幽门螺杆菌感染相关的非编码RNA筛

发布时间：2018-05-16 21:41

本文选题：非编码 + RNA　；参考：《大连医科大学》2016年博士论文

【摘要】：一般认为,幽门螺杆菌(Helicobacter pylori,H.pylori)导致胃癌的发病机理是由其触发的炎症引起的胃萎缩、肠上皮化生、异型增生并最终发展成腺癌的一个多级发展过程。H.pylori感染引起胃炎和胃萎缩,是促成癌前病变和最终导致胃癌的重要因素。机体对于H.pylori感染能够产生免疫应答,但是并不能完全有效地清除H.pylori。H.pylori对宿主的毒性作用、免疫逃逸作用以及宿主对H.pylori的杀灭清除作用存在着有机的动态平衡,但这种平衡的分子机制尚不明确。非编码RNA,即不能编码蛋白质的RNA,包括核糖体RNA(r RNA),转运RNA(t RNA),小干扰RNA(siRNA),核内小RNA(snRNA),核仁小RNA(small nuclear RNA,sno RNA),微小RNA(microRNA),piwi相互作用RNA(piwi-interacting RNAs,piRNA)及长链非编码RNA(long noncoding RNA,lncRNA)等多种RNA,其中大部分RNA的功能是已知的,但也有相当一部分的RNA类型,具有特殊的结构,其功能也没有得到广泛的研究。这些RNA的共同特点是都能从基因组上转录而来,但是不翻译成蛋白,在RNA水平上就能行使各自的生物学功能了。从不同层面调控基因表达的非编码RNA,根据分子长度划分为microRNA及lncRNA。目前约有8000多种microRNA得到鉴定,其中1500多种microRNA在哺乳动物体内表达。在人类基因组中已经明确标定的lncRNA有9640种。microRNA是一类带有18-24个核苷酸的RNA,影响多种基因的转录和表达,在炎症、细胞增殖、细胞凋亡和变异中起着重要作用。lncRNA在近年的研究中得到了极大的关注,越来越多的学者和实验室致力于研究其结构和功能。大多数的lncRNA参与的生物学功能包括基因印记、染色体修饰、蛋白质结合与干扰等,也有部分lncRNA行使其功能与microRNA相类似,通过激活或干扰转录本,实现对某个特定蛋白的靶向激活或抑制。同时,还有大量的lncRNA尚未知其功能。本研究通过H.pylori感染人胃上皮细胞模型,利用全基因组测序技术检测H.pylori感染前后胃上皮细胞的lncRNA表达谱变化,结合实时定量PCR,对差异lncRNA进行筛选与鉴定。选取差异表达最明显的XLOC_004122和XLOC_014388,检测其在临床H.pylori感染组织样本的表达特性,并探讨其与致肿瘤预后指标是否存在相关性。同时,针对同样差异表达的microRNA-146a和microRNA-99b,通过靶点鉴定和功能分析等手段,深入研究其调控H.pylori感染的分子机制。本研究分为三部分:(一)幽门螺杆菌侵袭胃上皮细胞引起的长链非编码RNA差异表达分析;(二)microRNA-146a靶向COX-2调控H.pylori引起胃癌细胞凋亡的研究;(三)microRNA-99b在胃癌上皮细胞中通过调控自噬发生抑制H.pylori感染及细胞增殖作用研究。第一部分幽门螺杆菌侵袭胃上皮细胞引起的长链非编码RNA差异表达分析本实验利用H.pylor感染人胃上皮GES-1细胞24小时后,提取细胞的total RNA,利用全基因组测序手段,筛选鉴定差异表达的lncRNA分子;收集临床病人胃上皮组织样本,应用Realtime RT-PCR技术检测lncRNA分子在H.pylor感染病人和非感染病人中的表达规律,探讨lncRNA分子在H.pylor致癌发生、发展的关系及其在临床预后判断中的应用意义。同时,利用GO分析及基因共表达分析等生物信息学分析手段,探索差异表达的lncRNA的生物学功能。结果显示,H.pylori感染引起GES-1细胞一系列非编码RNA的表达改变,其中表达上调的有24条lncRNA,表达下调的有22条lncRNA。其中五条lncRNA,XLOC_004562(p0.05),XLOC_005912,XLOC_000620,XLOC_004122(p0.05)和XLOC_014388(p0.05)的表达差异最显著,并已被PCR结果证实。同时发现,XLOC_004122和XLOC_014388在临床H.pylori感染的组织样本中显著差异表达,差异具有统计学意义,且与肠化生现象成线性相关性(p0.05)。这提示这两段基因在临床治疗及预后方面可能存在标志物的功能。GO分析和共表达分析发现,XLOC_004122共表达主要包括膜蛋白组份、跨膜蛋白、离子通道等相应功能的基因,这说明XLOC_004122可能参与H.pylori被细胞胞吞的过程;XLOC_014388共表达的主要包括细胞间的信号传递、激酶活性、神经发育等功能的基因。本研究的意义在于我们可能发现了治疗H.pylori感染及预防其癌变的新方法,有广阔的临床应用前景。第二部分 microRNA-146a靶向COX-2调控H.pylori引起胃癌细胞凋亡的研究本实验通过向H.pylori感染的胃癌细胞MKN7转染microRNA-146a mimics和inhibitor,建立microRNA-146a过表达及抑制表达的细胞模型,利用V-Fluos staining kit(凋亡检测试剂盒)测定细胞凋亡率,结合凋亡正相关标志物Bax和负相关标志物Bcl-2的表达,确定microRNA-146a是否可以影响癌细胞的发生并深入探讨microRNA-146a对癌细胞的凋亡发生影响是否通过抑制靶基因COX-2实现。同时,检测胃癌临床样本中,microRNA-146a表达与凋亡发生率的相关性并分析microRNA-146a与肿瘤预后的相关性。结果显示:H.pylori感染的癌细胞模型和临床样本中,microRNA-146a表达(p0.05)和细胞凋亡发生率(p0.05)均显著高于H.pylori阴性对照组;microRNA-146a过表达的肿瘤细胞较对照组凋亡率显著升高(p0.05),且凋亡正相关分子Bax显著上调(p0.05),负相关分子Bcl-2显著下调(p0.05);在此细胞模型中,对COX-2的过表达可抑制microRNA-146a对凋亡的上调作用(p0.05),说明microRNA-146a对细胞凋亡的调控是通过靶向抑制COX-2的表达实现的。进一步的临床样本分析发现,microRNA-146a与肿瘤组织细胞凋亡率正相关,且与肿瘤的淋巴转移具有显著相关性(p0.05)。本实验结果说明microRNA-146a可能通过调控COX-2表达,促进凋亡,进而抑制H.pylori感染引起的胃癌发生。这部分研究的发现进一步阐明H.pylori感染的致癌发生机制以及机体对H.pylori的致癌作用调节机制研究提供新的方向。第三部分 microRNA-99b在胃癌上皮细胞中通过调控自噬发生抑制H.pylori感染及细胞增殖作用研究本实验在H.pylori感染的胃癌细胞模型和临床样本中,检测microRNA-99b的表达。通过向H.pylori感染的胃癌细胞转染microRNA-99b mimics和inhibitor,建立microRNA-99b过表达及抑制表达的细胞模型,检测细胞内H.pylori菌荷菌量的变化;利用Western blot检测自噬相关蛋白LC3-II,转染GFP-LC3并利用激光共聚焦技术对LC3进行斑点计数,从而判定microRNA-99b是否可以调控自噬发生;同时,利用生物信息学软件预测microRNA-99b的靶基因,并通过靶基因荧光素酶报告基因实验,鉴定microRNA-99b的靶基因并分析microRNA-99b调控自噬的分子机制是否通过抑制靶基因实现的。结果显示,H.pylori感染的胃癌细胞模型和临床样本中,microRNA-99b表达(p0.05)显著高于H.pylori阴性对照组;microRNA-99b过表达的肿瘤细胞对H.pylori的杀菌作用强于对照组,结果具有统计学意义(p0.05);Western blot结果显示,LC3分子由LC3-I向LC3-II转化,说明microRNA-99b过表达可诱导细胞自噬的发生,对LC3斑点计数也印证了这一点(p0.05);利用targetscan软件预测到microRNA-99b的靶基因为m TOR。荧光素酶报告基因实验证实了microRNA-99b确实能与m TOR的3′-UTR结合;Western blot结果表明,microRNA-99b通过抑制m TOR蛋白表达促进自噬的发生。结论:综合本文的发现及相关数据,我们提出以下结论:1.筛选鉴定出H.pylori感染引起的差异表达的长链非编码RNA,并发现XLOC_004122和XLOC_014388在临床H.pylori感染的组织样本中显著差异表达,且与肠化生现象成线性相关性。提示这两段基因在临床治疗及预后方面可能存在标志物的功能。2.发现microRNA-146a在H.pylori感染中调控细胞的凋亡和癌变,并证实是通过靶向COX-2实现此功能。microRNA-146a也与胃癌的淋巴转移正相关。3.发现microRNA-99b在H.pylori感染中刺激细胞自噬发生并因此上调细胞杀菌能力。此功能是通过靶向抑制m TOR蛋白的功能实现的。
