、ENST00000501541、ENST00000457325、uc003owl.1相对表达量增加,ENST00000448834、ENST00000554458、ENST00000433377相对表达量降低,与基因芯片结果一致。更重要的是,ENST00000501541在CNE1和HONE1上表达量明显高于NP69表达量,具有明显统计学差异(p0.05)。2、CCK8增殖实验结果显示:与对照组(si RNA-NC)相比,实验组(si RNA-1)、实验组(si RNA-2)鼻咽癌细胞CNE1和HONE1增殖能力显著下降(P0.05)。3、Transwell迁移实验显示:与对照组(si RNA-NC)相比,实验组(si RNA-1)每高倍镜下穿过的细胞数较对照组明显减少(P0.05)。4、Boyden侵袭实验结果显示:与对照组(si RNA-NC)相比,实验组(si RNA-1)每高倍镜下穿过Matrigel膜的细胞数较对照组明显减少(P0.05)。5、划痕实验结果显示:与空白组及阴性对照组相比,在48小时和72小时检测,实验组(si RNA-1)细胞划痕愈合能力较对照组显著下降(P0.05)。6、凋亡实验结果显示:实验组(si RNA-1)相对于阴性对照组(sh RNA-NC),鼻咽癌细胞CNE1和HONE1凋亡细胞百分比显著增加(p0.05)。【结论】1、我们利用lnc RNA基因芯片研究发现了324个差异表达lnc RNAs,并在鼻咽癌细胞株上验证了基因芯片结果的可靠性。2、实验证明ENST00000501541在鼻咽癌组织及鼻咽癌细胞株中均上调表达,干扰细胞ENST00000501541表达水平能够抑制鼻咽癌细胞株增殖、迁移和侵袭能力以及促进细胞凋亡,结果提示ENST00000501541可能参与鼻咽癌的发生发展过程。综上所述,我们的实验初步研究了ENST00000501541在体外对鼻咽癌细胞株的影响,为后续鼻咽癌的功能机制研究打下了基础,也为局部晚期鼻咽癌的研究提供了新的视角。
[Abstract]:[background] long chain non coded RNA (long non-coding RNA, LNC RNA) is a class of RNA with a length exceeding 200nt. They do not encode proteins themselves, but regulate the expression of genes in the form of RNA (epigenetic regulation, transcriptional regulation and post transcriptional regulation). [1]. Recent studies have shown that Lnc RNA is widely involved in a variety of life. The abnormal expression of Lnc RNA is closely related to a variety of diseases including tumors. There is evidence that LNC RNA is involved in the development of a variety of malignant tumors, such as liver cancer, pancreatic cancer, and glioma [2-5], but the functional mechanism of LNC RNA in nasopharyngeal carcinoma is rarely reported. The incidence of nasopharyngeal carcinoma has a distinct regional difference. In the south of China, North Africa and some Mediterranean regions are high incidence areas, especially in the southern part of China. Nasopharyngeal carcinoma has a high incidence of nasopharyngeal carcinoma. The prognosis of nasopharyngeal carcinoma is closely related to the staging of the disease. There are no symptoms and signs in the early stage of nasopharyngeal tumor, and 70% of the patients are locally advanced nasopharyngeal carcinoma (6) at the stage of stage III or IV A. In recent years, although the level of diagnosis and treatment of nasopharyngeal carcinoma has been greatly improved, its specific mechanism is still not very clear, the clinical treatment is still lack of effective means, the 5 year survival rate is only about 70% [7], local treatment failure and distant transfer are important reasons for the failure of treatment. Therefore, the improvement of local control rate and the prevention of distant metastasis are locally late. LNC RNA has become a hot topic in the treatment of nasopharyngeal carcinoma in recent years. In recent years, more and more people have been the hot spot of research, but the relationship between LNC RNA and nasopharyngeal carcinoma remains to be studied. Especially, the relationship between LNC RNA and local advanced nasopharyngeal carcinoma is worth exploring the.LncRNA gene core is widely used and studied the RNA expression spectrum of 1nc at present. An ideal and effective method. Through the Lnc RNA gene chip, the expression of Lnc RNA related to specific biological processes or diseases can be obtained quickly and high fluxes, thus providing a great convenience for subsequent Lnc RNA function research or biomarker screening. [Objective] this lesson aims to study l related to locally advanced nasopharyngeal carcinoma. NC RNA and its related biological functions, and preliminarily explore its mechanism to provide theoretical and scientific basis for the study of subsequent nasopharyngeal carcinoma. [method] 1, Human Lnc RNA Array V4.0 was used to detect the adjacent tissues of locally advanced nasopharyngeal carcinoma and its control, and the differential expression profiles of LNC RNA and m RNA were obtained between the two tissues. CSC, RNAdb and Lnc RNAs and other database resources, carry out cluster analysis, GO analysis and Pathway analysis for the preliminary bioinformatics analysis,.2, and in vitro culture of nasopharyngeal carcinoma cells CNE1 and HONE1, and normal nasopharyngeal epithelial cells