长链非编码RNA在肿瘤发生、发展中的作用研究进展

非编码RNA是指一类不编码蛋白质,但在生物体生命活动中具有功能的RNA分子。非编码RNA在发育和疾病中有众多角色,而其中一个突出的作用是调节基因的表达。非编码RNA按其长度可分为短链非编码RNA和长链非编码RNA（long non coding RNA,lncRNA）。LncRNA是一类长度大于200 nt、广泛存在于哺乳动物细胞中的一类蛋白非编码序列。LncRNA通过剂量补偿效应、表观遗传调控和细胞分化调控等环节在生命活动中发挥重要作用,尤其在一些复杂疾病的发生、发展过程中发挥重要功能。近年来,有关lncRNA在肿瘤发生、发展中的作用研究日益增多,已经成为肿瘤发病机制的研究热点。作者就lncRNA在肿瘤发生、发展中的功能及作用机制进行综述。     

长链非编码RNA在癌症诊断中的应用

人类基因组中蛋白质非编码基因占大多数,这些非编码基因与癌症的发生、发展有密切联系。非编码基因转录形成的RNA称作非编码RNA。其中的长链非编码RNA(long non coding RNA,lncRNA)具有重要的基因调控功能并由此受到重视。由于部分lncRNA在各类型的癌症中表达异常,因此可以作为癌症的标志物用以诊断癌症。本文对lncRNA的分类、基因调控机制以及在癌症中的作用进行介绍,并对现有的以及部分潜在的癌症标志物lncRNA进行详细阐述。随着基因测序技术及微阵列技术的发展,更多的lncRNA会被发现并且能够成为重要的诊断肿瘤的标志物。     

微RNA:一类新的基因调控因子

微RNA是新发现的一类非编码RNA分子，在动植物细胞中广泛表达，它的基因是短的反向重复序列。一般认为微RNA参与调控mRNA的翻译，并与其稳定性有关。微RNA可能在细胞中起着非常重要的作用。     

长链非编码RNA H19与肿瘤耐药的相关性研究进展

长链非编码RNA (LncRNA)是一类由于缺乏开放阅读框而丧失蛋白编码能力的RNA序列,临床上与多种疾病的发生相关,大量研究表明LncRNA在肿瘤耐药过程中起着关键调控的作用,已经证实相关LncRNA如H19的异常表达与肿瘤的耐药有关,因此,LncRNA与肿瘤耐药的相关性研究意义重大。本文就LncRNA介导肿瘤耐药过程的发生机制及LncRNA H19与肿瘤耐药中的相关研究进展进行综述。     

14 q32 miRNA基因簇与肝细胞癌的发生发展

肝细胞癌（肝癌,hepatocellular carcinoma ,HCC ）是全球范围内发生率和死亡率较高的恶性肿瘤之一。微小RNA（ microRNA,miRNA）是一类内源性、非编码、高度保守的单链小分子RNA,主要在转录后水平抑制靶基因的表达。有些位置相近的miRNA基因在染色体上成簇排列,在一个多顺反子内形成miRNA基因簇,通常以共表达的形式协同作用。在人类14号染色体长臂端的14q32印迹基因区域约215 kb的基因组范围内,聚集了52个miRNA基因。已有研究发现此miRNA基因簇的异常表达与肝癌的发生发展密切相关。该文概括了14 q32 miRNA基因簇的结构特点,并对其在肝癌发生发展过程中所发挥的作用进行了综述。     

