非酒精性脂肪性肝纤维化小鼠肝组织中异常表达miRNAs的鉴定及功能研究
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摘要
非酒精性脂肪性肝病（non-alcoholic fatty liver disease，NAFLD）是指除外酒精和其他明确因素所致的以肝实质细胞脂肪变性和脂肪贮积为主要特征的临床病理综合征，是与胰岛素抵抗和遗传易感性密切相关的获得性代谢应激性肝损伤。NAFLD疾病谱包括单纯性脂肪肝（simple fattyliver，SFL）、非酒精性脂肪性肝炎（non-alcoholic steatohepatitis，NASH）、肝纤维化及相关肝硬化。其中，NASH为该病进展的重要病理阶段，以肝细胞脂肪变伴有坏死性炎症和/或纤维化为主要特征，进一步可进展为肝硬化，甚至肝细胞癌。
近年来，随着生活方式和饮食结构的改变，NAFLD的发病率逐年增高，并趋于年轻化。NAFLD患病率为欧美及澳大利亚等发达国家慢性肝病的首位，也是肝酶学异常的首要病因，普通人群患病率高达20%～40%。亚太地区NAFLD患病率为12%～24%，其中1/3～1/2为NASH，后者有15%～25%在10年内可进展为肝硬化，而非酒精性脂肪性肝硬化患者原发性肝细胞癌、肝功能衰竭的发生率高达30%～40%。近年来我国NAFLD患病率逐年上升，城市人口患病率达15.35%～31.3%。据2010年城市人口健康调查结果，男性脂肪肝患病率为19%，女性为15%，分别为危害健康第一位和第九位的危险因素。南月敏等对健康体检的综合医院医务工作者连续随访5年，患病率达31.6%。朗振为等对不明原因肝炎病理学研究发现NASH占15.5%。NAFLD已成为危害人类健康和生活质量的主要肝脏疾病之一。非酒精性脂肪性肝炎/肝纤维化为NAFLD进展中的关键环节。但是，目前非酒精性脂肪性肝纤维化的发病机制尚不十分清楚，亦缺乏准确判断病情的指标及特异有效的防治措施，对于非酒精性脂肪性肝纤维化发病机制、基因诊断标志及治疗新靶点的探索十分必要和迫切。
MicroRNAs（miRNAs）作为一种重要的转录后调节因子在肝脏的生成、分化及代谢过程中发挥着重要的调控作用。Sanyal等发现在NASH患者肝组织中miR-34a和miR-146b表达上调、miR-122下调。其中，miR-122可通过负性调控固醇调节元件结合蛋白-1c（Sterol ResponsiveElement Binding Protein-1c， SREBP-1c）、脂肪酸合成酶（fatty acidsynthetase，FAS）和HMG-CoA还原酶（HMG CoA reductase，HMGCR）的表达，参与肝细胞脂肪酸的生物合成过程。而miR-33a与miR-33b也可通过调控固醇调节元件结合蛋白（Sterol Responsive Element BindingProtein，SREBP）转录因子的表达，参与胆固醇和脂肪代谢的调控。miR-34a通过抑制酰基辅酶A合成酶长链家族成员1（acyl-CoA synthetaselong-chain family member1，ACSL1）的表达、去乙酰化酶-1的表达调控腺苷一磷酸激酶（AMP kinase）和HMG-CoA还原酶的脱磷酸化，参与胆固醇的生物合成过程。另外，miR-467b通过负性调控其靶蛋白肝脏脂蛋白酶表达，减少游离脂肪酸的生成，参与胰岛素抵抗（insulin resistance，IR）导致脂质沉积过程的调控。miR-103与miR-107可通过调控其靶基因胰岛素受体小窝蛋白-1（Caveolin-1，CAV1），调节胰岛素敏感性、维持血糖平衡。作为转录后调节因子，特定的miRNAs可能参与NAFLD的发生发展，但其具体调控机制有待进一步研究。
本研究首先应用胆碱-蛋氨酸缺乏（methionine and choline deficience，MCD）饮食8周建立非酒精性脂肪性肝纤维化动物模型，应用基因芯片技术检测小鼠肝组织中miRNAs表达谱的变化；采用实时定量PCR方法验证各组小鼠肝组织中差异表达的miRNAs，筛选与非酒精性脂肪性肝纤维化有关表达变化最明显的miRNA；通过生物信息学软件预测miRNA的靶基因，进行功能分类和信号通路预测；采用实时定量PCR及westernblot方法检测miRNA靶基因及靶蛋白在非酒精性肝纤维化小鼠肝组织中的表达变化。构建含有目标miRNA靶基因3′-UTR的报告载体，采用荧光素酶实验（Luciferase Reporter Assay）进一步验证miRNA对其靶基因的调控作用。靶向调控LX-2细胞中关键miRNA表达，观察非酒精性脂肪性肝纤维化发病关键基因表达变化，深入阐明miRNAs在非酒精性脂肪性肝纤维化发病过程中的作用、其作用的靶基因及主导分子信号通路，为非酒精性脂肪性肝纤维化的临床防治提供新的靶点及依据。
第一部分：非酒精性脂肪性肝纤维化小鼠肝组织中异常表达miRNAs的筛选与验证
目的：筛选非酒精性脂肪性肝纤维化形成过程中异常表达的miRNAs。
方法：采用MCD饮食喂养6~7周龄清洁级健康雄性C57BL/6J小鼠8周，建立非酒精性脂肪性肝纤维化模型，以胆碱蛋氨酸充足饲料喂养对照组小鼠。酶联免疫法测定小鼠血清中丙氨酸氨基转移酶（alanineaminotransferase， ALT）及天门冬氨酸氨基转移酶（aspartateaminotransferase，AST）的水平；HE及Masson染色观察肝脏组织脂肪变性、炎症活动及纤维化程度，并进行组织学评分；应用Qiagen公司的MiRNeasy Mini Kit试剂盒提取部分新鲜肝脏组织的总RNA，分光光度计测定RNA质量，1%琼脂糖凝胶电泳测定RNA纯度，采用AffymetrixmiRNA基因表达谱芯片GeneChip miRNA2.0Array进行杂交，进行miRNAs基因芯片检测，筛查在非酒精性脂肪性肝纤维化小鼠肝组织中异常表达的miRNAs并进行实时定量PCR验证。
结果：
1.一般情况：造模过程中，对照组小鼠活泼，毛发有光泽，体重逐渐增加；MCD模型组小鼠精神萎靡，少动，毛发紊乱、无光泽，摄食明显减少，体重增长缓慢，随着时间推移，模型组小鼠出现被毛脱落现象。
2.肝指数变化：MCD模型组小鼠体重显著低于对照组（P <0.05），肝湿重较对照组无明显变化（P>0.05），肝指数（肝湿重/体重×100%）较对照组明显升高（P <0.05）。
3.各组小鼠血生化指标的结果：MCD组血清中ALT及AST的水平明显高于对照组，差异有统计学意义（P <0.05）。
4.各组小鼠肝组织HE及Masson染色的结果：MCD组小鼠肝细胞出现重度脂肪变性，并伴点状和灶状坏死、炎性细胞浸润、汇管区纤维组织增生及窦周纤维化等。
5. miRNAs芯片结果：与对照组相比，MCD模型组有47个差异表达的miRNAs（P <0.05），其中15个变化在2倍之上。mmu-let-7i、mmu-mir-155、 mmu-mir-199a-5p、 hsa-mir-34a、 mmu-mir-221、mmu-mir-200c、 mmu-mir-297c、 mmu-mir-713、 mmu-mir-190b、mmu-mir-678，10个miRNAs在MCD模型组的肝脏组织中表达明显上调；mmu-mir-122、mmu-mir-103、mmu-mir-146、mmu-mir-101a、mmu-mir-466j，5个miRNAs在MCD模型组的肝脏组织中表达明显下调。
6.综合本实验基因芯片结果及相关文献报道，选择miR-122、miR-221及miR-199a-5p进行实时荧光定量RT-PCR验证，结果显示，与正常对照组相比，MCD组小鼠肝组织中miR-199a-5p的表达明显增高、miR-122明显降低（P <0.05）。
结论：
1在MCD饮食诱导的小鼠非酒精性脂肪性肝纤维化动物模型中，肝组织miRNAs表达谱明显改变，表明差异表达的miRNAs参与非酒精性脂肪性肝炎／肝纤维化的发生发展。
2miR-199a-5p可能为非酒精性脂肪性肝纤维化发生发展的重要调控基因之一。第二部分：miR-199a-5p生物信息学分析及靶基因验证
目的：探讨miR-199a-5p在非酒精性脂肪性肝纤维化发病中可能的作用及其潜在的作用靶点。
方法：动物分组及标本取得同第一部分。应用TargetScan6.2、miRanda及PITA软件共同预测miR-199a-5p的靶基因，应用DAVID软件分析miR-199a-5p可能的生物学功能及参与调节的信号通路。采用实时荧光定量RT-PCR及Western blot法分析在非酒精性脂肪性肝纤维化小鼠肝组织中miR-199a-5p靶基因mRNA及蛋白水平的表达变化情况。
