绒山羊70d胚胎皮肤小分子RNA文库构建及miRNA鉴定

目的:miRNA是一类21~23个核苷酸的单链小分子RNA,最近发现miRNA对哺乳动物毛囊的发育和维持具有重要作用。为了研究miRNA在绒山羊次级毛囊发育中的作用,可以为人工调节绒毛生长和提高羊绒产量提供新的线索。方法:该文构建了绒山羊70 d胎儿胚胎体测部皮肤小RNA文库,库容量为2.5×107。随机挑选192个克隆提取质粒进行序列分析,序列经GenBank Blastn和mirBase搜索比对。结果:结果显示有32个序列和绒山羊及其它动物的miRNA高度同源,可以确定是山羊的miRNA。这些miRNA代表19个不同的miRNA序列,其中9个已注册,其余10个代表新的绒山羊miRNA。在克隆的miRNA中,miR-199a*和miR-424的拷贝数最高,分别达到6个和3个,表明它他们在绒山羊次级毛囊发育期间的皮肤中非常活跃,可能有重要的生物学功能。结论:从绒山羊70d胚胎皮肤中鉴定了19个miRNA,这些miRNA可能参与绒山羊次级毛囊发育的调控。     

绒山羊70d胚胎与新生羊羔体侧部皮肤miRNA的比较分析

目的:最近发现miRNA对哺乳动物毛囊的发育和维持具有重要作用,研究miRNA在绒山羊次级毛囊发育中的作用,可以为人工调节绒毛生长和提高羊绒产量提供新的线索。方法:应用Exiqon miRNA8.1表达谱芯片对70d胎儿皮肤和出生2w羊羔体侧部皮肤组织差异表达的miRNA基因进行筛查,找出绒山羊皮肤发育不同阶段差异表达的miRNA。结果:绒山羊70d胎儿皮肤中相对于出生2w羊羔体侧部皮肤上调的miRNA 4个。绒山羊70d胎儿皮肤中相对于出生2w羊羔体侧部皮肤下调的miRNA 64个。结论:绒山羊70d胚胎皮肤中上调的miRNA远远少于出生后羊羔皮肤上调miRNAs,说明miRNA在皮肤毛囊不同发育阶段中发挥重要作用。     

鳜不同孵化时期miRNA转录组分析及生长相关miRNA鉴定

MicroRNA是一类20-24 nt核苷酸长度的参与基因转录后水平调控的非编码小分子RNA。利用高通量测序技术获得鳜(Siniperca chuatsi)孵化后第3天、第17天和第28天全鱼miRNA转录组,共鉴定出1 084个miRNAs,其中432个已知、624个保守、28个新发现。第17天时鉴定出的miRNA个数最多,为926个。在3个发育时期都表达的有628个miRNAs,只在各时期中特异表达的分别有71、104和70个miRNAs。一些与鳜发育相关的miRNAs在上述3个时期呈现持续上调或下调的表达特点。进一步的实验表明,miR-199-5p和miR-203可能与鳜生长发育相关,相应的靶基因分别是WnT和MyoD。     

青年羊驼耳部和背部皮肤组织中miRNA差异表达研究

羊驼是毛用型经济动物,其耳部和背部的毛发品质和生长速度存在差异.MicroRNA(miRNA)是新发现的一类在转录后水平调控基因表达的非编码RNA分子,为比较miRNA在羊驼耳部和背部皮肤的表达差异,从而探讨miRNA在羊驼皮肤和毛囊发育过程中的调控作用,本实验提取羊驼皮肤总RNA,制备了羊驼皮肤miRNA芯片,通过与Affymetrix多物种miRNA芯片跨物种杂交对耳部和背部皮肤的miRNA进行筛选,并通过实时荧光定量PCR进行了验证,同时利用在线生物信息软件预测miRNA靶基因.结果显示,羊驼耳部和背部皮肤中高表达差异2倍以上的miRNA有39个,实时荧光定量PCR检测let-7b和miR-24在2个部位皮肤中的差异表达量与miRNA基因芯片结果一致;预测到let-7b和miR-24的靶基因中包含有与毛囊生长发育和毛发品质相关的基因,提示这些miRNA可能参与羊驼皮肤和毛囊的生长发育、更新以及毛发品质的调控.     