NP69. After the A interference technique interference target LNC RNA expression, the Real-time PCR method was used to detect the transfection efficiency of Si RNA, and the effect of LNC RNA on target LNC RNA on the biological characteristics of nasopharyngeal carcinoma cells was detected after the target LNC RNA, and the interference test, the migration experiment, the cell scratch test and the cell apoptosis experiment were used to detect the interference, respectively. Target LNC RNA, the effect on proliferation, invasion, migration and apoptosis of nasopharyngeal carcinoma cell lines CNE1 and HONE1. [results] 1: 1, the differential expression of LNC RNA and m RNA were successfully screened by gene chip technology, and the screening criteria were set: P value less than 0.05, Foldchange > 1.5, 324 differential expression LNC RNAs, up 226, down 9 8, 188 differentially expressed m RNAs, up 98, and 90.2, and Pathway results showed that LNC RNA potential target gene m RNA may participate in 22 signal pathways, of which 6 of up regulated signaling pathway, and 13.GO analysis of down regulation indicated that the up regulation of M RNA biological process, cell composition, and the highest enrichment degree of molecular function The items are cell chemotaxis, collagen trimer and chemokine activity, respectively. The downregulated expressions are protein localization to cytoskeleton, cell cortex and second parts respectively. In addition, the relative expression of ENST00000448834, ENST00000554458 and ENST00000433377 decreased, which was consistent with the results of gene chip. More importantly, the expression of ENST00000501541 on CNE1 and HONE1 was significantly higher than that of NP69, with a significant statistical difference (P0.05).2, and the results of CCK8 colonization showed that compared with the control group (Si RNA-NC), the experimental group was compared with the control group (Si RNA-NC). RNA-1), the proliferation ability of CNE1 and HONE1 cells in the experimental group (Si RNA-2) decreased significantly (P0.05).3. The Transwell migration experiment showed that compared with the control group (Si RNA-NC), the number of cells passed under each high magnification microscope in the experimental group was significantly lower than that in the control group. The experimental results showed that compared with the control group, the experiment showed that the experiment group was compared with the control group. In group (Si RNA-1), the number of cells passing through Matrigel membrane under each high magnification was significantly lower than that of the control group (P0.05).5. The results of scratch test showed that compared with the blank group and negative control group, the scar healing ability of the experimental group (Si RNA-1) was significantly lower than that of the control group (P0.05).6 compared with the blank group and the negative control group (P0.05). The experimental group (Si RN) showed that the experimental group (Si RN) A-1) compared to the negative control group (SH RNA-NC), the percentage of CNE1 and HONE1 apoptotic cells in nasopharyngeal carcinoma cells increased significantly (P0.05). [Conclusion] 1, we found 324 differentially expressed LNC RNAs using LNC RNA gene chip study, and verified the reliability.2 of the gene chip results on the nasopharyngeal carcinoma cell line, and the experiment proved ENST00000501541 in nose. Both pharyngeal and nasopharyngeal carcinoma cell lines are all up expression. The interference of ENST00000501541 expression level can inhibit the proliferation, migration and invasion of nasopharyngeal carcinoma cell lines and promote cell apoptosis. The results suggest that ENST00000501541 may be involved in the development and development of nasopharyngeal carcinoma. In summary, our experiment preliminarily studied ENST00000501 The effect of 541 on nasopharyngeal carcinoma cell line in vitro lays a foundation for the study of the functional mechanism of the subsequent nasopharyngeal carcinoma and provides a new perspective for the study of local advanced nasopharyngeal carcinoma.

【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】：R739.63

【参考文献】