發(fā)布時間：2018-02-23 20:38

  本文關(guān)鍵詞： 線粒體病 線粒體腦肌病 非編碼RNA A3243G miRNA lncRNA 標(biāo)志物　出處：《山東大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：線粒體DNA(mtDNA)或/和編碼線粒體功能相關(guān)蛋白的核DNA發(fā)生基因缺失或點突變,造成編碼線粒體氧化代謝必需的酶或載體發(fā)生障礙,糖原和脂肪酸等不能進入線粒體充分利用,導(dǎo)致能量代謝障礙和產(chǎn)生復(fù)雜的臨床癥狀稱為線粒體疾病(mitochondrial disease)。線粒體疾病骨骼肌受累者稱為線粒體肌病(mitochondrial myopathy)。線粒體肌病涉及臨床類型復(fù)雜,包括線粒體肌病伴高乳酸血癥和卒中樣發(fā)作(MELAS)綜合征等多種臨床綜合征,不同臨床類型往往具有不同致病基因突變但亦有重疊。圍繞線粒體與疾病及衰老、細(xì)胞程序性死亡、氧自由基生成以及細(xì)胞信號轉(zhuǎn)導(dǎo)調(diào)控過程中的相互關(guān)系的研究一直是線粒體醫(yī)學(xué)的研究熱點�；驕y序的進步讓我們意識到,人類基因組存在著數(shù)以萬計的多種雖不具有編碼蛋白的功能但具有基因調(diào)控和表觀遺傳修飾功能的非編碼RNA(ncRNA),如微小非編碼 RNA(miRNA)、長鏈非編碼 RNA(long non-coding RNA,lncRNA)。近年來ncRNAs己被證明可以通過多種不同機制影響基因的表達水平,但絕大多數(shù)非編碼RNA的功能還是未知的。更為復(fù)雜的是各類RNA分子相互間存在復(fù)雜的調(diào)控網(wǎng)絡(luò)。得益于核酸測序等技術(shù)的飛速發(fā)展,越來越多研究發(fā)現(xiàn)在人類血清/血漿中存在大量穩(wěn)定的ncRNA。非編碼RNA分子具備可以作為疾病生物學(xué)標(biāo)志物的各類特征,有巨大潛力成為線粒體肌病等疾病的診治標(biāo)志物。線粒體功能相關(guān)ncRNA的研究還只是處于起步階段,ncRNA如何影響線粒體基因表達及其功能是一個全新的領(lǐng)域。越來越多的研究證實非編碼RNA可以通過影響核基因編碼線粒體相關(guān)蛋白基因表達或直接在線粒體中調(diào)節(jié)線粒體自身基因表達影響線粒體的功能。目前線粒體功能相關(guān)非編碼RNA研究仍大多局限在體外線粒體損傷細(xì)胞模型相關(guān)非編碼RNA的發(fā)現(xiàn)及功能驗證。盡管已發(fā)現(xiàn)多種影響線粒體功能的miRNAs和lncRNAs,但ncRNA調(diào)控線粒體功能詳細(xì)機制有待進一步闡明。ncRNA與線粒體肌病的相關(guān)研究尚未見報道,我們有必要探究ncRNA參與線粒體肌病發(fā)生發(fā)展的的詳細(xì)機制,或能更深入地揭示ncRNA與線粒體功能的密切聯(lián)系,且為線粒體肌病找到潛在的治療靶點。我們希望能在ncRNA調(diào)控層面,在線粒體肌病中發(fā)現(xiàn)調(diào)控線粒體功能或者參與線粒體肌病致病機制的某些關(guān)鍵ncRNAs,進而可以通過影響ncRNA實現(xiàn)異常線粒體的有效清除等影響線粒體肌病發(fā)生發(fā)展的關(guān)鍵病理過程的調(diào)控。具備疾病診治標(biāo)志物特征的ncRNA分子具備還可以作為線粒體肌病診治標(biāo)志物服務(wù)臨床。為此,本課題通過篩選線粒體肌病患者肌肉組織和血清中與健康對照者差異表達的ncRNA(miRNA/lncRNA),構(gòu)建不同RNA之間相互調(diào)控網(wǎng)絡(luò),探索ncRNA參與線粒體肌病發(fā)生發(fā)展的潛在通路及線粒體損傷對關(guān)鍵性IncRNA的表達調(diào)控機制,并尋找線粒體肌病發(fā)生發(fā)展過程中的關(guān)鍵ncRNA(miRNA/lncRNA),為ncRNA用于線粒體肌肉病的診斷和治療提供前期理論依據(jù)。本課題圍繞著線粒體病非編碼RNA參與線粒體肌病發(fā)生發(fā)展的調(diào)控機制進行了以下幾方面的工作:第一部分線粒體肌病肌肉非編碼RNA(mi RNA/IncRNA)差異表達譜研究研究目的采用高通量技術(shù)結(jié)合后續(xù)RT-PCR重復(fù)性驗證的前期篩選策略,篩選代表性線粒體腦肌病MELAS(A3243G突變患者)肌肉組織中非編碼RNA(miRNA/lncRNA)的差異表達譜,為后續(xù)尋找感興趣的ncRNA以及探究非編碼RNA參與線粒體肌病的復(fù)雜機制提供前期基礎(chǔ)。研究對象和方法1)首先選取MELAS(A3243G突變患者)及活檢排除肌肉病變的對照者各20例,采用miRNA表達譜芯片和IncRNA高通量測序技術(shù),篩選MELAS(A3243G突變患者)相比對照肌肉組織中非編碼RNA(miRNA/lncRNA)差異表達譜。2)由于高通量技術(shù)結(jié)果存在潛在假陽性及假陰性,通過RT-PCR對篩選出的關(guān)鍵差異非編碼RNA(miRNA/lncRNA)及mRNA再次驗證。再次在上述MELAS(A3243G突變患者)及活檢排除肌肉病變的對照者各20例單個肌肉樣本中進行RT-PCR實驗驗證高通量初篩結(jié)果,確認(rèn)關(guān)鍵性ncRNAs(miRNAs、IncRNAs)及相關(guān)靶mRNAs在MELAS(A3243G突變患者)肌肉組織中的表達差異。實驗結(jié)果1.我們首次發(fā)現(xiàn)MELAS A3243G突變患者肌肉組織中獨特的ncRNAs(miRNAs/lnc RNAs)表達譜,共篩選出具有2倍以上差異的microRNAs(31個上調(diào);41個下調(diào)),lncRNAs(57個上調(diào);52個下調(diào))以及mRNAs(167個上調(diào);162個下調(diào));2種線粒體肌病(mtDNA A3243G突變與核基因TK2突變)肌肉組織顯示二者共有41個共差異表達的mRNAs。2.我們通過對篩選出的 6 個 miRNAs(miR-6089、miR-27b-3p、miR-214-3p、mi R-150-5p、let-7e-5p 和 miR-145-5p)、5個IncRNAs(LINC01405、SNHG12、R P11-403P17.4、CTC-260E6.6 和 RP11-357D18.1)和 6 個 mRNAs(PDK4、CDKN1A、ATP2A2、SOD3、DDIT4和EEF1A1)進行RT-PCR驗證,結(jié)果顯示同高通量測序結(jié)果比較二者基本一致。