饲料硒含量对黄颡鱼肠系膜脂肪组织中脂类代谢和miRNAs表达水平的影响

使用3种不同硒含量的饲料饲养黄颡鱼12周, 研究饲料硒含量对黄颡鱼(Pelteobagrus fulvidraco)肠系膜脂肪组织脂类代谢和miRNAs表达水平的影响, 饲料硒含量分别为0.03(低硒组)、0.25(适宜硒组)和6.39 mg Se/kg饲料(高硒组)。结果表明, 相比较于适宜硒组, 高硒和低硒组中甘油三酯(TG)含量显著升高(P<0.05), 葡萄糖-6-磷酸脱氢酶(G6PD)和6-磷酸葡萄糖脱氢酶(6PGD)的酶活性在低硒组中活性显著下降(P<0.05), 在高硒组中活性显著上升(P<0.05); 异柠檬酸脱氢酶(ICDH)的酶活性在低硒组中没有显著性变化(P>0.05), 在高硒组中显著下降(P<0.05); 苹果酸酶(ME)和脂肪酸合成酶(FAS)的酶活性在低硒和高硒组中均显著上升(P<0.05)。相比于适宜硒组, 在高硒组miR-26a、miR-183、miR-135、let-7b、let-7c和let-7g的表达量显著上升(P<0.05), 而在低硒组中没有显著性差异(P>0.05); miR-181a-5p和let-7e在低硒组中显著上升(P<0.05), 在高硒组中没有显著性差异(P>0.05); miR-130和miR-203a在低硒和高硒组中表达量均显著上升(P<0.05); miR-200a、miR-143、let-7d和let-7f在低硒组中表达量显著下降同时在高硒组中表达量上升(P<0.05)。miR-143、miR-203a和miR-130在脂肪组织中高表达, 且对硒产生强响应, 通过TargetScanFish6.2和miRwalk3.0共预测3个miRNA的靶基因, 并对靶基因进行KEGG富集分析, 结果显示, miR-143的靶基因在矿物盐吸收、不饱和脂肪酸生物合成、胰岛素通路、自噬、脂肪酸代谢、鞘脂类代谢、调节脂肪细胞脂肪分解和甘油磷脂代谢等途径中显著富集(P<0.05, FDR≤0.05); miR-203a的靶基因在胰岛素抵抗、胰岛素通路、调节脂肪细胞脂肪分解和自噬等代谢途径、信号通路中显著富集(P<0.05, FDR≤0.05); miR-130的靶基因在胰岛素通路、胰岛素抵抗、自噬、鞘脂类代谢、调节脂肪细胞脂肪分解和mTOR通路中显著富集(P<0.05, FDR≤0.05)。综合分析发现, miR-143、miR-203a和miR-130的靶基因共同富集在脂肪酸合成、调节脂肪细胞脂肪分解、胰岛素抵抗等脂代谢相关的通路中, 选取共富集通路中的靶基因进行qPCR检测, 结果显示fas、碳水化合物反应元件结合蛋白α(chrebpα)、肉碱棕榈酰转移酶1α(cpt1α)、激素敏感酯酶(hsl)、固醇调节元件结合蛋白(srebp1)、过氧化物酶体增殖剂激活受体α(pparα)和脂肪组织甘油三酯酶(atgl)的表达趋势与miR-143、miR-203a和miR-130的表达趋势相符合, 推测饲料硒缺乏和过量诱导黄颡鱼肠系膜脂肪组织脂质沉积是通过诱导miR-143、miR-203a和miR-130的表达量上调, 进而负调控靶基因chrebpa、cpt1a、hsl、srebp1、ppara和atgl的表达实现。     