[Abstract]:It is generally believed that the pathogenesis of Helicobacter pylori (H.pylori) causes gastric atrophy caused by inflammation, intestinal metaplasia, dysplasia and eventually a multistage development of adenocarcinoma..H.pylori infection causes gastritis and gastric atrophy, which is important for leading to precancerous lesions and ultimately leading to gastric cancer. Factors. The body can produce an immune response to H.pylori infection, but it can not completely effectively eliminate the toxic effect of H.pylori.H.pylori on the host, the immune escape effect and the host's killing and scavenging effect on H.pylori, but the molecular mechanism of this balance is not clear. Non coded RNA, that is, can not be made up. RNA, including the ribosome RNA (R RNA), transshipment RNA (t RNA), small interference RNA (siRNA), small RNA (snRNA), small nucleolar RNA, and long chain non coding traditions, etc. It is known, but there are a considerable number of RNA types, with special structures, and their functions are not widely studied. The common characteristics of these RNA are to be transcribed from the genome, but not translated into proteins, and they can exercise their respective biological functions at the RNA level. The non coded R that regulates gene expression from different levels. NA, according to molecular length divided into microRNA and lncRNA., about 8000 kinds of microRNA have been identified, of which more than 1500 kinds of microRNA are expressed in mammals. In the human genome, 9640 kinds of.MicroRNA, which have been clearly demarcated in the human genome, are RNA with 18-24 nucleotides, affecting the transcription and expression of a variety of genes, and in inflammation. .lncRNA plays an important role in cell proliferation, apoptosis and mutation. More and more scholars and laboratories are working to study their structures and functions. Most of the biological functions of lncRNA include gene imprinting, chromosome modification, protein binding and interference, and some lncRNA lines. By activating or interfering with the transcriptional transcript, the function of the microRNA is activated or suppressed by activating or interfering with the transcriptional transcript. At the same time, a large number of lncRNA are still unknown. In this study, the human gastric epithelial cell model was infected by H.pylori and the whole genome sequencing technology was used to detect the lncRNA of the gastric epithelial cells before and after the H.pylori infection. The expression of lncRNA was screened and identified by real-time quantitative PCR. The most distinct XLOC_004122 and XLOC_014388 were selected, and the expression characteristics of the H.pylori infected tissue samples were detected, and the correlation with the tumor prognosis index was discussed. At the same time, the expression of the same differential microRNA-146a was also discussed. MicroRNA-99b, through target identification and functional analysis, the molecular mechanism of H.pylori infection control was studied in depth. This study was divided into three parts: (1) the differential expression analysis of long chain non coded RNA caused by Helicobacter pylori invasion of gastric epithelial cells; (two) the study of microRNA-146a targeting COX-2 to regulate the apoptosis of gastric cancer cells; Three) microRNA-99b in gastric epithelial cells by regulating autophagy to inhibit H.pylori infection and cell proliferation. First, Helicobacter pylori invasion of gastric epithelial cells caused by the long chain non coding RNA differential