第二部分線粒體肌病肌肉差異RNA分子互作網(wǎng)絡(luò)構(gòu)建及關(guān)鍵ncRNA(miRNA/lncRNA)篩選研究目的構(gòu)建mRNA及非編碼RNA(miRNA/lncRNA)相互間調(diào)控網(wǎng)絡(luò),并分析其參與的信號通路建立調(diào)節(jié)機制假說。研究方法對差異ncRNA(miRNA/lncRNA)進行生物信息學(xué)(聚類及GO和Pathway分析)、表達相關(guān)性等分析確定參與不同突變類型線粒體肌病患者發(fā)生發(fā)展的關(guān)鍵非編碼R NA(miRNA/lncRNA)相互作用分子。共表達互作網(wǎng)絡(luò)構(gòu)建包括miRNA-mRNA、miRNA-lncRNA 及 IncRNA-mRNA 兩兩互作網(wǎng)絡(luò)以及 miRNA-mRNA-lncRNA 和 mRNA-miRNA-lncRNA三分子互作網(wǎng)絡(luò)。應(yīng)用Cytoscape軟件計算網(wǎng)絡(luò)及各節(jié)點的拓?fù)涮匦�。通過miRecords預(yù)測差異miRNA的靶基因,對差異靶基因進行GO功能注釋、KEGG通路分析。實驗結(jié)果1.我們首次通過生物信息學(xué)靶基因預(yù)測構(gòu)建出miRNA-mmRNA-lncRNA和mRNA-mi RNA-lncRNA共表達互作網(wǎng)絡(luò),篩選出調(diào)控網(wǎng)絡(luò)中關(guān)鍵性ncRNA分子。2.GO分析發(fā)現(xiàn)差異miRNA/lncRNAs靶基因集合及總體差異mmRNAs集合共同的生物過程富集分析集中于細(xì)胞外基質(zhì)分子、肌肉收縮和蛋白結(jié)合分子。除此之外,總的差異表達mRNAs的顯著GO條目富集結(jié)果集中于小分子代謝、信號轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄分子等過程;miRNA靶mRNA網(wǎng)絡(luò)代表的顯著GO分類條目為信號轉(zhuǎn)導(dǎo)分子、細(xì)胞凋亡相關(guān)分子等;lncRNA靶mRNA對應(yīng)的相關(guān)聯(lián)的GO分析富集條目為小分子代謝過程和肌肉收縮相關(guān)分子。KEGG通路分析表明這些失調(diào)的mRNA主要參與小分子代謝過程、細(xì)胞外基質(zhì)生成、肌肉收縮、蛋白結(jié)合、信號轉(zhuǎn)導(dǎo)、轉(zhuǎn)錄等信號途徑和生物功能。綜上所述,MELAS患者mtDNA A3243G突變造成的下游差異表達的ncRNA譜可能通過免疫應(yīng)答、小分子代謝、信號轉(zhuǎn)導(dǎo)、凋亡和發(fā)育過程等多種信號途徑參與線粒體肌病的發(fā)生發(fā)展。第三部分線粒體肌病ncRNA(miRNA/lncRNA)生物學(xué)標(biāo)志物的潛在臨床應(yīng)用價值研究目的本課題進一步分析關(guān)鍵性ncRNAs在肌肉組織和血清對MELAS患者的診斷效能,尋找線粒體肌病潛在的ncRNA診斷標(biāo)志物。研究對象和方法1.選取54例MELAS(A3243G突變患者)與49例正常對照組獨立肌肉樣本再次驗證A3243G突變MELAS患者高通量結(jié)果;2.選取34例MELAS(A3243G突變患者)與34例對照獨立血清樣本驗證A3243G突變 MELAS 患者 miR-27b-3p 表達;3.選取22例MELAS(非A3243G突變患者)與34例對照獨立肌肉及血清樣本驗證非 A3243G 突變 MELAS 患者 miR-27b-3p 表達;3.軟件分析肌肉組織ncRNAs及血清miR-27b-3p對于MELAS患者診斷的敏感度、特異度;對血清miRNA-27b-3p表達水平與各類臨床參數(shù)如年齡、性別、BMI、NMDAS、疾病持續(xù)時間、血乳酸和肌肉A3243G突變率進行相關(guān)性分析。實驗結(jié)果1.我們再次證實 4 個 ncRNAs(miR-6089、miR-27b-3p、miR-214-3p 及 LINC01405)肌肉表達顯著下調(diào),而 7 個ncRNAs(miR-150-5p、let-7e-5p、miR-145-5p、SNHG12、RP11-403P17.4、CTC-260E6.6 及 RP11-357D18.11)在 MELAS 肌肉組織中顯著上調(diào);ROC曲線分析表明在所選擇的肌肉組織關(guān)鍵性ncRNA中miR-27b-3p對于MELAS患者診斷具有最高的靈敏度和特異性。2.關(guān)鍵性miR-27b-3p在MELAS患者(A3243G和非A3243G突變患者)肌肉和血清中表達均降低。3.ROC曲線分析表明,相比乳酸血清miR-27b-3p對于MELAS具有更好的診斷價值。相關(guān)性分析顯示血清miR-27b-3p和乳酸鹽及NMDAS之間存在顯著負(fù)相關(guān)。結(jié)論我們首次發(fā)現(xiàn)MELAS A3243G突變患者肌肉組織中獨特的ncRNAs(miRNAs/lncRNAs)表達譜;首次構(gòu)建出 miRNA-mRNA-lncRNA 和 mRNA-miRNA-lncRNA共表達互作網(wǎng)絡(luò),篩選出調(diào)控網(wǎng)絡(luò)中關(guān)鍵性ncRNA分子血清miR-27b-3p比乳酸對于MELAS具有更好的診斷價值,并通過功能富集分析明確了非編碼RNA分子參與線粒體肌病可能主要通過信號轉(zhuǎn)導(dǎo)、氧化應(yīng)激、凋亡等途徑,為進一步探究相應(yīng)ncRNA分子的功能提供理論依據(jù)。
[Abstract]:Mitochondrial DNA (mtDNA) DNA and / or nuclear mitochondrial function related protein encoding gene deletions or point mutations caused by encoding mitochondrial oxidative metabolism enzymes essential carrier or impairment of glycogen and fatty acids into mitochondria can not fully utilized, resulting in energy metabolism and has complex clinical symptoms called mitochondrial diseases (mitochondrial disease). Mitochondrial diseases of