旋毛虫肌幼虫期外泌体的分离和小RNA鉴定

目的 分离纯化旋毛虫肌幼虫分泌的外泌体,并进行鉴定,为研究旋毛虫免疫逃逸机制提供新的线索。方法 采用超速离心法提取旋毛虫肌幼虫期外泌体,通过透射电子显微镜、纳米颗粒跟踪分析、免疫印记和小RNA测序对其进行鉴定。结果 旋毛虫肌幼虫期外泌体为有膜的泡状物,直径约80～200 nm,表达外泌体特异性标记蛋白CD63和Enolase,含1 266个已知的miRNA。结论 经形态、粒径及标记蛋白分析证实成功分离旋毛虫肌幼虫期外泌体,同时构建旋毛虫肌幼虫期外泌体的小RNA文库,鉴定出多种功能性小RNA,为深入研究旋毛虫肌幼虫期外泌体内小RNA分子的应用提供数据。     

Detection of apoptosis-associated microRNA in human apheresis platelets during storage by quantitative real-time polymerase chain reaction analysis

Background

Platelet transfusion is an essential part of the treatment of a variety of conditions such as thrombocytopenia and qualitative platelet disorders. As indicated in previous reports, during in vitro storage, platelets undergo morphological and physiological changes collectively known as the platelet storage lesion. Apoptosis is a programmed process of cell death, which has been considered as an important cause of platelet storage lesion under the common storage conditions in standard blood banks. Platelets are anucleate blood cells, but contain significant amounts of microRNA (miRNA, miR), which may play an important role in the regulation of gene expression. Drawing on previously published reports on cell apoptosis, we selected 49 miRNA for analysis to explore whether miRNA are of importance during the storage of platelets.

Materials and methods

We used quantitative real-time polymerase chain reaction analysis to determine the levels of expression of miRNA in apheresis platelets at different times of storage. Bioinformatics analysis was applied to explore target genes and the main functions of the selected miRNA.

Results

Our observations suggest that apheresis platelets contain large amounts of apoptosis-associated miRNA. The levels of expression of 25 miRNA remained high and ten of these miRNA showed different expression from that at day 0. Of these ten miRNA, hsa-miR-326, hsa-miR-96, hsa-miR-16, hsa-miR-155 and hsa-miR-150 were up-regulated, while hsa-miR-7, hsa-miR-145, hsa-miR-24, hsa-miR-25 and hsa-miR-15a were down-regulated. The markedly increased expression of hsa-miR-326 in all platelets is noteworthy (p<0.001).

Discussion

Since Bcl-xl and Bak1, members of the Bcl-2 family, are the targets of hsa-miR-326, our findings suggest that hsa-miR-326 may be involved in platelet apoptosis during storage.     

microRNA let-7a对乙型肝炎病毒复制的影响

目的 研究microRNA let-7a(以下简称let-7a)对乙型肝炎病毒(HBV)复制的影响.方法 (1)收集23例伴有慢性活动性乙型肝炎的肝细胞癌(HCC)患者的手术标本,用实时荧光定量PCR(real-time qRT-PCR)检测let-7a在HCC组织中的表达,分析let-7a的表达和HBV复制状态之间的关系.(2)用real-time qRT-PCR法检测HBV稳定复制的HCC细胞系HepG2.2.15及其亲本HCC细胞HepG2的let-7a表达.(3)通过细胞转染,利用let 7a特异的反义寡核苷酸抑制物下调HepG2.2.15细胞中let-7a的表达,通过实时PCR(real time PCR)检测HBV DNA水平的变化.结果 let-7a在HBV高复制的临床HCC标本中明显高于HBV低复制者(P＜0.05).与HepG2相比,let-7a在HBV稳定复制的HepG2.2.15细胞系中表达上调(P＜0.05).let-7a抑制剂能显著抑制HBV DNA的水平,并且具有时间依赖性(P＜0.05).结论 let-7a的表达和HBV的复制状态呈正相关,let-7a能促进HBV复制.     

BclAF1 restriction factor is neutralized by proteasomal degradation and microRNA repression during human cytomegalovirus infection

Cell proteins can restrict the replication of viruses. Here, we identify the cellular BclAF1 protein as a human cytomegalovirus restriction factor and describe two independent mechanisms the virus uses to decrease its steady-state levels. Immediately following infection, the viral pp71 and UL35 proteins, which are delivered to cells within virions, direct the proteasomal degradation of BclAF1. Although BclAF1 reaccumulates through the middle stages of infection, it is subsequently down-regulated at late times by miR-UL112-1, a virus-encoded microRNA. In the absence of BclAF1 neutralization, viral gene expression and replication are inhibited. These data identify two temporally and mechanistically distinct functions used by human cytomegalovirus to down-regulate a cellular antiviral protein.     

