本文選題：lncRNA + U90926��； 參考：《山東大學》2017年博士論文

【摘要】：肥胖癥是體內的脂肪組織過度蓄積所導致的一種疾病。研究證實,肥胖癥是許多慢性疾病的主要危險因素,可以增加糖尿病、心血管疾病和癌癥的發(fā)病率。近幾年內肥胖癥有急劇增長的趨勢,尤其在發(fā)達國家,也包括經濟迅速增長的發(fā)展中國家。研究肥胖發(fā)生、發(fā)展的機制可為人類防治肥胖、提高生活質量提供有價值的理論依據(jù)。肥胖癥通�？杀憩F(xiàn)為脂肪細胞數(shù)量增多和/或體積增大。因此,脂肪細胞和肥胖癥的發(fā)生發(fā)展密切相關。從前脂肪細胞到成熟脂肪細胞生成的過程稱為成脂分化,而此過程對脂肪組織蓄積和肥胖的形成至關重要。因而,脂肪細胞的分化過程及相關的調控機制已經成為研究肥胖及相關疾病的核心。長鏈非編碼RNA(long noncoding RNA,lncRNA)是一類長度大于200個核苷酸(nucleotide,nt)的RNA分子,通常具有polyA尾巴,并且具有以下幾個特點:1)表達豐度低于蛋白質編碼基因;2)物種間保守性差;3)缺乏開放閱讀框(open reading frames,ORFs)從而無編碼蛋白質的能力;4)組織特異性表達。由于對其功能認識的不充分,在20世紀90年代,lncRNA 一度被認為是轉錄過程中的"噪音",是RNA聚合酶Ⅱ轉錄的副產物。隨著分子生物學實驗技術的發(fā)展,近年來,lncRNA與多種復雜疾病發(fā)生之間的關系逐漸受到關注。研究顯示,lncRNA可以在多種生物學過程中發(fā)揮豐富多樣的調控作用,比如可以調控mRNA的降解、X染色體失活、剪接調控、轉錄激活、染色質重塑等,通過以上作用方式最終參與到細胞增殖、分化、凋亡,物質代謝,腫瘤發(fā)生等過程中。也有文獻報道,lncRNA可以調控前脂肪細胞的分化,這為人們研究肥胖的發(fā)病機制乃至預防和治療肥胖癥提供了新的思路。本課題研究內容分為以下兩個部分:第一部分長鏈非編碼RNAU90926(IncRNAU90926)在3T3-L1前脂肪細胞分化過程中的變化及其與肥胖癥的關聯(lián)研究目的:探討lncRNAU90926在小鼠3T3-L1前脂肪細胞分化為成熟脂肪細胞過程中的表達變化及其與肥胖癥的關聯(lián)研究內容:1.以3T3-L1前脂肪細胞為模型,用脂肪細胞分化誘導劑誘導其分化。用油紅O鑒定脂滴形成,并在分化的第0、2、4、13天收集細胞做包含編碼基因和非編碼基因的全轉錄組圖譜分析,即microarray分析,尋找差異表達基因。收集誘導分化不同時間(第0、4、8、12天)的脂肪細胞并提取RNA,用qPCR技術驗證microarray分析所得結果。2.采用熒光原位雜交(fluorescent in situ hybridization,FISH)實驗檢測長鏈非編碼RNAU90926(lncRNAU90926)在3T3-L1前脂肪細胞中的亞細胞定位。3.肥胖動物模型的建立:選用6周齡雄性C57BL/6J小鼠、ob/ob小鼠和db/db小鼠為實驗材料。適應性喂養(yǎng)一周之后,C57BL/6J小鼠隨機分為3組,一組給予基礎飼料(脂肪含量5%)喂養(yǎng),另兩組給予高脂飼料(脂肪含量20%)喂養(yǎng),其中一組高脂飲食的小鼠用來做為ob/ob小鼠和db/db小鼠的對照。ob/ob小鼠和db/db小鼠喂養(yǎng)高脂飼料以期快速達到肥胖狀態(tài),每周稱動物體重一次,4周之后,ob/ob小鼠和db/db小鼠體重增長超過對照組小鼠體重的30%,造模成功;另一組高脂飲食的C57BL/6J小鼠亦每周稱重一次,直至體重超過基礎飼料喂養(yǎng)的C57BL/6J小鼠體重的30%,此時即造模成功。造模成功之后,深度麻醉處死小鼠,提取其皮下脂肪組織和內臟脂肪組織(腎周脂肪和附睪脂肪),同時提取心臟、肝臟、脾臟、腎臟、肺臟、睪丸、棕色脂肪組織進行下述相關檢測:1)取小鼠心臟、肝臟、脾臟、腎臟、肺臟、睪丸、棕色脂肪組織和附睪脂肪組織提取RNA進行實時熒光定量核酸擴增實驗,分析lncRNAU90926在不同組織間的表達差異。