结果：
1.生物信息学分析结果：GO分析结果显示，miR-199a-5p参与了转录调控、转录因子复合物组成、丝苏氨酸蛋白激酶活化等多种生物学调控；KEGG分析显示，miR-199a-5p参与胰岛素信号通路、Wnt信号通路、MAPK信号通路等多条通路的调控。
2. miR-199a-5p的靶基因预测：应用三种miroRNA靶基因预测软件（TargetScan、PITA和miRanda）共同预测miR-199a-5p的靶基因，得到26个与miR-199a-5p相关的基因。
3.各组小鼠肝脏组织中miR-199a-5p靶基因核受体共抑制剂1（nuclear receptor corepressor，NCOR1)的表达情况：与对照组相比，MCD组肝组织中NCOR1蛋白的表达明显减少（p <0.05），与miR-199a-5p的表达负相关；模型组与对照组肝组织NCOR1mRNA表达无明显差异。
结论：
1生物信息学分析结果示，miR-199a-5p参与胰岛素信号通路、Wnt信号通路、MAPK信号通路等多条与非酒精性脂肪性肝纤维化发生发展相关信号通路的调控。
2在MCD饮食诱导的肝纤维化模型中，miR-199a-5p可能通过调控NCOR1蛋白的表达参与疾病的发生与进展。
第三部分：miR-199a-5p与其靶基因NCOR1的相互作用机制
目的：验证miR-199a-5p与NCOR13′-UTR靶向调节关系。
方法：根据Gene Bank公布的人NCOR1基因3′-UTR序列设计含有miR-199a-5p结合位点的NCOR13′-UTR PCR引物，并在引物中加入Not I和Xho I两个限制性酶切位点。将扩增的NCOR13′-UTR序列插入携带萤火虫荧光与海肾荧光双荧光报告基因的psiCHECKTM-2质粒的Xho I和Not I位点之间，构建NCOR13′-UTR-luciferase报告载体，将NCOR13′-UTR-luciferase报告载体与pre-miR-199a-5p共同转染至HEK293细胞，48h后检测荧光强度，并计算萤火虫荧光与海肾荧光的比值。分析miR-199a-5p对NCOR1的靶向调控作用。
结果：
1.查询“Ensembl Mouse database”，得到全长2202bp的NCOR13′-UTR序列，其中，在NCOR13′-UTR的790bp～796bp处存在可能与miR-199a-5p结合的位点。
2.从血液中提取基因组DNA经PCR扩增后，扩增产物在1.0%的琼脂糖凝胶上进行电泳显示出一条约2000bp特异性片段，实验与预期结果相符。重组质粒p3′UTR-NCOR1经上海生工进一步测序鉴定质粒构建正确。
3.将构建的p3′UTR-NCOR1质粒与pre-miR-199a-5p或阴性对照序列共转染至HEK293细胞，48h后检测荧光素酶活性。结果显示，与对照组相比，miR-199a-5p可以直接作用于NCOR13′-UTR序列，并可抑制荧光素酶活性。。
结论：miR-199a-5p可靶向作用于NCOR13′-UTR，负性调控NCOR1的表达。第四部分：miR-199a-5p靶向调控对LX-2细胞活力与功能的影响
目的：靶向调控LX-2细胞中miR-199a-5p表达，观察非酒精性脂肪性肝纤维化发病关键基因表达变化，深入阐明miRNAs在非酒精性脂肪性肝纤维化发病过程中的作用、其作用的靶基因及主导分子信号通路，为非酒精性脂肪性肝纤维化的临床防治提供新的靶点及科学依据。
方法：应用人肝星状细胞系LX-2细胞，培养于含10%胎牛血清、100U/ml青霉素和100μg/ml链霉素的DMEM培养液中，37℃、5%CO2孵箱中进行培养。当细胞融合度达到60%左右时，用不同浓度miR-199a-5p的模拟物及抑制剂转染LX-2细胞，采用MTT法测定各组细胞活力，选择合适的转染浓度。采用实时荧光定量PCR及Western-blot法分析miR-199a-5p及其靶基因NCOR1mRNA及蛋白的表达情况。同时应用Western-blot方法观察miR-199a-5p对非酒精性脂肪性肝纤维化发病关键因子的影响。
结果：
1. MTT结果显示，转染不同浓度的对照序列对LX-2细胞活力无明显影响；转染100nM及150nM的miR-199a-5p模拟物后，LX-2细胞活力明显降低，而转染50nM的模拟物，则无明显变化；转染100nM及150nM的miR-199a-5p抑制剂后，LX-2细胞活力明显增强，而50nM的miRNA抑制剂则对细胞活力无明显影响；
2.各转染组与未转染组LX-2细胞相比，miR-199a-5p的表达量有不同程度改变，miR-199a-5p靶基因NCOR1mRNA的表达量则无明显变化。与未转染组比较，NC组miR-199a-5p的表达量无明显变化，Mimic组miR-199a-5p的表达量显著增高（P <0.01），Inhibitor组miR-199a-5p表达量明显降低（P <0.05）；
3.转染后细胞miR-199a-5p靶蛋白NCOR1及过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor gamma，PPARγ）蛋白的表达量有不同程度改变，与未转染组比较，NC组NCOR1和PPARγ蛋白的表达量均无明显变化，Mimic组NCOR1蛋白的表达量显著降低，PPARγ蛋白的表达量明显增高（P <0.05），Inhibitor组NCOR1蛋白表达量明显增高，PPARγ蛋白的表达量则降低（P <0.05）。PPARγ蛋白与转染各组细胞中NCOR1蛋白的表达变化相反；
4.转染后细胞肝纤维化相关蛋白转化生长因子β1（transforminggrowth factor beta，TGFβ1）、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）的表达量有不同程度改变，与未转染组比较，NC组TGF-β1及α-SMA蛋白的表达量无明显变化，Mimic组TGF-β1及α-SMA蛋白的表达量显著降低（P <0.05），Inhibitor组TGF-β1及α-SMA蛋白表达量明显增高（P <0.05）。TGF-β1及α-SMA蛋白与转染各组细胞中NCOR1蛋白的表达变化一致。
结论：
1转染miR-199a-5p模拟物或抑制剂可引起LX-2细胞活力的改变，提示miR-199a-5p可能通过影响HSC细胞增殖及活化参与非酒精性脂肪性肝纤维化的发病过程。
2miR-199a-5p可能通过负性调控NCOR1蛋白表达影响PPARγ蛋白活性，参与肝星状细胞的激活。
3靶向调控miR-199a-5p表达可阻止或逆转肝纤维化的发生与进展。
Nonalcoholic fatty liver disease (NAFLD), a hepatic manifestation of themetabolic syndrome, has similar pathological changes with alcoholic liverdisease, but the patients suffer from it without excessive alcohol intake andother definite etiological factor. It has been reported that NAFLD is relatedwith insulin resistance and hereditary susceptibility. The disease spectrum ofNAFLD includes simple fatty liver (SFL) non-alcoholic steatohepatitis(NASH), NASH-related hepatic fibrosis and cirrhosis, hepatocellularcarcinoma. NASH is the key stage in the spectrum of NAFLD which ischaracterized by non-alcoholic steatohepatitis／fibrosing steatohepatitis andcan progress to fibrosis, cirrhosis, liver failure, and hepatocellular carcinoma.
