长链非编码RNA U90926在3T3-L1前脂肪细胞分化中的作用及其机制研究

发布时间：2018-06-18 03:05

  本文选题：lncRNA + U90926　； 参考：《山东大学》2017年博士论文

【摘要】：肥胖症是体内的脂肪组织过度蓄积所导致的一种疾病。研究证实,肥胖症是许多慢性疾病的主要危险因素,可以增加糖尿病、心血管疾病和癌症的发病率。近几年内肥胖症有急剧增长的趋势,尤其在发达国家,也包括经济迅速增长的发展中国家。研究肥胖发生、发展的机制可为人类防治肥胖、提高生活质量提供有价值的理论依据。肥胖症通常可表现为脂肪细胞数量增多和/或体积增大。因此,脂肪细胞和肥胖症的发生发展密切相关。从前脂肪细胞到成熟脂肪细胞生成的过程称为成脂分化,而此过程对脂肪组织蓄积和肥胖的形成至关重要。因而,脂肪细胞的分化过程及相关的调控机制已经成为研究肥胖及相关疾病的核心。长链非编码RNA(long noncoding RNA,lncRNA)是一类长度大于200个核苷酸(nucleotide,nt)的RNA分子,通常具有polyA尾巴,并且具有以下几个特点:1)表达丰度低于蛋白质编码基因;2)物种间保守性差;3)缺乏开放阅读框(open reading frames,ORFs)从而无编码蛋白质的能力;4)组织特异性表达。由于对其功能认识的不充分,在20世纪90年代,lncRNA 一度被认为是转录过程中的"噪音",是RNA聚合酶Ⅱ转录的副产物。随着分子生物学实验技术的发展,近年来,lncRNA与多种复杂疾病发生之间的关系逐渐受到关注。研究显示,lncRNA可以在多种生物学过程中发挥丰富多样的调控作用,比如可以调控mRNA的降解、X染色体失活、剪接调控、转录激活、染色质重塑等,通过以上作用方式最终参与到细胞增殖、分化、凋亡,物质代谢,肿瘤发生等过程中。也有文献报道,lncRNA可以调控前脂肪细胞的分化,这为人们研究肥胖的发病机制乃至预防和治疗肥胖症提供了新的思路。本课题研究内容分为以下两个部分:第一部分长链非编码RNAU90926(IncRNAU90926)在3T3-L1前脂肪细胞分化过程中的变化及其与肥胖症的关联研究目的:探讨lncRNAU90926在小鼠3T3-L1前脂肪细胞分化为成熟脂肪细胞过程中的表达变化及其与肥胖症的关联研究内容:1.以3T3-L1前脂肪细胞为模型,用脂肪细胞分化诱导剂诱导其分化。用油红O鉴定脂滴形成,并在分化的第0、2、4、13天收集细胞做包含编码基因和非编码基因的全转录组图谱分析,即microarray分析,寻找差异表达基因。收集诱导分化不同时间(第0、4、8、12天)的脂肪细胞并提取RNA,用qPCR技术验证microarray分析所得结果。2.采用荧光原位杂交(fluorescent in situ hybridization,FISH)实验检测长链非编码RNAU90926(lncRNAU90926)在3T3-L1前脂肪细胞中的亚细胞定位。3.肥胖动物模型的建立:选用6周龄雄性C57BL/6J小鼠、ob/ob小鼠和db/db小鼠为实验材料。适应性喂养一周之后,C57BL/6J小鼠随机分为3组,一组给予基础饲料(脂肪含量5%)喂养,另两组给予高脂饲料(脂肪含量20%)喂养,其中一组高脂饮食的小鼠用来做为ob/ob小鼠和db/db小鼠的对照。ob/ob小鼠和db/db小鼠喂养高脂饲料以期快速达到肥胖状态,每周称动物体重一次,4周之后,ob/ob小鼠和db/db小鼠体重增长超过对照组小鼠体重的30%,造模成功;另一组高脂饮食的C57BL/6J小鼠亦每周称重一次,直至体重超过基础饲料喂养的C57BL/6J小鼠体重的30%,此时即造模成功。造模成功之后,深度麻醉处死小鼠,提取其皮下脂肪组织和内脏脂肪组织(肾周脂肪和附睾脂肪),同时提取心脏、肝脏、脾脏、肾脏、肺脏、睾丸、棕色脂肪组织进行下述相关检测:1)取小鼠心脏、肝脏、脾脏、肾脏、肺脏、睾丸、棕色脂肪组织和附睾脂肪组织提取RNA进行实时荧光定量核酸扩增实验,分析lncRNAU90926在不同组织间的表达差异。2)取肥胖小鼠和各自对照小鼠皮下脂肪和内脏脂肪组织,提取RNA,检测 lncRNAU90926、PPARy2(peroxisome proliferator-activated receptor gamma 2,过氧化物酶体增殖物激活受体γ2)、FABP4(fatty acid binding protein 4,脂肪酸结合蛋白4)和AdipoQ(adiponectin,脂联素)在不同肥胖动物中的mRNA表达变化。研究结果:1.3T3-L1前脂肪细胞经过诱导能分化成为成熟脂肪细胞,油红O染色可以看到大量的脂滴聚积。分化不同天数细胞的microarray结果显示,随着3T3-L1前脂肪细胞的分化,lncRNAU90926的表达呈逐渐下降趋势。收集分化过程中的细胞提取RNA进行qPCR验证,其结果与microarray实验相一致。2.FISH实验表明,lncRNAU90926主要分布于3T3-L1前脂肪细胞胞质中,同时细胞核中也有少量lncRNAU90926存在。3.经过4周喂养,ob/ob小鼠和db/db小鼠体重增长超过对照组小鼠30%,造模成功;经过12周喂养,高脂饮食的C57BL/6J小鼠体重超过基础饲料喂养的C57BL/6J小鼠体重的30%,亦造模成功。4.对小鼠包括附睾脂肪在内的8个脏器组织提取RNA,qPCR扩增lncRNA U90926,结果显示lncRNAU90926主要存在但不局限于白色脂肪组织中,在其他组织也有表达,但其表达丰度较低。5.分别提取各组小鼠皮下、肾周和附睾脂肪组织,分离获得成熟脂肪细胞,检测lncRNAU90926表达,结果表明无论是高脂喂养肥胖小鼠还是ob/ob和db/db肥胖小鼠,其脂肪组织中lncRNAU90926含量均显著低于各自对照组小鼠。而各肥胖组小鼠脂肪组织中PPARγ2、FABP4和AdipoQ的表达量均明显高于对照组小鼠。研究结论:1.3T3-L1前脂肪细胞分化过程中lncRNAU90926的表达量逐渐降低。2.lncRNAU90926主要分布于小鼠白色脂肪组织中,且在脂肪细胞中绝大部分定位于细胞质。3.lncRNA U90926在肥胖小鼠中表达低于对照小鼠,即lncRNA U90926与肥胖负相关。第二部分长链非编码RNA U90926(IncRNA U90926)对3T3-L1前脂肪细胞分化的影响及其相关机制研究研究目的:研究lncRNAU90926对3T3-L1前脂肪细胞分化过程的影响及其相关机制研究内容:1.用过表达lncRNAU90926的慢病毒和对照病毒分别感染3T3-L1前脂肪细胞,经嘌呤霉素筛选建立稳定过表达lncRNAU90926的3T3-L1细胞克隆和对照细胞,诱导分化,以油红O鉴定脂滴聚积,收集分化第0、6天的细胞提取RNA,鉴定 PPARy2、FABP4 和 AdipoQ 的表达。收集分化第 0、2、4、6、8、10、12天的细胞进行Western Blotting实验,检测PPARγ2和FABP4的蛋白表达变化。2.设计靶向抑制小鼠lncRNA U90926基因的shRNA序列,构建重组慢病毒载体GV118-U90926-shRNA(以GV118-CON为对照)并包装慢病毒。