2)取肥胖小鼠和各自對照小鼠皮下脂肪和內臟脂肪組織,提取RNA,檢測 lncRNAU90926、PPARy2(peroxisome proliferator-activated receptor gamma 2,過氧化物酶體增殖物激活受體γ2)、FABP4(fatty acid binding protein 4,脂肪酸結合蛋白4)和AdipoQ(adiponectin,脂聯(lián)素)在不同肥胖動物中的mRNA表達變化。研究結果:1.3T3-L1前脂肪細胞經過誘導能分化成為成熟脂肪細胞,油紅O染色可以看到大量的脂滴聚積。分化不同天數(shù)細胞的microarray結果顯示,隨著3T3-L1前脂肪細胞的分化,lncRNAU90926的表達呈逐漸下降趨勢。收集分化過程中的細胞提取RNA進行qPCR驗證,其結果與microarray實驗相一致。2.FISH實驗表明,lncRNAU90926主要分布于3T3-L1前脂肪細胞胞質中,同時細胞核中也有少量lncRNAU90926存在。3.經過4周喂養(yǎng),ob/ob小鼠和db/db小鼠體重增長超過對照組小鼠30%,造模成功;經過12周喂養(yǎng),高脂飲食的C57BL/6J小鼠體重超過基礎飼料喂養(yǎng)的C57BL/6J小鼠體重的30%,亦造模成功。4.對小鼠包括附睪脂肪在內的8個臟器組織提取RNA,qPCR擴增lncRNA U90926,結果顯示lncRNAU90926主要存在但不局限于白色脂肪組織中,在其他組織也有表達,但其表達豐度較低。5.分別提取各組小鼠皮下、腎周和附睪脂肪組織,分離獲得成熟脂肪細胞,檢測lncRNAU90926表達,結果表明無論是高脂喂養(yǎng)肥胖小鼠還是ob/ob和db/db肥胖小鼠,其脂肪組織中l(wèi)ncRNAU90926含量均顯著低于各自對照組小鼠。而各肥胖組小鼠脂肪組織中PPARγ2、FABP4和AdipoQ的表達量均明顯高于對照組小鼠。研究結論:1.3T3-L1前脂肪細胞分化過程中l(wèi)ncRNAU90926的表達量逐漸降低。2.lncRNAU90926主要分布于小鼠白色脂肪組織中,且在脂肪細胞中絕大部分定位于細胞質。3.lncRNA U90926在肥胖小鼠中表達低于對照小鼠,即lncRNA U90926與肥胖負相關。第二部分長鏈非編碼RNA U90926(IncRNA U90926)對3T3-L1前脂肪細胞分化的影響及其相關機制研究研究目的:研究lncRNAU90926對3T3-L1前脂肪細胞分化過程的影響及其相關機制研究內容:1.用過表達lncRNAU90926的慢病毒和對照病毒分別感染3T3-L1前脂肪細胞,經嘌呤霉素篩選建立穩(wěn)定過表達lncRNAU90926的3T3-L1細胞克隆和對照細胞,誘導分化,以油紅O鑒定脂滴聚積,收集分化第0、6天的細胞提取RNA,鑒定 PPARy2、FABP4 和 AdipoQ 的表達。收集分化第 0、2、4、6、8、10、12天的細胞進行Western Blotting實驗,檢測PPARγ2和FABP4的蛋白表達變化。2.設計靶向抑制小鼠lncRNA U90926基因的shRNA序列,構建重組慢病毒載體GV118-U90926-shRNA(以GV118-CON為對照)并包裝慢病毒。用適宜滴度的慢病毒感染小鼠3T3-L1前脂肪細胞,鑒定并誘導其分化,用油紅O染色鑒定脂肪細胞分化程度,并收集分化第0、12天的細胞提取RNA,檢測PPARγ2、FABP4、C/EBPα(CCAAT/enhancer binding protein α,CCAAT/增強子結合蛋白αα)和AdipoQ的表達。收集分化過程中第0、2、4、6天的細胞進行Western Blotting實驗,檢測PPARy和FABP4的蛋白水平變化。3.用雙熒光素酶實驗檢測lncRNAU90926對轉錄因子C/EBPα,C/EBPβ和PPAR γ2 轉錄活性的影響。