With the changes of life style and diet, the prevalence of NAFLDincreases rapidly in modern society. NAFLD has became the most commonchronic liver diseases in the United States and worldwide. Recent studiesusing different methodologies indicate that in the general population theprevalence of NAFLD is approximately20%~40%. Of greater concern is therecognition that cirrhosis and liver-related deaths occur in approximately20%and8%of these NASH patients over a10-year period. Among more affluentregions of China, the community prevalence of non-alcoholic fatty liverdisease (NAFLD) is approximately15%. With the increasing pandemic ofobesity, the prevalence of NAFLD has approximately doubled in the pastdecade. Nonalcoholic steatohepatitis/fibrosis, one type of a larger spectrum ofnonalcoholic fatty liver disease, is now recognized as a cause of progressivefibrotic liver disease with adverse clinical sequelae. However, thepathogenesis of nonalcoholic steatohepatitis/fibrosis leading to diseaseprogression remains poorly understood and definitive therapies for thesepatients are lacking．
MicroRNAs (miRNAs) are a class of important factors in the regulation ofprotein expression at the posttranscriptional level. miRNAs are associatedwith many pathophysiologic events such as hepatic tissue development anddifferentiation, cell proliferation, apoptosis. In the study of Sanyal and hiscolleagues, they found that NASH was associated with altered hepaticmiRNAs expression including the upregulation of miR-34a and miR-146b,downregulation of miR-122. miR-122has been found to be required for thelipid homeostasis through the regulation of sterol-regulatory element-bindingprotein (SREBP), fatty acid synthetase (FAS) and HMG CoA reductase(HMGCR). miR-33a and miR-33b have a crucial role in controllingcholesterol and lipid metabolism in concert with their host genes, the SREBPtranscription factors. miR-34family has been found to control cholesterolsynthesis by targeting acyl-CoA synthetase long-chain family member1(ACSL1) and inhibiting sirtuin-1with downstream dephosphorylation ofAMP kinase and HMGCR. Downregulation of miR-467b is involved in thedevelopment of hepatic steatosis by modulating the expression of its targethepatic lipoprotein lipase (LPL). miR-103and miR-107regulate insulin andglucose homeostasis through insulin receptor CAV1. Dysregulation ofmiRNAs may contribute to the development of NAFLD, however, the exactroles of miRNAs in the pathogenesis of NASH are not fully understood.
In this study， we aimed to establish a non-alcoholic fibrosingsteatohepatitis model caused by methionine and choline deficient (MCD) diet8weeks. Serum alanine aminotransferase (ALT) and aspartateaminotransferase (AST), reliable indicators of liver inflammatory injury, weremeasured. Histological scoring for NAFLD was carried out to evaluate hepaticsteatosis and fibrosis. Using microarray experiments, we found that feedingC57BL/6J mice a methyl-deficient diet8weeks to induce non-alcoholicfibrosing steatohepatitis could result in aberrant expression of miRNAs. Toclarify the roles of miRNAs in the process of non-alcoholic fibrosingsteatohepatitis and its molecular mechanism, and provided a basis for NAFLDprevention and the development of miRNA targeting drugs, the different expressed miRNAs were performed with the bioinformatic analysis. Thederegulated miRNA and miRNA targeting gene was validated usingquantitative real-time PCR and western blot assay.
To study the role of these miRNAs in the NASH, levels of miR-122,miR-199a-5p and miR-221were determined in the livers extracted from micefed MCD and control diets, using quantitative real-time PCR experiments.To investigate the potential interaction experimentally, the wild-type3′-UTRof NCOR1gene was subcloned downstream of the Renilla luciferase codingsequence and cotransfected into293T cells with the pre-miR-199a-5p andnegative control sequence. The luciferase activity was assayed at48hpost-transfection. To examine the effect of miR-199a-5p, we transfected itsmimic, inhibitor and Ncontrol (NC) into a hepatic stellate cellline named LX-2cells and observed the expression of several fibrosis-associated genes,providing new insights to the pathogenesis of NASH.
Part1：Alterations and validation of microRNA expression profile innon-alcoholic fibrosing steatohepatitis model
Objective: To establish a non-alcoholic fibrosing steatohepatitis modeland study the expression profiles of fibrosis related miRNAs.
Methods: Experimental non-alcoholic fibrosing steatohepatitis modelswere established by feeding mice with MCD diet8weeks (MCD group).Control mice were fed with choline–methionine supplemented diet (controlgroup). Serum alanine aminotransferase (ALT) and aspartate aminotransferase(AST) were measured by enzymic method with automatic biochemistryanalyzer. Hepatic inflammation and fibrosis were graded under HE andMasson staining. The stage of steatosis and fbrosis was assessed according tothe guidelines for diagnosis and treatment of non-alcoholic fatty liver diseases.RNAs from experimental mice hepatic tissue were isolated with a MiRNeasyMini Kit (Qiagen). The miRNAs profiles were determined using GeneChipmiRNA2.0Array (Affymetrix). The result of microarray was validated byreal-time quantitative PCR. Results:
1. General observation: The mice of MCD group showed less activity,sikness and dim hair than the mice of Control group.
2. Liver index changes: The body weights of MCD group mice weresignificantly lower than the control group at the8th week body (P <0.05).Liver wet weight of MCD group mice had no significant change comparedwith the control group (P>0.05). Liver index (liver weight/body weight×100%) was significantly higher than the control group (P <0.05).