Let-7家族在阿尔茨海默病发病机制中的作用

阿尔茨海默病（AD）是一类病因未明的神经系统退行性疾病。近来研究发现,AD发生、发展可能与微小RNA（miRNA）的异常表达相关。Let-7家族作为第2个被发现的miRNA,参与调控细胞增殖、分化、凋亡、免疫应答、肿瘤发生及转移等多种生理病理过程。就Let-7家族在AD发病机制中的作用进行综述,旨在为探索AD新的生物标记及药物靶点提供参考。     

Down-regulation of MeCP2 in Hirschsprung's disease

Background/Purpose

Hirschsprung's disease (HSCR) is a congenital disorder characterized by the absence of intramural ganglion cells which are highly associated with impaired proliferation and migration of neural crest cells. Whether methyl CpG binding protein 2 (MeCP2) is related with HSCR still remains unknown. This study investigates the involvement of MeCP2 in HSCR.

Methods

Quantitative real time PCR and western blot were used to detect the expression level of MeCP2 both in the aganglionic/diseased segment and the ganglionic/normal segment. In vitro assays we used siRNAs to knock-down the expression of MeCP2 in SH-SY5Y cell lines, and furthermore, MTT and transwell assays were used to detect the proliferation and migration ability, respectively. In addition, bisulfite sequencing (BSP) and miRNA analysis were used to examine why MeCP2 is decreased in HSCR samples.

Results

MeCP2 exhibited a lower expression level in tissues of HSCR patients compared with the controls. The down-regulation may also suppress the proliferative ability of the cells. However, there was no significant difference in the MeCP2 methylation level between cases and controls. Similarly, there was no difference between cases and controls in miRNA-34b (miR-34b) which is predicted to regulate MeCP2 through complementary binding to the 3′-untranslated region of MeCP2.

Conclusion

Our results indicated that an aberrant decreased level of MeCP2 may play an important role in the pathogenesis of HSCR.     

塞来昔布联合p65miRNA抑制人乳腺癌移植瘤的生长

目的探讨下调核因子NF-κB p65亚基的表达联合塞来昔布对人乳腺癌裸鼠移植瘤生长的协同抑制效应及其机制。方法建立人乳腺癌裸鼠移植瘤模型,随机将裸鼠分为6组,control组、Neg-miRNA组、p65miRNA组、塞来昔布组、塞来昔布+Neg-miRNA组、塞来昔布+p65miRNA组。观察治疗前后裸鼠的一般状态、肿瘤体积和瘤重,计算平均抑瘤率。免疫组化法检测p65miRNA干扰后肿瘤组织中p65蛋白的表达。RT-PCR、Western印迹法检测肿瘤组织中血管内皮生长因子(VEGF)、金属基质蛋白酶(MMP-2)基因mRNA及蛋白表达的变化。结果塞来昔布组、p65miRNA组和塞来昔布+p65miRNA组肿瘤生长受到明显抑制,抑瘤率分别为43.23%、40.72%及61.69%。p65miRNA组较对照组p65蛋白表达明显减少。塞来昔布组、p65miRNA组和塞来昔布+p65miRNA组VEGF、MMP-2 mRNA及蛋白表达受到不同程度地抑制。结论塞来昔布及p65miRNA均具有明显的抗肿瘤作用,联合应用时具有一定的协同作用,可显著抑制人乳腺癌裸鼠移植瘤的生长,并可显著下调VEGF、MMP-2的表达。     

非编码RNA调控晶状体上皮细胞死亡在白内障中的作用

非编码RNA（ncRNA）是不编码蛋白质的RNA,其中包括微小RNA（miRNA）、长链非编码RNA（lncRNA）和环状RNA（circRNA）等。ncRNA可以广泛参与细胞增殖、分化、生长以及细胞死亡等重要的生物学过程。ncRNA的异常表达可导致晶状体上皮细胞发生细胞凋亡、焦亡以及自噬异常,使得晶状体透明度下降,从而导致白内障的发生。本文主要对8种miRNA（miR-125b、let-7a、miR-4328、miR-34a、miR-133b、miR138、miR-23b-3p、miRNA-30a）、9种lncRNA（lncRNA MIAT、lncRNA ANRIL、lncRNA PLCD3-OT1、lncRNA GPX3-AS、H19、TUG1、lncRNA PVT1、lncRNA ALB、KCNQ1OT1）以及一种circRNA （circHIPK3）调控晶状体上皮细胞死亡在白内障发生发展中的作用机制最新进展予以综述。