用适宜滴度的慢病毒感染小鼠3T3-L1前脂肪细胞,鉴定并诱导其分化,用油红O染色鉴定脂肪细胞分化程度,并收集分化第0、12天的细胞提取RNA,检测PPARγ2、FABP4、C/EBPα(CCAAT/enhancer binding protein α,CCAAT/增强子结合蛋白αα)和AdipoQ的表达。收集分化过程中第0、2、4、6天的细胞进行Western Blotting实验,检测PPARy和FABP4的蛋白水平变化。3.用双荧光素酶实验检测lncRNAU90926对转录因子C/EBPα,C/EBPβ和PPAR γ2 转录活性的影响。检索 NCBI(National Center for Biotechnology Information)和Ensemble数据库,查找小鼠lncRNA U90926基因序列以及C/EBPα,C/EBPβ和PPARγ2基因的启动子序列,用基因体外重组的方法构建过表达lncRNA U90926的载体以及C/EBPα,C/EBPβ和PPARγ2包含全长及部分截短片段启动子的报告基因,共转染Hela细胞之后,检测荧光素酶活性以判断 lncRNA U90926 对 C/EBPα,C/EBPβ 和 PPARγ2 转录活性的作用。研究结果:1.分别用过表达lncRNAU90926的慢病毒和对照病毒感染3T3-L1前脂肪细胞,经过嘌呤霉素筛选,扩增,得到稳定过表达lncRNAU90926的细胞株,经过qPCR检测发现过表达细胞株(OV)lncRNAU90926比对照细胞株(NC)显著升高,选出3对克隆进行后续实验。分别对两组细胞(OV及NC)进行诱导分化,于分化不同时期进行油红O染色,染色结果显示,过表达组细胞脂滴聚积能力明显低于对照组。分别收取两组细胞在分化的第0及12天的RNA并进行qPCR检测,实验结果表明,过表达组前脂肪细胞(即第0天的细胞)中PPARγ2和FABP4 mRNA水平与对照组相比表达显著降低,成熟脂肪细胞(即分化第12天的细胞)中PPARγ2、FABP4和AdipoQ的表达亦明显低于对照组细胞。对两组细胞分化不同时期收取的蛋白进行Western Blotting分析,发现两组细胞中PPARγ和FABP4蛋白表达均随分化逐渐升高,但过表达组中二者蛋白表达显著低于对照组。2.分别用3条靶向lncRNAU90926的shRNA序列的慢病毒及对照病毒感染3T3-L1前脂肪细胞,用感染后的细胞群体进行实验观察,选择一条lncRNA U90926敲低效率最高的shRNA病毒用于建立稳转细胞株,即敲低组细胞(KD),同时筛选用对照病毒感染所得到的对照组细胞(CON)。分别对敲低组(KD)和对照组(CON)细胞进行诱导分化,油红O染色鉴定分化程度,结果显示敲低组脂滴聚积明显高于对照组。分别收取两组细胞分化至第0、6天的RNA进行qPCR分析,实验结果表明,未分化前(第0天),敲低组细胞中PPARγ2、FABP4和AdipoQ mRNA水平比对照组高,分化6天时敲低组细胞中PPARγ2、FABP4、C/EBPα和AdipoQ的表达亦明显高于对照组细胞。分别收取两组细胞在诱导分化的第0、2、4、6天的样品提取蛋白进行Western Blotting分析,发现两组细胞中PPARγ和FABP4蛋白表达均随分化逐渐升高,但敲低组细胞中二者表达明显高于对照组细胞。3.荧光素酶实验分析表明,在Hela细胞中过表达lncRNAU90926对C/EBPα和C/EBPβ的转录活性没有影响,但可以降低PPARγ2全长启动子转录活性的50%,进一步进行PPARγ 2启动子截短分析之后发现,人为去除PPARγ2启动子-2000bp到-1500bp的片段之后,过表达lncRNA U90926失去抑制PPARγ2全长启动子转录活性的能力。研究结论:1.在小鼠3T3-L1前脂肪细胞中过表达lncRNAU90926可以抑制其分化。2.敲低lncRNAU90926可以促进小鼠3T3-L1前脂肪细胞分化。3.lncRNAU90926可以抑制PPARy2启动子的转录活性,从而抑制3T3-L1前脂肪细胞分化。4.lncRNA U90926还可能通过其它未知的方式去改变PPARy2在mRNA及蛋白的表达水平,最终改变前脂肪细胞的成脂分化。
[Abstract]:Obesity is a disease caused by excessive accumulation of fatty tissue in the body. Studies have shown that obesity is a major risk factor for many chronic diseases, which can increase the incidence of diabetes, cardiovascular disease and cancer. In recent years, obesity has a rapid growth trend, especially in developed countries, including rapid economic growth. Chinese. Research on the mechanism of obesity and development can provide a valuable theoretical basis for the prevention and control of obesity and the improvement of quality of life. Obesity is usually manifested by the increase in the number and / or volume of fat cells. Therefore, the development of adipocytes and obesity is closely related. This process is called lipid differentiation, which is essential for the accumulation of fat tissue and the formation of obesity. Therefore, the differentiation process and related regulatory mechanisms of adipocytes have become the core of the study of obesity and related diseases. Long chain non coded RNA (long noncoding RNA, lncRNA) is a class of RNA minutes longer than 200 nucleotides (nucleotide, NT). Children, usually having the polyA tail, and have the following characteristics: 1) the expression abundance is lower than the protein encoding gene; 2) the conservatism of the species is