3. Serum transaminases in C57BL/6J mice fed control and MCD diet for8weeks: MCD diet increased the level of serum transaminases, both ALT andAST.
4. HE and Masson staining of liver sections of C57BL/6J mice fedcontrol and MCD diets: MCD treated animals showed disordered lobulestructure, severe macrosteatosis, focal hepatocyte necrosis, inflammatoryinfiltration, portal fibrosis and fibrous septum.
5. The result of GeneChip miRNA2.0Array showed that there were47deregulated miRNAs in mice hepatic tissue treated with MCD or control diet,in which15miRNAs changed above2times. The result of microarray showedthat10miRNAs were upregulated in MCD group including mmu-let-7i,mmu-mir-155, mmu-mir-199a-5p, mmu-mir-34a, mmu-mir-221,mmu-mir-200c, mmu-mir-297c, mmu-mir-713, mmu-mir-190b, mmu-mir-678;5miRNAs downregulated including mmu-mir-122, mmu-mir-103,mmu-mir-146, mmu-mir-101a and mmu-mir-466j.
6. Refer to the relevant literature and the results of microarray, weselected miR-122, miR-199a-5p and miR-221to perform quantitativereal-time PCR experiments. We found that MCD diet increased miR-199a-5pand decreased miR-122in mice livers (P <0.05).
Conclusion:
1. MCD diet could induce characteristic pathology of steatosis, mixedhepatic inflammatory and fibrosis, representing a successful model ofnon-alcoholic fibrosing steatohepatitis.
2. Non-alcoholic fibrosing steatohepatitis in mice significantly change the expression of miRNA profiles, which may be involved in non-alcoholicsteatohepatitis/fibrosing steatohepatitis.
3. We found a dramatic upregulation of miR-199a-5p in the non-alcoholicfibrosing steatohepatitis mouse model. These data indicated that dysregulatedexpression of miR-199a-5p might be critical in non-alcoholic fibrosingsteatohepatitis pathogenesis.
Part2：Functional analysis and evaluation of miR-199a-5p target genes
Objective: To searche the target genes of miR-199a-5p and detect therole of miR-199a-5p in non-alcoholic fibrosing steatohepatitis etiology.
Methods: The DAVID (Database for Annotation, Visualization andInterrogated Discovery) bioinformatic resources were performed to analysisthe function of miR-199a-5p. Three most commonly prediction algorithmsTargetScan6.2, miRanda and PITA was used to find the exact target genes ofmiR-199a-5p. Target gene of miR-199a-5p in the experimental mice hepatictissue was evaluated by real-time quantitative PCR and western blot assay.
Results:
1. Enrichment analysis on predicted target genes: Result obtained fromGO-term analysis revealed a relationship between miR-199a-5p and regulationof transcription, transcription factor complex, protein serine/threonine kinaseactivity, and others. KEGG analysis showed the involvement of miR-199a-5pin a variety of pathways, including insulin signaling, Wnt signaling andMAPK signaling pathways.
2. Prediction of miR-199a-5p target genes:26target genes ofmiR-199a-5p were predicted by all of the three algorithms. NCOR1(nuclearreceptor corepressor1), one of the predicted target genes, was reported to berequired for the integration of inflammatory and anti-inflammatory signals,which is associated with the pathogenesis of non-alcoholic fibrosingsteatohepatitis.
3. Expression of NCOR1in hepatic tissues: Increased hepaticmiR-199a-5p upon MCD diet is associated with decreased NCOR1expressionat the protein, but not mRNA level.
Conclusion:
1. Results of bioinformatic analysis showed that miR-199a-5p may takepart in the pathogenesis of non-alcoholic fibrosing steatohepatitis throughinsulin signaling, Wnt signaling and MAPK signaling pathways.
2. miR-199a-5p may regulate the translation of NCOR1mRNA.
Part3：The interaction between miR-199a-5p and its target gene NCOR1
Objective: To investigate the potential interaction between miR-199a-5pand its target gene NCOR1mRNA3′-UTR experimentally.
Methods: The3′-UTR of NCOR1was PCR amplified and cloneddownstream of the renilla luciferase stop codon in psiCHECKTM-2vector,giving rise to NCOR13′-UTR-luciferase. This construct luciferase reportervectors were transfected into HEK293cells with pre-miR-199a-5p ornegative control. Cells were harvested48h after transfection and assayed withDual Luciferase Reporter Assay System according to the manufacturer’sinstructions.
Results:
1. From “Ensembl Mouse database”, we obtained a full length2202bpNCOR13'-UTR sequences, where may be binding sites with miR-199a-5p inNCOR13'-UTR of790bp~796bp place.
2. Genomic DNA was extracted from the blood and amplified by PCRassay. The PCR products showed a specific treaty2000bp fragment on a1.0%agarose gel electrophoresis which was in line with expectations. Therecombinant plasmid p3'UTR-NCOR1was confirmed by Shanghai Sangonfurther sequencing of plasmid.
3. The luciferase activity assays at48h post-transfection showed that theoverexpression of miR-199a-5p could decrease relative luciferase activitysignificantly.
Conclusion: miR-199a-5p may play important roles in the pathogenesisof non-alcoholic fibrosing steatohepatitis by influencing protein translation ofNCOR1.
Part4The regulation of NCOR1and fibrosis-related genes bymiR-199a-5p was examined by silencing or overexpressing this miRNA inLX-2cells
Objective: To observe the expression of several fibrosis-associated genesby regulating the expression of miR-199a-5p in LX-2cells, further detect therole of miR-199a-5p in the pathogenesis of non-alcoholic fibrosingsteatohepatitis and prevention and provide a new target basis to treatnon-alcoholic fibrosing steatohepatitis.
Methods: Human hepatic stellate cells (LX-2cells) were cultured inDMEM supplemented with10%fetal bovine serum,100U/mL penicillin, and100g/L streptomycin at37°C in a humidified environment with5%CO2.Cells were grown to60%confluency prior to transfection with differentconcentrations of miR-199a-5p mimic, inhibitor, or their correspondingcontrols using Lipofectamine2000. Cell viability was determined by MTTassay in each group to select the appropriate concentration of transfection.Transfected cells were cultured for48hours and harvested for total RNA andprotein extraction. The samples were stored at80°C for q-PCR or Westernblot assays.
Results:
1. The result of MTT assay showed that transfection with controlsequences of different concentrations without significant effect on cellviability; transfection of100nM and150nM miR-199a-5p mimic, cell viabilitywas significantly reduced, but no significant changes with50nM mimic;transfection of100nM and150nM miR-199a-5p inhibitor, cell viability wassignificantly enhanced, but no significant changes with50nM inhibitor.
2. Compared with the control group, the expression of miR-199a-5pdramatically increased in mimic group and significantly reduced in inhibitorgroup. NCOR1mRNA was not affected by miR-199a-5p mimic, inhibitor andNcontrol (NC).
3. NCOR1protein was decreased by miR-199a-5p overexpression andincreased by miR-199a-5p knockdown (p <0.05) in LX-2cells, while PPAR γprotein expressed inversely of NCOR1protein in each group.
4. miR-199a-5p inversely correlated with the expression of severalfibrosis-associated genes, including transforming growth factor-β (TGF-β) andα-smooth muscle actin (α-SMA).