expression analysis of the experiment using H.pylor infected human gastric epithelial GES-1 24 hours after the extraction of GES-1 cells, the extraction of cell total RNA, Screening and identification of differentially expressed lncRNA molecules by whole genome sequencing, collecting samples of gastric epithelial tissue from clinical patients, using Realtime RT-PCR technique to detect the expression of lncRNA molecules in H.pylor infected patients and non infected patients, and to explore the relationship between the carcinogenesis of lncRNA molecules in H.pylor and the relationship between the development of the H.pylor and the clinical prognosis. At the same time, using GO analysis and gene co expression analysis and other bioinformatics analysis methods to explore the biological functions of differentially expressed lncRNA. The results show that H.pylori infection causes a series of non coded RNA expression changes in GES-1 cells, of which 24 lncRNA are up regulated, and 22 lncRNA. of five ln are down regulated. The difference in expression of cRNA, XLOC_004562 (P0.05), XLOC_005912, XLOC_000620, XLOC_004122 (P0.05) and XLOC_014388 (P0.05) is most significant and has been confirmed by PCR results. Meanwhile, the significant difference between XLOC_004122 and XLOC_014388 in the tissue samples infected by the clinic is found, and the difference is statistically significant and is linearly related to the intestinal metaplasia. P0.05. This suggests that the functional.GO analysis and co expression analysis of the two genes may exist in clinical treatment and prognosis. The co expression of XLOC_004122 mainly includes the genes of membrane protein, transmembrane protein, ion channel and so on, which indicates that XLOC_004122 may be involved in the process of H.pylori by cell endocytosis; XLOC_01 4388 co expression mainly includes genes of signaling, kinase activity, and nerve development among cells. The significance of this study is that we may find a new method for the treatment of H.pylori infection and the prevention of its carcinogenesis. The second part of the study has a broad prospect of clinical application. The second part of the target COX-2 regulates H.pylori to induce apoptosis of gastric cancer cells In this study, microRNA-146a mimics and inhibitor were transfected into the gastric cancer cell MKN7 infected with H.pylori, and the cell model of microRNA-146a overexpression and inhibition expression was established. The apoptosis rate of cells was measured by V-Fluos Staining Kit (apoptosis detection kit), and the expression of Bax and negative correlation markers Bcl-2 on the positive correlation marker of apoptosis was combined. Determine whether microRNA-146a can affect the occurrence of cancer cells and explore whether the effect of microRNA-146a on the apoptosis of cancer cells is realized by inhibiting the target gene COX-2. At the same time, the correlation between the expression of microRNA-146a and the incidence of apoptosis in the clinical samples of gastric cancer is detected and the correlation between the microRNA-146a and the prognosis of the tumor is analyzed. In the cancer cell model and clinical samples of H.pylori infection, microRNA-146a expression (P0.05) and apoptosis rate (P0.05) were significantly higher than those in the H.pylori negative control group; the apoptosis rate of microRNA-146a overexpressed tumor cells was significantly higher than that in the control group (P0.05), and the positive correlation molecule Bax increased significantly (P0.05), and the negative correlation molecule Bcl-2 showed Bcl-2. Down regulation (P0.05); in this cell model, overexpression of COX-2 inhibits the up-regulated action of microRNA-146a on apoptosis (P0.05), indicating that