肝细胞癌患者血清外泌体microRNA表达鉴定

目的:探讨肝癌细胞外泌体中差异表达的microRNAs(miRNAs)在肝细胞癌(HCC)诊断中的应用价值。方法:通过高通量测序筛选肝癌细胞外泌体中差异表达的miRNAs。实时定量PCR验证差异表达分子;检测差异表达的miRNAs在健康人(Health)、慢性乙型肝炎患者(CHB)、肝硬化患者(LC)及乙型肝炎病毒阳性的肝细胞癌患者(HCC)血清外泌体中的表达。结果:高通量测序筛选到肝癌细胞外泌体中差异表达的miRNA共88种,其中58种表达上调,30种表达下调。选择其中8种差异表达的miRNAs进行q RT-PCR验证,结果显示,此8种miRNAs在细胞上清外泌体、细胞内、癌与癌旁组织中的表达趋势与测序结果一致。miR-221-3p和miR-224-5p在HCC组外泌体中的表达水平显著高于Health组、CHB组和LC组(P0.01),miR-124-3p和let-7a-5p在HCC组外泌体中的表达水平显著低于其他各组(P0.05)。四个组中,miR-21-5p、miR-191-5p、miR-34a-5p和miR-122-5p的表达水平不存在显著性差异(P0.05)。结论:血清外泌体中的miR-221-3p、miR-224-5p、miR-124-3p和let-7a-5p可能成为肝细胞癌的候选标志物。     

基于通用荧光标签的microRNA芯片检测平台

microRNA（miRNA）是一类生物内源性非编码小RNA分子,在细胞的生长、发育、增殖和细胞凋亡中具有重要的调控作用,并且与癌症的发生息息相关. miRNA表达谱是miRNA研究中的一个关键环节,使用芯片技术分析miRNA表达谱通常要求对miRNA样品进行耗时耗力的荧光标记,并且常规的芯片技术难以精确识别高度同源的miRNAs分子. 为了解决以上问题,本实验室建立了一个被命名为SHUT（Stacking-hybridized Universal Tag）的新型非标记miRNA芯片分析方法. 本文通过分别使用两步杂交法、一步杂交法对SHUT平台的miRNAs特异性检测进行优化. 研究结果表明,SHUT平台在48℃下杂交18 h,除了let-7b和P-let-7c,let-7c和P-let-7a分别存在超过32.5%和83.3%的假阳性信号之外,其它交叉杂交信号都低于10%,具有较好的特异性. 在该杂交条件下,靶标分子let-7d的最低检测浓度为20 fmol/L,并且在20 fmol/L至200 pmol/L之间具有较好的线性关系.     

缺血后处理对大鼠肾脏miRNAs表达谱的影响

目的：观察缺血后处理对Wistar大鼠肾组织miRNAs表达的影响。方法：将20 只Wistar 大鼠随机分为缺血再灌注组（I/R 组）和缺血后处理组（IPO 组）,每组10 只。I/R 组行剖腹手术,分离双肾动脉后切除右肾,夹闭左肾动脉阻断血供,45 min 后恢复; IPoC组行剖腹手术,分离左肾动脉后将其夹闭,在阻断血液供应45 min 后恢复血供时采取10 s再灌、10 s停灌处理,反复5个周 期。在所有实验完成6 小时后,处死大鼠切除左肾,分别提取两组肾组织中的总RNA和miRNA,利用miRNA微阵列对其进行杂 交检测,通过芯片扫描和数据聚类分析,获取两组大鼠肾脏组织miRNAs 表达谱,并进行qRT-PCR 验证,筛选差异表达的 miRNAs。结果：通过qRT-PCR 验证共筛选出7 种表达差异较大的miRNAs,其中下调的三种,分别是miR-27a,miR-665, miR-let7f,上调的四种,分别为miR-532-3p,miR-205,miR-122,miR-291b。结论：差异表达miRNAs 可能参与缺血后处理减弱缺血 再灌注损伤的作用机制中。     