Conclusion:
1. miR-199a-5p may influence cell proliferation or apoptosis involved inthe pathogenesis of non-alcoholic fibrosing steatohepatitis.
2. miR-199a-5p may affect PPAR γ involved in the activation of hepaticstellate cells through negative regulation the expression of NCOR1.
3. Targeted regulation of miR-199a-5p expression may prevent or reversethe occurrence and development of hepatic fibrosis.
近年来，随着生活方式和饮食结构的改变，NAFLD的发病率逐年增高，并趋于年轻化。NAFLD患病率为欧美及澳大利亚等发达国家慢性肝病的首位，也是肝酶学异常的首要病因，普通人群患病率高达20%～40%。亚太地区NAFLD患病率为12%～24%，其中1/3～1/2为NASH，后者有15%～25%在10年内可进展为肝硬化，而非酒精性脂肪性肝硬化患者原发性肝细胞癌、肝功能衰竭的发生率高达30%～40%。近年来我国NAFLD患病率逐年上升，城市人口患病率达15.35%～31.3%。据2010年城市人口健康调查结果，男性脂肪肝患病率为19%，女性为15%，分别为危害健康第一位和第九位的危险因素。南月敏等对健康体检的综合医院医务工作者连续随访5年，患病率达31.6%。朗振为等对不明原因肝炎病理学研究发现NASH占15.5%。NAFLD已成为危害人类健康和生活质量的主要肝脏疾病之一。非酒精性脂肪性肝炎/肝纤维化为NAFLD进展中的关键环节。但是，目前非酒精性脂肪性肝纤维化的发病机制尚不十分清楚，亦缺乏准确判断病情的指标及特异有效的防治措施，对于非酒精性脂肪性肝纤维化发病机制、基因诊断标志及治疗新靶点的探索十分必要和迫切。
MicroRNAs（miRNAs）作为一种重要的转录后调节因子在肝脏的生成、分化及代谢过程中发挥着重要的调控作用。Sanyal等发现在NASH患者肝组织中miR-34a和miR-146b表达上调、miR-122下调。其中，miR-122可通过负性调控固醇调节元件结合蛋白-1c（Sterol ResponsiveElement Binding Protein-1c， SREBP-1c）、脂肪酸合成酶（fatty acidsynthetase，FAS）和HMG-CoA还原酶（HMG CoA reductase，HMGCR）的表达，参与肝细胞脂肪酸的生物合成过程。而miR-33a与miR-33b也可通过调控固醇调节元件结合蛋白（Sterol Responsive Element BindingProtein，SREBP）转录因子的表达，参与胆固醇和脂肪代谢的调控。miR-34a通过抑制酰基辅酶A合成酶长链家族成员1（acyl-CoA synthetaselong-chain family member1，ACSL1）的表达、去乙酰化酶-1的表达调控腺苷一磷酸激酶（AMP kinase）和HMG-CoA还原酶的脱磷酸化，参与胆固醇的生物合成过程。另外，miR-467b通过负性调控其靶蛋白肝脏脂蛋白酶表达，减少游离脂肪酸的生成，参与胰岛素抵抗（insulin resistance，IR）导致脂质沉积过程的调控。miR-103与miR-107可通过调控其靶基因胰岛素受体小窝蛋白-1（Caveolin-1，CAV1），调节胰岛素敏感性、维持血糖平衡。作为转录后调节因子，特定的miRNAs可能参与NAFLD的发生发展，但其具体调控机制有待进一步研究。
本研究首先应用胆碱-蛋氨酸缺乏（methionine and choline deficience，MCD）饮食8周建立非酒精性脂肪性肝纤维化动物模型，应用基因芯片技术检测小鼠肝组织中miRNAs表达谱的变化；采用实时定量PCR方法验证各组小鼠肝组织中差异表达的miRNAs，筛选与非酒精性脂肪性肝纤维化有关表达变化最明显的miRNA；通过生物信息学软件预测miRNA的靶基因，进行功能分类和信号通路预测；采用实时定量PCR及westernblot方法检测miRNA靶基因及靶蛋白在非酒精性肝纤维化小鼠肝组织中的表达变化。构建含有目标miRNA靶基因3′-UTR的报告载体，采用荧光素酶实验（Luciferase Reporter Assay）进一步验证miRNA对其靶基因的调控作用。靶向调控LX-2细胞中关键miRNA表达，观察非酒精性脂肪性肝纤维化发病关键基因表达变化，深入阐明miRNAs在非酒精性脂肪性肝纤维化发病过程中的作用、其作用的靶基因及主导分子信号通路，为非酒精性脂肪性肝纤维化的临床防治提供新的靶点及依据。
第一部分：非酒精性脂肪性肝纤维化小鼠肝组织中异常表达miRNAs的筛选与验证
目的：筛选非酒精性脂肪性肝纤维化形成过程中异常表达的miRNAs。
方法：采用MCD饮食喂养6~7周龄清洁级健康雄性C57BL/6J小鼠8周，建立非酒精性脂肪性肝纤维化模型，以胆碱蛋氨酸充足饲料喂养对照组小鼠。酶联免疫法测定小鼠血清中丙氨酸氨基转移酶（alanineaminotransferase， ALT）及天门冬氨酸氨基转移酶（aspartateaminotransferase，AST）的水平；HE及Masson染色观察肝脏组织脂肪变性、炎症活动及纤维化程度，并进行组织学评分；应用Qiagen公司的MiRNeasy Mini Kit试剂盒提取部分新鲜肝脏组织的总RNA，分光光度计测定RNA质量，1%琼脂糖凝胶电泳测定RNA纯度，采用AffymetrixmiRNA基因表达谱芯片GeneChip miRNA2.0Array进行杂交，进行miRNAs基因芯片检测，筛查在非酒精性脂肪性肝纤维化小鼠肝组织中异常表达的miRNAs并进行实时定量PCR验证。
结果：
1.一般情况：造模过程中，对照组小鼠活泼，毛发有光泽，体重逐渐增加；MCD模型组小鼠精神萎靡，少动，毛发紊乱、无光泽，摄食明显减少，体重增长缓慢，随着时间推移，模型组小鼠出现被毛脱落现象。
2.肝指数变化：MCD模型组小鼠体重显著低于对照组（P <0.05），肝湿重较对照组无明显变化（P>0.05），肝指数（肝湿重/体重×100%）较对照组明显升高（P <0.05）。
3.各组小鼠血生化指标的结果：MCD组血清中ALT及AST的水平明显高于对照组，差异有统计学意义（P <0.05）。
4.各组小鼠肝组织HE及Masson染色的结果：MCD组小鼠肝细胞出现重度脂肪变性，并伴点状和灶状坏死、炎性细胞浸润、汇管区纤维组织增生及窦周纤维化等。
5. miRNAs芯片结果：与对照组相比，MCD模型组有47个差异表达的miRNAs（P <0.05），其中15个变化在2倍之上。mmu-let-7i、mmu-mir-155、 mmu-mir-199a-5p、 hsa-mir-34a、 mmu-mir-221、mmu-mir-200c、 mmu-mir-297c、 mmu-mir-713、 mmu-mir-190b、mmu-mir-678，10个miRNAs在MCD模型组的肝脏组织中表达明显上调；mmu-mir-122、mmu-mir-103、mmu-mir-146、mmu-mir-101a、mmu-mir-466j，5个miRNAs在MCD模型组的肝脏组织中表达明显下调。
6.综合本实验基因芯片结果及相关文献报道，选择miR-122、miR-221及miR-199a-5p进行实时荧光定量RT-PCR验证，结果显示，与正常对照组相比，MCD组小鼠肝组织中miR-199a-5p的表达明显增高、miR-122明显降低（P <0.05）。
结论：
1在MCD饮食诱导的小鼠非酒精性脂肪性肝纤维化动物模型中，肝组织miRNAs表达谱明显改变，表明差异表达的miRNAs参与非酒精性脂肪性肝炎／肝纤维化的发生发展。
2miR-199a-5p可能为非酒精性脂肪性肝纤维化发生发展的重要调控基因之一。第二部分：miR-199a-5p生物信息学分析及靶基因验证
目的：探讨miR-199a-5p在非酒精性脂肪性肝纤维化发病中可能的作用及其潜在的作用靶点。
方法：动物分组及标本取得同第一部分。应用TargetScan6.2、miRanda及PITA软件共同预测miR-199a-5p的靶基因，应用DAVID软件分析miR-199a-5p可能的生物学功能及参与调节的信号通路。采用实时荧光定量RT-PCR及Western blot法分析在非酒精性脂肪性肝纤维化小鼠肝组织中miR-199a-5p靶基因mRNA及蛋白水平的表达变化情况。