poor; 3) the lack of the open reading frame (open reading frames, ORFs) and the ability to not encode protein; 4) the organization specific table is not sufficient, in 1990s, lncRNA 1 As the "noise" in the transcriptional process, it is a by-product of RNA polymerase II transcription. With the development of molecular biological experimental techniques, the relationship between lncRNA and a variety of complex diseases has been gradually paid attention in recent years. Research shows that lncRNA can play a variety of regulatory roles in a variety of biological processes, such as Regulation of mRNA degradation, X chromosome inactivation, splicing regulation, transcriptional activation, chromatin remodeling, and so on. Through the above methods, it is ultimately involved in cell proliferation, differentiation, apoptosis, substance metabolism, and tumorigenesis. It has also been reported that lncRNA can regulate the differentiation of pre fat cells, which can be used to study the pathogenesis and prevention of obesity. It provides new ideas for the treatment of obesity. The research content of this study is divided into two parts: the first part is the change of long chain non coded RNAU90926 (IncRNAU90926) in the differentiation of preadipocytes and its association with obesity. The purpose of this study is to explore the differentiation of lncRNAU90926 into mature adipocytes in the preadipocytes of 3T3-L1 in mice. Expression changes during the process and its association with obesity: 1. using 3T3-L1 preadipocytes as models and adipocyte differentiation inducers to induce differentiation. The formation of lipid droplets was identified with oil red O, and cells were collected for the full transcriptional mapping of the encoding and non coding genes on day 0,2,4,13 of differentiation, that is, the microarray score. Analyze the differentially expressed genes, collect the adipocytes of different time (day 0,4,8,12) and extract RNA, and verify the result of microarray analysis by qPCR technique, and.2. uses fluorescence in situ hybridization (fluorescent in situ hybridization, FISH) to test the long chain non coding RNAU90926 (lncRNAU90926) in the preadipocytes. The establishment of a subcellular localization.3. obesity animal model: 6 weeks old male C57BL/6J mice, ob/ob mice and db/db mice were selected as experimental materials. After one week of adaptive feeding, C57BL/6J mice were randomly divided into 3 groups, one group was given basal diet (fat content 5%), and the other two groups were fed with high fat diet (20% fat content), of which a group of high fat drinks were given. Feeding mice were used to feed ob/ob mice and db/db mice to.Ob/ob mice and db/db mice to feed high fat feed in order to quickly achieve obesity, once a week, and after 4 weeks, ob/ob mice and db/db mice increased more than 30% of the control group, and the other group of high fat diet C57BL/6J mice were also successful. After weighing more than 30% of the body weight of the C57BL/6J mice fed on the base diet once a week, the model was successful. After the success of