干扰HERG钾通道表达可抑制成神经细胞瘤细胞增殖

目的：采用RNA干扰（RNA interference，RNAi）技术抑制HERG基因的表达，探讨HERG基因编码钾通道对人成神经细胞瘤细胞（SH-SY5Y）存活及增殖的影响。方法：构建发卡状RNA干扰质粒（shRNA-HERG）转染SH-SY5Y细胞，再分别使用半定量RT-PCR和Western印迹方法观察shRNA-HERG对SH-SY5Y细胞中HERG基因mRNA及其编码蛋白表达的干扰效果。利用SH-SY5Y细胞的生长曲线和集落形成实验，观察转染shRNA-HERG质粒对SH-SY5Y细胞存活及增殖能力的影响。结果：转染shRNA-HERG干扰质粒后，SH—SY5Y细胞中HERG基因的mRNA水平及其编码的钾通道蛋白表达水平显著下降。与对照组相比，shRNA—HERG使SH-SY5Y细胞生长曲线明显下压，细胞倍增时间延长136．8％。集落形成实验结果显示，无论接种细胞数为50，100或200，shRNA-HERG组克隆形成率均显著下降，与shRNA-control组和未干扰组相比，差异有统计学意义（P〈0．01）。结论：所构建的shRNA-HERG表达质粒能有效抑制HERG基因编码钾通道蛋白的表达，明显抑制SH-SY5Y细胞的生长和增殖。     

非编码RNA参与昼夜节律调控研究进展

微小RNA(microRNA)、长链非编码RNA(lncRNA)、环状RNA(circRNA)等非编码RNA(noncoding RNAs,ncRNA)属于表观遗传调控因子,通过在染色质水平、转录及转录后水平、翻译水平调控基因表达和功能,进而影响各种细胞生物学过程。在人体生理活动中,昼夜节律受到多种内外因素的调节,包括ncRNA。本文总结了参与调节昼夜节律的ncRNA,并讨论了在钟基因功能中ncRNA的作用机制,为进一步探究ncRNA在昼夜节律调控中的作用提供新的研究思路,为节律相关疾病的研究和诊治提供借鉴。     

miR-93在肿瘤发生发展中的作用机制

MicroRNAs(miRNAs)是长约20~25个核苷酸的非编码RNA,在肿瘤中表达异常,发挥癌基因或抑癌基因的作用。miR-93是miR-106b-93-25簇的成员之一,在胃癌、乳腺癌和肺癌等肿瘤中高表达。miR-93通过调节靶基因的表达,参与调控肿瘤细胞增殖、转移和凋亡等过程,因此其表达水平与肿瘤的发生、发展、转移和预后等相关。此外,miR-93的表达还与抗肿瘤药物的耐药性相关。因此miR-93是肿瘤治疗的一个潜在的重要靶点。     

MicroRNA在神经损伤后基因表达调控和神经修复中的作用

MicroRNA(miRNA)是一系列能通过序列特异性方式来调节基因表达的非编码小分子RNA.它是由基因编码形成的核蛋白复合物,通常由19～25个核苷酸组成[1],能与信使RNA(mRNA)通过分子互补配对结合诱导其降解或抑制其翻译.     

人源汉坦病毒H8205株M基因序列分析及其分类地位的研究

目的 :测定汉坦病毒H82 0 5株M基因全序列并对其分类地位进行探讨。方法 :从感染H82 0 5株病毒的鼠脑中提取RNA ,经RT PCR扩增M基因全序列 ,克隆到 pGEM TEasy载体中测序 ,与汉坦病毒属的各型标准株进行同源比较 ,构建系统进化树 ,并与汉滩型中已知的 7株病毒进一步比较 ,确定H82 0 5株的分类地位。结果 :H82 0 5株M基因由 36 15个核苷酸组成 ,编码1135个氨基酸 ;同源性分析显示该株病毒属于布尼亚病毒科汉坦病毒属中的汉滩型 (HTN) ,但与同型中其他毒株的亲缘关系较远。结论 :H82 0 5株是不同于已知汉滩型毒株的一个新亚型 ,表明我国的汉滩型病毒存在不同亚型     

Parentally imprinted allele (PIA) typing in the differentially methylated region upstream of the human H19 gene