结果：
1.生物信息学分析结果：GO分析结果显示，miR-199a-5p参与了转录调控、转录因子复合物组成、丝苏氨酸蛋白激酶活化等多种生物学调控；KEGG分析显示，miR-199a-5p参与胰岛素信号通路、Wnt信号通路、MAPK信号通路等多条通路的调控。
2. miR-199a-5p的靶基因预测：应用三种miroRNA靶基因预测软件（TargetScan、PITA和miRanda）共同预测miR-199a-5p的靶基因，得到26个与miR-199a-5p相关的基因。
3.各组小鼠肝脏组织中miR-199a-5p靶基因核受体共抑制剂1（nuclear receptor corepressor，NCOR1)的表达情况：与对照组相比，MCD组肝组织中NCOR1蛋白的表达明显减少（p <0.05），与miR-199a-5p的表达负相关；模型组与对照组肝组织NCOR1mRNA表达无明显差异。
结论：
1生物信息学分析结果示，miR-199a-5p参与胰岛素信号通路、Wnt信号通路、MAPK信号通路等多条与非酒精性脂肪性肝纤维化发生发展相关信号通路的调控。
2在MCD饮食诱导的肝纤维化模型中，miR-199a-5p可能通过调控NCOR1蛋白的表达参与疾病的发生与进展。
第三部分：miR-199a-5p与其靶基因NCOR1的相互作用机制
目的：验证miR-199a-5p与NCOR13′-UTR靶向调节关系。
方法：根据Gene Bank公布的人NCOR1基因3′-UTR序列设计含有miR-199a-5p结合位点的NCOR13′-UTR PCR引物，并在引物中加入Not I和Xho I两个限制性酶切位点。将扩增的NCOR13′-UTR序列插入携带萤火虫荧光与海肾荧光双荧光报告基因的psiCHECKTM-2质粒的Xho I和Not I位点之间，构建NCOR13′-UTR-luciferase报告载体，将NCOR13′-UTR-luciferase报告载体与pre-miR-199a-5p共同转染至HEK293细胞，48h后检测荧光强度，并计算萤火虫荧光与海肾荧光的比值。分析miR-199a-5p对NCOR1的靶向调控作用。
结果：
1.查询“Ensembl Mouse database”，得到全长2202bp的NCOR13′-UTR序列，其中，在NCOR13′-UTR的790bp～796bp处存在可能与miR-199a-5p结合的位点。
2.从血液中提取基因组DNA经PCR扩增后，扩增产物在1.0%的琼脂糖凝胶上进行电泳显示出一条约2000bp特异性片段，实验与预期结果相符。重组质粒p3′UTR-NCOR1经上海生工进一步测序鉴定质粒构建正确。
3.将构建的p3′UTR-NCOR1质粒与pre-miR-199a-5p或阴性对照序列共转染至HEK293细胞，48h后检测荧光素酶活性。结果显示，与对照组相比，miR-199a-5p可以直接作用于NCOR13′-UTR序列，并可抑制荧光素酶活性。。
结论：miR-199a-5p可靶向作用于NCOR13′-UTR，负性调控NCOR1的表达。第四部分：miR-199a-5p靶向调控对LX-2细胞活力与功能的影响
目的：靶向调控LX-2细胞中miR-199a-5p表达，观察非酒精性脂肪性肝纤维化发病关键基因表达变化，深入阐明miRNAs在非酒精性脂肪性肝纤维化发病过程中的作用、其作用的靶基因及主导分子信号通路，为非酒精性脂肪性肝纤维化的临床防治提供新的靶点及科学依据。
方法：应用人肝星状细胞系LX-2细胞，培养于含10%胎牛血清、100U/ml青霉素和100μg/ml链霉素的DMEM培养液中，37℃、5%CO2孵箱中进行培养。当细胞融合度达到60%左右时，用不同浓度miR-199a-5p的模拟物及抑制剂转染LX-2细胞，采用MTT法测定各组细胞活力，选择合适的转染浓度。采用实时荧光定量PCR及Western-blot法分析miR-199a-5p及其靶基因NCOR1mRNA及蛋白的表达情况。同时应用Western-blot方法观察miR-199a-5p对非酒精性脂肪性肝纤维化发病关键因子的影响。
结果：
1. MTT结果显示，转染不同浓度的对照序列对LX-2细胞活力无明显影响；转染100nM及150nM的miR-199a-5p模拟物后，LX-2细胞活力明显降低，而转染50nM的模拟物，则无明显变化；转染100nM及150nM的miR-199a-5p抑制剂后，LX-2细胞活力明显增强，而50nM的miRNA抑制剂则对细胞活力无明显影响；
2.各转染组与未转染组LX-2细胞相比，miR-199a-5p的表达量有不同程度改变，miR-199a-5p靶基因NCOR1mRNA的表达量则无明显变化。与未转染组比较，NC组miR-199a-5p的表达量无明显变化，Mimic组miR-199a-5p的表达量显著增高（P <0.01），Inhibitor组miR-199a-5p表达量明显降低（P <0.05）；
3.转染后细胞miR-199a-5p靶蛋白NCOR1及过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor gamma，PPARγ）蛋白的表达量有不同程度改变，与未转染组比较，NC组NCOR1和PPARγ蛋白的表达量均无明显变化，Mimic组NCOR1蛋白的表达量显著降低，PPARγ蛋白的表达量明显增高（P <0.05），Inhibitor组NCOR1蛋白表达量明显增高，PPARγ蛋白的表达量则降低（P <0.05）。PPARγ蛋白与转染各组细胞中NCOR1蛋白的表达变化相反；
4.转染后细胞肝纤维化相关蛋白转化生长因子β1（transforminggrowth factor beta，TGFβ1）、α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）的表达量有不同程度改变，与未转染组比较，NC组TGF-β1及α-SMA蛋白的表达量无明显变化，Mimic组TGF-β1及α-SMA蛋白的表达量显著降低（P <0.05），Inhibitor组TGF-β1及α-SMA蛋白表达量明显增高（P <0.05）。TGF-β1及α-SMA蛋白与转染各组细胞中NCOR1蛋白的表达变化一致。
结论：
1转染miR-199a-5p模拟物或抑制剂可引起LX-2细胞活力的改变，提示miR-199a-5p可能通过影响HSC细胞增殖及活化参与非酒精性脂肪性肝纤维化的发病过程。
2miR-199a-5p可能通过负性调控NCOR1蛋白表达影响PPARγ蛋白活性，参与肝星状细胞的激活。
3靶向调控miR-199a-5p表达可阻止或逆转肝纤维化的发生与进展。
Nonalcoholic fatty liver disease (NAFLD), a hepatic manifestation of themetabolic syndrome, has similar pathological changes with alcoholic liverdisease, but the patients suffer from it without excessive alcohol intake andother definite etiological factor. It has been reported that NAFLD is relatedwith insulin resistance and hereditary susceptibility. The disease spectrum ofNAFLD includes simple fatty liver (SFL) non-alcoholic steatohepatitis(NASH), NASH-related hepatic fibrosis and cirrhosis, hepatocellularcarcinoma. NASH is the key stage in the spectrum of NAFLD which ischaracterized by non-alcoholic steatohepatitis／fibrosing steatohepatitis andcan progress to fibrosis, cirrhosis, liver failure, and hepatocellular carcinoma.