the model, the mice were killed by deep anesthesia, and the subcutaneous adipose tissue and visceral adipose tissue (Shen Zhou fat and epididymal fat) were extracted, and the heart, liver, spleen, kidney, lungs, testis, brown fat group were extracted. The following related tests were carried out: 1) taking mouse heart, liver, spleen, kidney, lungs, testis, brown adipose tissue and epididymal adipose tissue extraction RNA to carry out real-time quantitative nucleic acid amplification test, analyze the difference in expression of lncRNAU90926 between different tissues.2) and take the subcutaneous fat and visceral adipose tissue of the obese mice and their control mice. RNA, detection of lncRNAU90926, PPARy2 (peroxisome proliferator-activated receptor gamma 2, peroxisome proliferator activated receptor gamma 2), FABP4 (fatty acid binding protein 4, fatty acid binding protein 4) and expression changes in different fat animals. After the induction can differentiate into mature adipocytes, a large number of lipid droplets can be seen with oil red O staining. The microarray results of different days of differentiation of cells show that the expression of lncRNAU90926 is gradually decreasing with the differentiation of 3T3-L1 preadipocytes, and the cells in the process of differentiation and differentiation of RNA are verified by qPCR, and the results are with microarr. The ay experiment consistent.2.FISH experiment showed that lncRNAU90926 was mainly distributed in the cytoplasm of 3T3-L1 preadipocytes, and a small amount of lncRNAU90926 existed in the nucleus for 4 weeks. The weight growth of ob/ob mice and db/db mice exceeded the control group by 30%, and the model was successful. After 12 weeks feeding, the high fat diet of C57BL/6J mice exceeded the weight of the mice. 30% of the body weight of the C57BL/6J mice fed on the base diet was also made by.4., and RNA was extracted from the 8 organs of the mice including epididymal fat and qPCR amplification of lncRNA U90926. The results showed that lncRNAU90926 mainly existed but not in the white adipose tissue, and also expressed in other tissues, but the expression abundance was lower than that of.5., respectively. In mice subcutaneous, peri renal and epididymal adipose tissue, mature adipocytes were isolated and lncRNAU90926 expression was detected. The results showed that the content of lncRNAU90926 in fat tissues of high fat fed obese mice, ob/ob and db/db obese mice were significantly lower than those of the control group, but PPAR 2, FABP4 and FABP4 in adipose tissue of all obese mice. The expression of AdipoQ was significantly higher than that of the control group. The study concluded that the expression of lncRNAU90926 in the process of 1.3T3-L1 preadipocyte differentiation gradually decreased in the white adipose tissue of mice, and most of the cytoplasm.3.lncRNA U90926 expressed in fat cells was lower than the control in obese mice. The negative correlation between lncRNA U90926 and obesity. Second the effect of long chain non coding RNA U90926 (IncRNA U90926) on the differentiation of 