the regulation of microRNA-146a to cell apoptosis is achieved through the expression of target inhibition COX-2. Further clinical sample analysis shows that microRNA-146a is positively related to the apoptosis rate of tumor tissue and is associated with the apoptosis rate of the tumor tissue. The lymphatic metastasis of the tumor has a significant correlation (P0.05). The results of this study suggest that microRNA-146a may promote apoptosis by regulating COX-2 expression, and then inhibit the occurrence of gastric cancer caused by H.pylori infection. The findings of this study further elucidate the pathogenesis of H.pylori infection and the mechanism of the organism's regulatory mechanism for the carcinogenesis of H.pylori. The study provides a new direction. Part third microRNA-99b in gastric cancer epithelial cells through the regulation of autophagy inhibition of H.pylori infection and cell proliferation in this experiment in H.pylori infected gastric cancer cell model and clinical samples to detect the expression of microRNA-99b. Transfection of microRNA-99b MIM to H.pylori infected gastric cancer cells. ICs and inhibitor, a cell model of microRNA-99b overexpression and inhibition of expression was established to detect the changes in the amount of H.pylori bacteria in the cells; the autophagy related protein LC3-II was detected by Western blot, GFP-LC3 was transfected and the dot counting was used to determine LC3 by laser confocal technique, thus determining whether microRNA-99b could regulate autophagy; meanwhile, Using the bioinformatics software to predict the target gene of microRNA-99b, and identify the target gene of microRNA-99b by the target gene luciferase reporter gene test, and analyze whether the molecular mechanism of autophagy by microRNA-99b is realized by inhibiting the target gene. The results show that the gastric cancer cell model and clinical samples of H.pylori infection, microRNA-9 The expression of 9b (P0.05) was significantly higher than that of the H.pylori negative control group; the antiseptic effect of microRNA-99b overexpressed tumor cells on H.pylori was stronger than that of the control group, and the results were statistically significant (P0.05). The Western blot results showed that the LC3 molecules were converted from LC3-I to LC3-II, indicating that the expression of microRNA-99b over expression could induce autophagy. This was also confirmed (P0.05); the targetscan software predicted that the target gene of microRNA-99b was m TOR. luciferase reporter gene experiment confirmed that microRNA-99b was able to combine with the 3 '-UTR of M TOR; Western blot results showed that microRNA-99b by inhibiting the expression of protein to promote autophagy. Relevant data, we propose the following conclusions: 1. screening and identification of the differential expression of long chain noncoding RNA caused by H.pylori infection, and found that XLOC_004122 and XLOC_014388 are significantly different in the tissue samples of the clinical H.pylori infection, and have a linear correlation with the intestinal metaplasia. These results suggest that these two segments are in clinical treatment and prognosis. The possible sign of the function.2. found that microRNA-146a regulates cell apoptosis and canceration in H.pylori infection, and confirms that the function of.MicroRNA-146a is also positively related to the lymphatic metastasis of gastric cancer by targeting COX-2, and it is found that microRNA-99b stimulates cell autophagy in H.pylori infection and thus up-regulates the cell bactericidal ability. The function is achieved by inhibiting the function of M TOR protein.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378

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