With the changes of life style and diet, the prevalence of NAFLDincreases rapidly in modern society. NAFLD has became the most commonchronic liver diseases in the United States and worldwide. Recent studiesusing different methodologies indicate that in the general population theprevalence of NAFLD is approximately20%~40%. Of greater concern is therecognition that cirrhosis and liver-related deaths occur in approximately20%and8%of these NASH patients over a10-year period. Among more affluentregions of China, the community prevalence of non-alcoholic fatty liverdisease (NAFLD) is approximately15%. With the increasing pandemic ofobesity, the prevalence of NAFLD has approximately doubled in the pastdecade. Nonalcoholic steatohepatitis/fibrosis, one type of a larger spectrum ofnonalcoholic fatty liver disease, is now recognized as a cause of progressivefibrotic liver disease with adverse clinical sequelae. However, thepathogenesis of nonalcoholic steatohepatitis/fibrosis leading to diseaseprogression remains poorly understood and definitive therapies for thesepatients are lacking．
MicroRNAs (miRNAs) are a class of important factors in the regulation ofprotein expression at the posttranscriptional level. miRNAs are associatedwith many pathophysiologic events such as hepatic tissue development anddifferentiation, cell proliferation, apoptosis. In the study of Sanyal and hiscolleagues, they found that NASH was associated with altered hepaticmiRNAs expression including the upregulation of miR-34a and miR-146b,downregulation of miR-122. miR-122has been found to be required for thelipid homeostasis through the regulation of sterol-regulatory element-bindingprotein (SREBP), fatty acid synthetase (FAS) and HMG CoA reductase(HMGCR). miR-33a and miR-33b have a crucial role in controllingcholesterol and lipid metabolism in concert with their host genes, the SREBPtranscription factors. miR-34family has been found to control cholesterolsynthesis by targeting acyl-CoA synthetase long-chain family member1(ACSL1) and inhibiting sirtuin-1with downstream dephosphorylation ofAMP kinase and HMGCR. Downregulation of miR-467b is involved in thedevelopment of hepatic steatosis by modulating the expression of its targethepatic lipoprotein lipase (LPL). miR-103and miR-107regulate insulin andglucose homeostasis through insulin receptor CAV1. Dysregulation ofmiRNAs may contribute to the development of NAFLD, however, the exactroles of miRNAs in the pathogenesis of NASH are not fully understood.
In this study， we aimed to establish a non-alcoholic fibrosingsteatohepatitis model caused by methionine and choline deficient (MCD) diet8weeks. Serum alanine aminotransferase (ALT) and aspartateaminotransferase (AST), reliable indicators of liver inflammatory injury, weremeasured. Histological scoring for NAFLD was carried out to evaluate hepaticsteatosis and fibrosis. Using microarray experiments, we found that feedingC57BL/6J mice a methyl-deficient diet8weeks to induce non-alcoholicfibrosing steatohepatitis could result in aberrant expression of miRNAs. Toclarify the roles of miRNAs in the process of non-alcoholic fibrosingsteatohepatitis and its molecular mechanism, and provided a basis for NAFLDprevention and the development of miRNA targeting drugs, the different expressed miRNAs were performed with the bioinformatic analysis. Thederegulated miRNA and miRNA targeting gene was validated usingquantitative real-time PCR and western blot assay.
To study the role of these miRNAs in the NASH, levels of miR-122,miR-199a-5p and miR-221were determined in the livers extracted from micefed MCD and control diets, using quantitative real-time PCR experiments.To investigate the potential interaction experimentally, the wild-type3′-UTRof NCOR1gene was subcloned downstream of the Renilla luciferase codingsequence and cotransfected into293T cells with the pre-miR-199a-5p andnegative control sequence. The luciferase activity was assayed at48hpost-transfection. To examine the effect of miR-199a-5p, we transfected itsmimic, inhibitor and Ncontrol (NC) into a hepatic stellate cellline named LX-2cells and observed the expression of several fibrosis-associated genes,providing new insights to the pathogenesis of NASH.
Part1：Alterations and validation of microRNA expression profile innon-alcoholic fibrosing steatohepatitis model
Objective: To establish a non-alcoholic fibrosing steatohepatitis modeland study the expression profiles of fibrosis related miRNAs.
Methods: Experimental non-alcoholic fibrosing steatohepatitis modelswere established by feeding mice with MCD diet8weeks (MCD group).Control mice were fed with choline–methionine supplemented diet (controlgroup). Serum alanine aminotransferase (ALT) and aspartate aminotransferase(AST) were measured by enzymic method with automatic biochemistryanalyzer. Hepatic inflammation and fibrosis were graded under HE andMasson staining. The stage of steatosis and fbrosis was assessed according tothe guidelines for diagnosis and treatment of non-alcoholic fatty liver diseases.RNAs from experimental mice hepatic tissue were isolated with a MiRNeasyMini Kit (Qiagen). The miRNAs profiles were determined using GeneChipmiRNA2.0Array (Affymetrix). The result of microarray was validated byreal-time quantitative PCR. Results:
1. General observation: The mice of MCD group showed less activity,sikness and dim hair than the mice of Control group.
2. Liver index changes: The body weights of MCD group mice weresignificantly lower than the control group at the8th week body (P <0.05).Liver wet weight of MCD group mice had no significant change comparedwith the control group (P>0.05). Liver index (liver weight/body weight×100%) was significantly higher than the control group (P <0.05).
3. Serum transaminases in C57BL/6J mice fed control and MCD diet for8weeks: MCD diet increased the level of serum transaminases, both ALT andAST.
4. HE and Masson staining of liver sections of C57BL/6J mice fedcontrol and MCD diets: MCD treated animals showed disordered lobulestructure, severe macrosteatosis, focal hepatocyte necrosis, inflammatoryinfiltration, portal fibrosis and fibrous septum.