3T3-L1 preadipocytes and its related mechanisms: the study of the effect of lncRNAU90926 on the differentiation of pre 3T3-L1 preadipocytes and the related mechanisms: 1. the slow disease expressing lncRNAU90926 is used. The virus and the control virus were infected with 3T3-L1 preadipocytes respectively. Through the screening of 3T3-L1 cell clones and control cells that stably overexpressed lncRNAU90926 by purinomycin, the differentiation was induced. The oil red O was used to identify the accumulation of lipid droplets, and the cells from 0,6 days of differentiation were collected to extract RNA, and the expression of PPARy2, FABP4 and AdipoQ were identified. The differentiation of 0,2,4,6,8,10,12 was collected. Western Blotting experiment of day cells, detection of protein expression changes of PPAR gamma 2 and FABP4,.2. designed to target shRNA sequence of lncRNA U90926 gene in mice, construct recombinant lentivirus vector GV118-U90926-shRNA (GV118-CON as control) and package lentivirus. The differentiation was induced and the differentiation degree of adipocytes was identified by oil red O staining, and RNA was extracted from the cells of 0,12 day differentiation. The expression of PPAR gamma 2, FABP4, C/EBP alpha (CCAAT/enhancer binding protein alpha, CCAAT/ enhancer binding protein alpha) and AdipoQ expression were detected. The protein levels of PPARy and FABP4 were measured by.3., and the effects of lncRNAU90926 on transcription factor C/EBP alpha, C/EBP beta and PPAR gamma 2 transcriptional activity were detected by double luciferase test. The promoter sequence of lncRNA U90926 and C/EBP a, C/EBP beta and PPAR gamma 2 contain the reporter gene of the full length and partial truncated promoter, and after CO transfection of Hela cells, the activity of luciferase is detected to determine the transcriptional activity of lncRNA U90926 against C/EBP a, C/EBP beta and PPAR gamma 2. The results were as follows: 1. the 3T3-L1 preadipocytes were infected by the lentivirus and the control virus that overexpressed lncRNAU90926, and the cells were amplified and amplified by purinamycin. After qPCR detection, the overexpressed cell line (OV) lncRNAU90926 was found to be significantly higher than the control cell line (NC), and 3 pairs of clones were selected. Two groups of cells (OV and NC) were induced and differentiated, and the oil red O staining was performed at different stages of differentiation. The staining results showed that the accumulation of lipid droplets in the overexpressed group was significantly lower than that of the control group. Two groups of cells were collected for zeroth and 12 days of differentiation, respectively, and qPCR was carried out. The results showed that the preadipocytes were overexpressed. The expression of PPAR gamma 2 and FABP4 mRNA in the cells of zeroth days was significantly lower than that in the control group. The expression of PPAR gamma 2 in mature adipocytes (the cells of twelfth days of differentiation) and the expression of FABP4 and AdipoQ were also significantly lower than those of the control