skeletal muscle involvement called mitochondrial myopathy (mitochondrial myopathy). The clinical type of mitochondrial myopathy involving complex, including mitochondrial myopathy with lactic acidosis and stroke like episodes (MELAS) syndrome and other clinical syndromes, different clinical types tend to have different mutations but also on mitochondria and diseases overlap. And senescence, programmed cell death, the relationship between oxygen free radicals and cellular signal transduction in the process of a Has been the research hotspot in mitochondrial medicine. Gene sequencing progress makes us aware of the human genome, there are tens of thousands of kinds of does not have the function of encoding protein but with gene regulation and apparent non genetically modified RNA encoding function (ncRNA), such as small non encoding RNA (miRNA), RNA (long chain non encoding long non-coding RNA, lncRNA). In recent years, ncRNAs has been proved by several different mechanisms influencing gene expression level, but the vast majority of non RNA encoding function is unknown. More complicated is that all kinds of RNA molecules in the presence of a complex regulatory network between. Thanks to the rapid development of nucleic acid sequencing techniques, more and more the study found that in human serum / plasma in the presence of a large number of stable ncRNA. non RNA encoding can be used as molecular biological markers of disease have all kinds of features, there is a huge potential to become mitochondrial myopathy The diagnosis and treatment of markers of disease. Research on mitochondrial function of ncRNA is still at the start stage, the influence of ncRNA to the mitochondrial gene expression and function is a new field. More and more studies have confirmed that RNA can express non encoding or regulation of mitochondrial genes directly in their mitochondria affects mitochondrial function by affecting the expression of nuclear genes encoding mitochondrial egg white. The current study of mitochondrial function related gene RNA encoding is still mostly limited in non related mitochondrial damage in vitro cell model of non encoding RNA discovery and functional verification. Although many have been found to affect mitochondrial function of miRNAs and lncRNAs, but the regulation of mitochondrial function with ncRNA mechanism to be further research shows that.NcRNA has not been reported with mitochondrial myopathy it is necessary for us to study, ncRNA is involved in the occurrence and development mechanism of mitochondrial myopathy with, or Close contact can reveal ncRNA and mitochondrial function and mitochondrial myopathy, find potential therapeutic targets. We hope that the regulation of ncRNA level, ncRNAs found some key control of mitochondrial function or participate in the pathogenesis of mitochondrial myopathy in mitochondrial myopathy, and could achieve the abnormal mitochondria by affecting ncRNA