檢索 NCBI(National Center for Biotechnology Information)和Ensemble數(shù)據(jù)庫,查找小鼠lncRNA U90926基因序列以及C/EBPα,C/EBPβ和PPARγ2基因的啟動子序列,用基因體外重組的方法構建過表達lncRNA U90926的載體以及C/EBPα,C/EBPβ和PPARγ2包含全長及部分截短片段啟動子的報告基因,共轉染Hela細胞之后,檢測熒光素酶活性以判斷 lncRNA U90926 對 C/EBPα,C/EBPβ 和 PPARγ2 轉錄活性的作用。研究結果:1.分別用過表達lncRNAU90926的慢病毒和對照病毒感染3T3-L1前脂肪細胞,經過嘌呤霉素篩選,擴增,得到穩(wěn)定過表達lncRNAU90926的細胞株,經過qPCR檢測發(fā)現(xiàn)過表達細胞株(OV)lncRNAU90926比對照細胞株(NC)顯著升高,選出3對克隆進行后續(xù)實驗。分別對兩組細胞(OV及NC)進行誘導分化,于分化不同時期進行油紅O染色,染色結果顯示,過表達組細胞脂滴聚積能力明顯低于對照組。分別收取兩組細胞在分化的第0及12天的RNA并進行qPCR檢測,實驗結果表明,過表達組前脂肪細胞(即第0天的細胞)中PPARγ2和FABP4 mRNA水平與對照組相比表達顯著降低,成熟脂肪細胞(即分化第12天的細胞)中PPARγ2、FABP4和AdipoQ的表達亦明顯低于對照組細胞。對兩組細胞分化不同時期收取的蛋白進行Western Blotting分析,發(fā)現(xiàn)兩組細胞中PPARγ和FABP4蛋白表達均隨分化逐漸升高,但過表達組中二者蛋白表達顯著低于對照組。2.分別用3條靶向lncRNAU90926的shRNA序列的慢病毒及對照病毒感染3T3-L1前脂肪細胞,用感染后的細胞群體進行實驗觀察,選擇一條lncRNA U90926敲低效率最高的shRNA病毒用于建立穩(wěn)轉細胞株,即敲低組細胞(KD),同時篩選用對照病毒感染所得到的對照組細胞(CON)。分別對敲低組(KD)和對照組(CON)細胞進行誘導分化,油紅O染色鑒定分化程度,結果顯示敲低組脂滴聚積明顯高于對照組。分別收取兩組細胞分化至第0、6天的RNA進行qPCR分析,實驗結果表明,未分化前(第0天),敲低組細胞中PPARγ2、FABP4和AdipoQ mRNA水平比對照組高,分化6天時敲低組細胞中PPARγ2、FABP4、C/EBPα和AdipoQ的表達亦明顯高于對照組細胞。分別收取兩組細胞在誘導分化的第0、2、4、6天的樣品提取蛋白進行Western Blotting分析,發(fā)現(xiàn)兩組細胞中PPARγ和FABP4蛋白表達均隨分化逐漸升高,但敲低組細胞中二者表達明顯高于對照組細胞。3.熒光素酶實驗分析表明,在Hela細胞中過表達lncRNAU90926對C/EBPα和C/EBPβ的轉錄活性沒有影響,但可以降低PPARγ2全長啟動子轉錄活性的50%,進一步進行PPARγ 2啟動子截短分析之后發(fā)現(xiàn),人為去除PPARγ2啟動子-2000bp到-1500bp的片段之后,過表達lncRNA U90926失去抑制PPARγ2全長啟動子轉錄活性的能力。研究結論:1.在小鼠3T3-L1前脂肪細胞中過表達lncRNAU90926可以抑制其分化。2.敲低lncRNAU90926可以促進小鼠3T3-L1前脂肪細胞分化。3.lncRNAU90926可以抑制PPARy2啟動子的轉錄活性,從而抑制3T3-L1前脂肪細胞分化。4.lncRNA U90926還可能通過其它未知的方式去改變PPARy2在mRNA及蛋白的表達水平,最終改變前脂肪細胞的成脂分化。
[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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R589.2
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本文編號：2033751