调节猪TNF-α表达的miRNAs鉴定

肿瘤坏死因子α(TNF-α)是脂肪细胞的分泌产物之一,在脂肪中行使着复杂的调节功能,为了研究猪脂肪组织中TNF-α的表达受到哪些miRNA的调控,从猪脂肪组织基因组中获得猪TNF-α3'端非翻译区(UTR)序列,与荧光素酶质粒PGL3-control连接构建猪TNF-α的萤光素酶表达质粒PGL3-TNF-α3'UTR。生物信息学预测miR-19a,miR-124,miR-130a,miR-301,miR-506等miRNAs均靶向猪TNF-α,将这些miRNA分别与PGL3-TNF-α3'UTR质粒共转到细胞中,以乱序序列作为阴性对照(NC),检测miRNA对质粒荧光素酶活性的作用。结果发现miR-19a,miR-124和miR-130a均能够显著抑制萤光素酶的活性(P0.01),为了验证这3个miRNA是否通过各自种子序列起调控作用,突变了PGL3-TNF-α3'UTR中这3个miRNA种子序列的结合位点,结果发现miRNAs对突变质粒中的荧光素酶均无明显抑制作用(P0.05)。结果证明,miR-19a,miR-124和miR-130a与猪TNF-α均有直接的靶向关系并通过各自种子序列抑制TNF-α的表达。     

胰腺癌细胞株和胰腺癌组织中miRNAs的表达检测

目的:探明miRNAs在胰腺癌细胞株及组织中的表达情况,证实miRNAs的差异表达与胰腺癌的发生有相关性.方法:对胰腺癌细胞株和胰腺癌组织进行总RNA的提取,通过Real-time PCR方法检测17种miRNAs(miR-16、miR-20a、miR-21、miR-26a、miR-142-3p、miR-155、miR-210、miR-181a、miR-181b、miR-196a、miR-939、miR-223、miR-1202、miR-1207-5p、miR-1825、miR-1228、miR-1915)在胰腺癌细胞株及胰腺癌组织中的表达,分析miRNAs的表达与胰腺癌患者临床表现之间有无相关性.结果:胰腺癌细胞株和胰腺癌组织中miRNAs的表达明显较正常胰腺组织高.癌组织及癌旁组织中miRNAs表达量在不同性别的胰腺癌患者中差异不大(P＞0.05),并且miRNAs的表达与患者年龄及其血清CA19-9关系不大.结论:经筛选的miRNAs可以作为胰腺癌的诊断标志.胰腺癌组织中的miRNAs表达并不是一成不变的,而是随着肿瘤的生长而不断发生变化.     

Effect of melatonin administration to lactating cashmere goats on milk production of dams and on hair follicle development in their offspring