effective removal effect the key to the development of mitochondrial myopathy occurred in the pathological process of regulation. NcRNA molecular markers have the characteristics of disease diagnosis and treatment can also be used as a marker of mitochondrial myopathy diagnosis and treatment of the clinical service. For this, this topic through screening the differentially expressed ncRNA and healthy control in patients with mitochondrial myopathy muscle tissue and serum (miRNA/lncRNA), RNA between different construction control network, explore the potential pathway and mitochondrial ncRNA damage in mitochondrial myopathy on occurrence and development of the key The mechanisms that regulate the expression of IncRNA and ncRNA, to find the key process in the development of mitochondrial myopathy (miRNA/lncRNA), to provide theoretical basis for the diagnosis and treatment of the disease is ncRNA for muscle mitochondria. The issue is about the non regulation mechanism of mitochondrial disease encoding RNA involved in mitochondrial myopathy and development are as follows: first part of muscle mitochondrial myopathy (MI RNA/IncRNA) RNA encoding pre screening strategy to study the spectrum of high throughput technology combined with subsequent RT-PCR repetitive test of differential expression screening of representative mitochondrial encephalomyopathy MELAS (A3243G mutation) encoding RNA non muscle tissues (miRNA/lncRNA) expression profile differences, provide a basis for interested to follow the ncRNA and complex mechanism of non encoding RNA involved in mitochondrial myopathy. Subjects and methods 1 selected MELAS (A3243G) first Mutation) and biopsy to exclude muscle disease control group 20 cases, using miRNA microarray and high-throughput IncRNA sequencing technology, MELAS screening (A3243G mutation) - encoding RNA compared with control muscle tissue (miRNA/lncRNA) expression difference of.2) due to the high throughput technology the potential false positive and false negative. The RT-PCR of the key differences between the selected non encoding RNA (miRNA/lncRNA) and mRNA in the MELAS. Verify again again (A3243G mutation) were excluded and biopsy of muscle lesions in 20 cases of single muscle samples of RT-PCR experimental verification of high-throughput screening results, confirm the key ncRNAs (miRNAs, IncRNAs) and related the target mRNAs in MELAS (A3243G mutation) differentially expressed in muscle tissue. The experimental results of 1. we first found that MELAS A3243G mutation unique muscle tissues of patients with ncRNAs (miRNAs/lnc RNAs) expression, Co Screening is more than 2 times the difference of microRNAs (31 up-regulated; 41 down), lncRNAs (57 up-regulated and 52 down regulated; (mRNAs) 167 up-regulated; 162 down regulated); 2 (mtDNA A3243G TK2 mutation of mitochondrial and nuclear genes mutation) muscle tissue shows that the two total 41 a total of mRNAs.2. of the difference through our 