5. The result of GeneChip miRNA2.0Array showed that there were47deregulated miRNAs in mice hepatic tissue treated with MCD or control diet,in which15miRNAs changed above2times. The result of microarray showedthat10miRNAs were upregulated in MCD group including mmu-let-7i,mmu-mir-155, mmu-mir-199a-5p, mmu-mir-34a, mmu-mir-221,mmu-mir-200c, mmu-mir-297c, mmu-mir-713, mmu-mir-190b, mmu-mir-678;5miRNAs downregulated including mmu-mir-122, mmu-mir-103,mmu-mir-146, mmu-mir-101a and mmu-mir-466j.
6. Refer to the relevant literature and the results of microarray, weselected miR-122, miR-199a-5p and miR-221to perform quantitativereal-time PCR experiments. We found that MCD diet increased miR-199a-5pand decreased miR-122in mice livers (P <0.05).
Conclusion:
1. MCD diet could induce characteristic pathology of steatosis, mixedhepatic inflammatory and fibrosis, representing a successful model ofnon-alcoholic fibrosing steatohepatitis.
2. Non-alcoholic fibrosing steatohepatitis in mice significantly change the expression of miRNA profiles, which may be involved in non-alcoholicsteatohepatitis/fibrosing steatohepatitis.
3. We found a dramatic upregulation of miR-199a-5p in the non-alcoholicfibrosing steatohepatitis mouse model. These data indicated that dysregulatedexpression of miR-199a-5p might be critical in non-alcoholic fibrosingsteatohepatitis pathogenesis.
Part2：Functional analysis and evaluation of miR-199a-5p target genes
Objective: To searche the target genes of miR-199a-5p and detect therole of miR-199a-5p in non-alcoholic fibrosing steatohepatitis etiology.
Methods: The DAVID (Database for Annotation, Visualization andInterrogated Discovery) bioinformatic resources were performed to analysisthe function of miR-199a-5p. Three most commonly prediction algorithmsTargetScan6.2, miRanda and PITA was used to find the exact target genes ofmiR-199a-5p. Target gene of miR-199a-5p in the experimental mice hepatictissue was evaluated by real-time quantitative PCR and western blot assay.
Results:
1. Enrichment analysis on predicted target genes: Result obtained fromGO-term analysis revealed a relationship between miR-199a-5p and regulationof transcription, transcription factor complex, protein serine/threonine kinaseactivity, and others. KEGG analysis showed the involvement of miR-199a-5pin a variety of pathways, including insulin signaling, Wnt signaling andMAPK signaling pathways.
2. Prediction of miR-199a-5p target genes:26target genes ofmiR-199a-5p were predicted by all of the three algorithms. NCOR1(nuclearreceptor corepressor1), one of the predicted target genes, was reported to berequired for the integration of inflammatory and anti-inflammatory signals,which is associated with the pathogenesis of non-alcoholic fibrosingsteatohepatitis.
3. Expression of NCOR1in hepatic tissues: Increased hepaticmiR-199a-5p upon MCD diet is associated with decreased NCOR1expressionat the protein, but not mRNA level.
Conclusion:
1. Results of bioinformatic analysis showed that miR-199a-5p may takepart in the pathogenesis of non-alcoholic fibrosing steatohepatitis throughinsulin signaling, Wnt signaling and MAPK signaling pathways.
2. miR-199a-5p may regulate the translation of NCOR1mRNA.
Part3：The interaction between miR-199a-5p and its target gene NCOR1
Objective: To investigate the potential interaction between miR-199a-5pand its target gene NCOR1mRNA3′-UTR experimentally.
Methods: The3′-UTR of NCOR1was PCR amplified and cloneddownstream of the renilla luciferase stop codon in psiCHECKTM-2vector,giving rise to NCOR13′-UTR-luciferase. This construct luciferase reportervectors were transfected into HEK293cells with pre-miR-199a-5p ornegative control. Cells were harvested48h after transfection and assayed withDual Luciferase Reporter Assay System according to the manufacturer’sinstructions.
Results:
1. From “Ensembl Mouse database”, we obtained a full length2202bpNCOR13'-UTR sequences, where may be binding sites with miR-199a-5p inNCOR13'-UTR of790bp~796bp place.
2. Genomic DNA was extracted from the blood and amplified by PCRassay. The PCR products showed a specific treaty2000bp fragment on a1.0%agarose gel electrophoresis which was in line with expectations. Therecombinant plasmid p3'UTR-NCOR1was confirmed by Shanghai Sangonfurther sequencing of plasmid.
3. The luciferase activity assays at48h post-transfection showed that theoverexpression of miR-199a-5p could decrease relative luciferase activitysignificantly.
Conclusion: miR-199a-5p may play important roles in the pathogenesisof non-alcoholic fibrosing steatohepatitis by influencing protein translation ofNCOR1.
Part4The regulation of NCOR1and fibrosis-related genes bymiR-199a-5p was examined by silencing or overexpressing this miRNA inLX-2cells
Objective: To observe the expression of several fibrosis-associated genesby regulating the expression of miR-199a-5p in LX-2cells, further detect therole of miR-199a-5p in the pathogenesis of non-alcoholic fibrosingsteatohepatitis and prevention and provide a new target basis to treatnon-alcoholic fibrosing steatohepatitis.
Methods: Human hepatic stellate cells (LX-2cells) were cultured inDMEM supplemented with10%fetal bovine serum,100U/mL penicillin, and100g/L streptomycin at37°C in a humidified environment with5%CO2.Cells were grown to60%confluency prior to transfection with differentconcentrations of miR-199a-5p mimic, inhibitor, or their correspondingcontrols using Lipofectamine2000. Cell viability was determined by MTTassay in each group to select the appropriate concentration of transfection.Transfected cells were cultured for48hours and harvested for total RNA andprotein extraction. The samples were stored at80°C for q-PCR or Westernblot assays.
Results:
1. The result of MTT assay showed that transfection with controlsequences of different concentrations without significant effect on cellviability; transfection of100nM and150nM miR-199a-5p mimic, cell viabilitywas significantly reduced, but no significant changes with50nM mimic;transfection of100nM and150nM miR-199a-5p inhibitor, cell viability wassignificantly enhanced, but no significant changes with50nM inhibitor.
2. Compared with the control group, the expression of miR-199a-5pdramatically increased in mimic group and significantly reduced in inhibitorgroup. NCOR1mRNA was not affected by miR-199a-5p mimic, inhibitor andNcontrol (NC).
3. NCOR1protein was decreased by miR-199a-5p overexpression andincreased by miR-199a-5p knockdown (p <0.05) in LX-2cells, while PPAR γprotein expressed inversely of NCOR1protein in each group.
4. miR-199a-5p inversely correlated with the expression of severalfibrosis-associated genes, including transforming growth factor-β (TGF-β) andα-smooth muscle actin (α-SMA).
Conclusion:
1. miR-199a-5p may influence cell proliferation or apoptosis involved inthe pathogenesis of non-alcoholic fibrosing steatohepatitis.
2. miR-199a-5p may affect PPAR γ involved in the activation of hepaticstellate cells through negative regulation the expression of NCOR1.
3. Targeted regulation of miR-199a-5p expression may prevent or reversethe occurrence and development of hepatic fibrosis.
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