Melatonin treatment in adult cashmere goats can increase cashmere yield and improve cashmere fibre quality by inducing cashmere growth during cashmere non-growth period, of which time cashmere goats are in the mid and late stages of lactation. However, whether melatonin treatment in adult cashmere goats affects their offspring’s growth performance remains unknown. Therefore, the objectives of the current study were to determine the effects of melatonin treatment in adult cashmere goats on cashmere and milk production performance in dams and on hair follicle development and subsequent cashmere production in their offspring. Twenty-four lactating Inner Mongolian Cashmere goat dams (50 ± 2 days in milk, mean ± SD) and their single-born female offspring (50 ± 2 days old, mean ± SD) were randomly assigned to one of two groups supplemented with melatonin implants (MEL; n = 12) or without (CON; n = 12). The melatonin implants were subcutaneously implanted behind the ear at a dose of 2 mg/kg live weight on two occasions – 30 April and 30 June 2016. The results demonstrated that melatonin treatment in adult cashmere goats increased cashmere production and improved cashmere fibre quality as indicated by greater cashmere yield, longer cashmere fibre staple length, finer cashmere fibre diameter and thicker cashmere fibre density. The milk fat content was higher in MEL compared with CON cashmere goats. The daily yields of milk production, milk protein and milk lactose were lower in MEL compared with CON cashmere goats. Serum melatonin concentrations were greater, serum prolactin concentrations were lower and milk melatonin concentrations and yields were greater in MEL compared with CON cashmere goats. With regard to offspring, there were no differences in cashmere yield, fibre staple length, fibre diameter and fibre density at yearling combing, and the primary and secondary hair follicles population and maturation between treatments. In conclusion, melatonin treatment in adult cashmere goats during cashmere non-growth period is a practical and an effective way in cashmere industry as indicated by not only increasing cashmere yield and improving cashmere fibre quality in adult cashmere goat dams but also having no impairment in hair follicle development and the subsequent cashmere production in their single-born offspring.     

褪黑素对内蒙阿白山羊在非生绒季节促绒生长及绒产量的影响

用简易的外科手术在阿白山羊梳绒后的非长绒季节，给羊体颈部皮下埋植一种含褪黑素的胶囊试验，经５周后受试羊群长出了新绒，对照群则未见新绒生长。在８月下旬天然生绒季节快下到时，试验组羊绒已长至近３公分；而对照群才开始生绒，但绒长不足１分分。表明含上述激素胶囊促绒生长首次成功。翌年对上述羊群跟踪检测结果：受试羊群个体产绒量的特级（≥４５０克／头）率。提高为５０－６８．４％，与对照群（３５％）相比，提高１５     

A subset of skin-expressed microRNAs with possible roles in goat and sheep hair growth based on expression profiling of mammalian microRNAs

The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18-25 nucleotides) with probable roles in the regulation of gene expression. Due to the fact that miRNAs are conserved in closely related eukaryotes and some are also conserved across a larger evolutionary distance, their potential functions in mammalian development are under active study. In order to identify mammalian miRNAs that might function in hair growth, we characterized the expression of 159 miRNAs in adult body side skin and ear skin from goat and sheep using microarray analysis. Of these, 19 miRNAs were specifically expressed or greatly enriched in body side skin in goats and sheep. This suggests hair growth-specific functions for miRNAs. Of the coexpressed 105 miRNAs, the degree of correlation within species is higher than interspecies. Nine of the expressed miRNAs were detected exclusively in the goat body side skin area where more cashmere was grown than coat hair; mmu-miR-720 and mmu-miR-199b were expressed primarily in goat skin. The identification of 105 of skin-expressed miRNAs whose expression behavior is conserved in goats and sheep differentiating hair follicles implicates these miRNAs have functions in mammalian hair follicles growth and development. We demonstrate that miRNAs previously associated with hair follicles in the mouse are also expressed in the adult skin of goats and sheep. In addition, 69 more conserved miRNAs cross-species were discerned in the study. Of them, the let-7, mir-17, mir-30, mir-15, and mir-8 gene families were expressed in high frequency. These results reveal critical roles of them in skin and hair follicle development and function.     

Plasma miRNAs as Diagnostic and Prognostic Biomarkers for Ovarian Cancer

Background

Most (70%) epithelial ovarian cancers (EOCs) are diagnosed late. Non-invasive biomarkers that facilitate disease detection and predict outcome are needed. The microRNAs (miRNAs) represent a new class of biomarkers. This study was to identify and validate plasma miRNAs as biomarkers in EOC.