6 selected by miRNAs (miR-6089, miR-27b-3p, miR-214-3p, MI, R-150-5p, let-7e-5p and miR-145-5p), 5 IncRNAs (LINC01405, SNHG12, R, P11-403P17.4, CTC-260E6.6 and RP11-357D18.1) and 6 mRNAs (PDK4, CDKN1A, ATP2A2, SOD3, DDIT4 and EEF1A1) RT-PCR verification the results show, with high-throughput sequencing of the two are basically the same. The second part of mitochondrial myopathy muscle differences of RNA molecular interaction network construction and key ncRNA (miRNA/lncRNA) screening research objective to construct mRNA and non RNA encoding (miRNA/lncRNA) between control network The network, and the signal pathway involved in the establishment of regulation mechanism hypothesis. Research methods on the difference of ncRNA (miRNA/lncRNA) by Bioinformatics (cluster analysis and GO and Pathway), correlation analysis to determine the expression in different key types of mutation in patients with mitochondrial myopathy of the occurrence and development of non encoding R NA (miRNA/lncRNA) co expression of molecular interactions. The interaction network including miRNA-mRNA, miRNA-lncRNA and IncRNA-mRNA 22 interaction network as well as miRNA-mRNA-lncRNA and mRNA-miRNA-lncRNA three molecular interaction network. Computing the topological properties of the network and the nodes using Cytoscape software by miRecords. The predicted target gene differences in miRNA, GO functional annotation of different target genes, KEGG pathway analysis. The experimental results of 1. for the first time bioinformatics predicted target gene construct the interaction network of co expression of miRNA-mmRNA-lncRNA and mRNA-mi RNA-lncRNA, Selected key ncRNA molecular regulatory network.2.GO analysis showed that the difference of miRNA/lncRNAs target gene set and mmRNAs set the overall difference of biological processes common enrichment analysis focused on extracellular matrix molecules, muscle contraction and protein binding molecules. In addition, mRNAs was a GO entry enrichment were focused on small molecule metabolite expression total difference signal molecular transduction and transcription process; significant GO classification entry miRNA mRNA network on behalf of the target for signal transduction, apoptosis related molecules; analysis of corresponding lncRNA target mRNA associated GO enrichment entry into small molecule metabolism and muscle contraction related molecules in.KEGG pathway analysis showed that the imbalance of mRNA mainly involved in small molecule metabolism, extracellular matrix formation, muscle contraction, protein binding, signal transduction, transcription, signal pathways and biological functions. In conclusion, MELAS in patients with MT The expression of downstream differences caused by mutations in the DNA A3243G ncRNA spectrum by immune response, small molecule metabolism, signal transduction, apoptosis and development process of the occurrence and development of a variety of signaling pathways