group. The Western Blotting analysis of the proteins collected at different stages of the two groups of cell differentiation and the detection of PPAR in the two groups of cells was found. The expression of gamma and FABP4 protein increased gradually with the differentiation, but the expression of two proteins in the overexpressed group was significantly lower than that of the control group.2., which were infected with the 3T3-L1 preadipocytes with 3 shRNA sequences targeting lncRNAU90926, and the infected cells were observed by the infected cell population, and a lncRNA U90926 knockout rate was the highest. ShRNA virus was used to establish a stable cell line, that is, to knock low group cells (KD). At the same time, the control group cells (CON) obtained from the control virus infection were screened. The cells of the low group (KD) and the control group (CON) were differentiated, respectively, and the oil red O staining was used to identify the differentiation degree. The results showed that the accumulation of lipid droplets in the knock low group was significantly higher than that of the control group. Two groups were collected. The cells differentiated to RNA on day 0,6 for qPCR analysis. The results showed that before the undifferentiation (zeroth days), PPAR gamma 2, FABP4 and AdipoQ mRNA levels were higher than those of the control group. The expression of PPAR gamma 2, FABP4, C/EBP A and AdipoQ in the low group of knockout groups at 6 days was also significantly higher than that in the control group. Two groups of cells were induced to induce differentiation, respectively. Western Blotting analysis of the sample extraction protein on day 0,2,4,6 showed that the expression of PPAR gamma and FABP4 protein in the two groups increased gradually with the differentiation, but the expression of the two in the knockout group was significantly higher than the.3. luciferase test of the control group, and the transcriptional activity of lncRNAU90926 to C/EBP A and C/EBP beta was overexpressed in the Hela cells. Sex does not affect, but can reduce the transcriptional activity of PPAR gamma 2 full length promoter 50%. After further analysis of PPAR gamma 2 promoter truncation, it is found that after human removal of PPAR gamma 2 promoter -2000bp to -1500bp fragments, overexpression lncRNA U90926 loses its ability to inhibit the transcriptional activity of PPAR gamma 2 full length promoter. Conclusion: 1. in mice 3T3-L1 Overexpression of lncRNAU90926 in preadipocytes can inhibit the differentiation of.2. knockdown lncRNAU90926, which can promote the differentiation of 3T3-L1 preadipocyte differentiation.3.lncRNAU90926 to inhibit the transcriptional activity of PPARy2 promoter, thus inhibiting the differentiation of.4.lncRNA U90926 from 3T3-L1 preadipocytes may also pass other unknown ways to change PPARy2 in mRNA. And the level of protein expression ultimately changes the adipogenic differentiation of preadipocytes.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R589.2
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本文编号：2033751