Methodology/Principal Findings

We evaluated plasma samples of 360 EOC patients and 200 healthy controls from two institutions. All samples were grouped into screening, training and validation sets. We scanned the circulating plasma miRNAs by TaqMan low-density array in the screening set and identified/validated miRNA markers by real-time polymerase chain reaction assay in the training set. Receiver operating characteristic and logistic regression analyses established the diagnostic miRNA panel, which were confirmed in the validation sets. We found higher plasma miR-205 and lower let-7f expression in cases than in controls. MiR-205 and let-7f together provided high diagnostic accuracy for EOC, especially in patients with stage I disease. The combination of these two miRNAs and carbohydrate antigen-125 (CA-125) further improved the accuracy of detection. MiR-483-5p expression was elevated in stages III and IV compared with in stages I and II, which was consistent with its expression pattern in tumor tissues. Furthermore, lower levels of let-7f were predictive of poor prognosis in EOC patients.

Conclusions/Significance

Our findings indicate that plasma miR-205 and let-7f are biomarkers for ovarian cancer detection that complement CA-125; let-7f may be predictive of ovarian cancer prognosis.     

褪黑素对绒山羊皮肤FGF5等绒毛生长相关基因的影响

目的:旨在分析埋植褪黑素后对绒山羊皮肤绒毛生长相关基因表达的影响,进而探讨褪黑素促绒毛生长的机理。方法:以内蒙古白绒山羊为研究对象,选取体况相近的4月龄母羔羊10只,按试验要求随机分为2组,处理组按照2mg/kg BW(体重)的剂量每两个月耳后皮下埋植褪黑素,对照组不埋植,试验时间为1年。实验开始后每月采集绒山羊体侧皮肤和毛样,利用荧光定量PCR技术检测9个绒毛生长相关基因的表达变化。结果:(1)埋植褪黑素后各个基因的平均表达水平都有所上调,其中显著上调FGF5(P<0.0001),而对β-catenin、MSX-2、Wnt10b、BMP2、BMPRIB、NTRK3、Delta1、hairless等无显著影响(P>0.05);(2)埋植褪黑素明显提高了FGF5与hairless的相关性,并且使FGF5与β-catenin、NTRK3的相关性由负相关变成正相关,与Wnt10b的相关性降低,FGF5与其余4个基因的相关性变化不大。结论:结果提示,褪黑素上调绒山羊皮肤FGF的表达,影响FGF5与hairless、β-catenin、NTRK3之间的相关性;山羊中FGF5基因对被毛表型的作用与其他动物不同;褪黑素对复杂组织和单个细胞的作用不同;MAPK信号通路是受褪黑素影响最大的一个信号途径。推测褪黑素可能通过调节绒山羊皮肤中RORα来发挥作用,影响FGF5与hairless、β-catenin、NTRK3和Wnt10b等的相关性,同时抑制MSX2和BMP2的功能,进而促进绒毛的生长。     

Gene Expression and Localization of LAMTOR3 in the Skin Cells of Liaoning Cashmere Goats

Liaoning cashmere goats are the most precious genetic resources in China. The function of LAMTOR3 [late endosomal/lysosomal adaptor, mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin activator 3/MAPK scaffold protein 1] gene is expressed in the skin of Liaoning cashmere goats. In situ hybridization (ISH) found that LAMTOR3 is expressed in the inner root sheath (IRS) of hair follicles. During the anagen or catagen phase, the expression of LAMTOR3 is higher in secondary hair follicles than in primary hair follicles. Expression of LAMTOR3 in skin cells treated with melatonin or insulin-like growth factor-1 (IGF-1) is lower than in untreated cells. In addition, the simultaneous treatment of fibroblast growth factor 5 and melatonin decrease the expression of LAMTOR3 in skin cells. The simultaneous treatment with melatonin and 10^?5?g/L IGF-1 or 10^?4?g/L IGF-1 increases the expression of LAMTOR3 gene in skin cells. If Noggin expression is decreased, then LAMTOR3 expression is increased. This hypothesis suggested that LAMTOR3 influences the character of cashmere fiber, and it may regulate the development of hair follicle and cashmere growth by inducing the MAPK signaling pathway.