involved in mitochondrial myopathy. The third part ncRNA of mitochondrial myopathy (miRNA/lncRNA) objective to study the clinical application value of potential biological markers of this topic for further analysis of key ncRNAs the diagnostic efficacy of muscle tissue and serum of MELAS patients, looking for mitochondrial potential biomarker for diagnosis of ncRNA. The research object and methods 1. a total of 54 cases of MELAS (A3243G mutation) and 49 cases of normal control group independent muscle samples tested again A3243G mutation in patients with MELAS high throughput results; 2. (a total of 34 cases of MELAS patients with A3243G mutations) and 34 cases of control independent verification of A3243G mutations in MELAS patients serum samples from 22 cases of miR-27b-3p expression; 3. (non MELAS patients with A3243G mutation) 34 cases of control independent muscle and serum samples to verify the non A3243G mutation in patients with MELAS miR-27b-3p expression; analysis of muscle tissue ncRNAs and serum miR-27b-3p for the diagnosis of patients with MELAS sensitivity, specificity 3. software; expression levels and various clinical parameters such as age and gender on serum miRNA-27b-3p, BMI, NMDAS, disease duration, blood lactic acid and muscle A3243G relationship between the mutation rate were analyzed. The experimental results confirmed that 1. of our 4 ncRNAs (miR-6089, miR-27b-3p, miR-214-3p and LINC01405) muscle expression were significantly reduced, while the 7 ncRNAs (miR-150-5p, let-7e-5p, miR-145-5p, SNHG12, RP11-403P17.4, CTC-260E6.6 and RP11-357D18.11) were significantly up-regulated in MELAS muscle tissue; ROC curve analysis showed that the key of ncRNA the selected muscle tissue miR-27b-3p with miR- key sensitivity and specificity for the diagnosis of MELAS patients with the highest.2. 27b-3p in MELAS patients (A3243G and non A3243G mutation) in serum and muscle decreased the expression of.3.ROC curve analysis showed that, compared with the diagnostic value of serum lactic acid miR-27b-3p is better for MELAS. Correlation analysis showed that there is significant negative correlation between serum miR-27b-3p and lactate and NMDAS. Conclusion we first found that MELAS A3243G mutation ncRNAs unique muscle tissue patients (miRNAs/lncRNAs) expression profile; for the first time to construct miRNA-mRNA-lncRNA and mRNA-miRNA-lncRNA co expression interaction network, selected key ncRNA molecular network in serum miR-27b-3p has better diagnostic value than lactic acid for MELAS, and through the functional enrichment analysis identified non encoding RNA molecules involved in mitochondrial myopathy may be mainly through signal transduction, oxidative stress. Apoptosis, provide a theoretical basis for the further exploration of the corresponding ncRNA molecular function.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R596

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本文編號：1527532