The H19 gene is a paternally imprinted gene located on chromosome 11p15.5. In this study, the H19FR1 and H19FR2 haplotype polymorphisms including four and three SNPs, respectively, upstream of the H19 gene according to the GenBank sequence (accession no. AF125183) were investigated. Five haplotypes and nine genotypes were detected for H19FR1 in the Chinese Han population by means of PCR and subsequent denaturing gradient gel electrophoresis (DGGE). The power of discrimination (Dp), polymorphism information content (PIC) and probability of paternity exclusion (PE) were estimated to be 0.803, 0.58 and 0.322, respectively. For the H19FR2, two haplotypes and three genotyes were observed, and the Dp, PIC and PE were 0.626, 0.37 and 0.162, respectively. Sequencing results showed that only two of the four reported SNPs, a7342g and g7547a, were detected in H19FR1 in the Chinese Han population, and two new SNPs, g7351c and a7357g, were found. In the H19FR2 region, only one of the three reported SNPs, a8097g, was detected. Based on the methylation status of the genomic DNA, selective detection of the parental alleles for H19FRs was examined by using two types of enzymes, the methylation-sensitive restriction enzyme (msRE) HpaII or HhaI and McrBC. Genomic DNA digested by either HpaII or HhaI, revealed a single band derived from the paternal allele, as a result of cleavage of unmethylated recognition sites on the maternal allele. On the contrary, the use of McrBC, which can digest a methylated paternal sequence, resulted in exclusively amplifying the maternal allele. This parentally imprinted allele (PIA) typing method could be one of the useful techniques for discriminating the parental origin of alleles.     

A parent-of-origin detectable polymorphism in the hypermethylated region upstream of the human<Emphasis Type="Italic"> H19</Emphasis> gene

The H19 gene is a paternally imprinted gene located on chromosome 11p15.5. In this study the H19FR haplotype polymorphism including three SNPs upstream of the H19 gene was investigated. Six genotypes derived from three alleles were detected in the Japanese population by means of PCR and subsequent constant denaturing gel electrophoresis. Based on the methylation status of the genomic DNA from blood samples, selective detection of the parental allele for H19FR was examined by using two types of enzyme, the methylation-sensitive restriction enzymes HpaII or HhaI and McrBC. Genomic DNA digested by either HpaII or HhaI, revealed a single band derived from the paternal allele, as a result of cleavage of unmethylated recognition sites on the maternal allele. On the contrary, the use of McrBC, which can digest a methylated paternal sequence, resulted in exclusively amplifying the maternal allele. This method could be one of the useful techniques for discriminating the parental origin of alleles.     

Applicability of the parentally imprinted allele (PIA) typing of a VNTR upstream the H19 gene to forensic samples of different tissues

The parentally imprinted allele (PIA) typing that we have recently developed determines parental alleles at a VNTR locus in the differentially methylated region upstream of the human H19 gene. The usefulness of this typing was demonstrated by its application to blood samples in paternity cases. However, its applicability to other tissue DNA remains to be tested. DNA samples from fifteen different postmortem tissues such as cerebrum, skeletal muscle and skin were examined, all of which were obtained from three autopsy cases 2-11h after death. DNA was digested with a methylation-sensitive HhaI enzyme and diluted solutions of the digests were subjected to the first PCR amplification, providing amplification of only the paternal H19 methylated allele. Subsequent VNTR typing was carried out for the amplified products to determine which allele was of paternal origin. No tissue-dependent difference was observed and all the samples examined, though degraded, were successfully used for determining the paternal allele. These results substantiate the usefulness of PIA typing in forensic examinations. Its application to two identity cases, a burned male body and a male body with adipocere formation, was also shown.     

宫颈癌中H19基因的特异表达模式及印记缺失分析

目的探讨H19等位基因在宫颈癌中特异性表达的模式,H19基因的基因组印记缺失与宫颈癌两者之间的关系。方法结合PCR和限制性片段长度多态性分析（RFLP）技术以及DNA甲基化检测技术分析我院收集的宫颈癌标本H19等位基因进行杂合子筛选,并进一步研究基因组印记缺失与宫颈癌两者之间的关系。结果在36例宫颈癌中筛选出16例杂合子现象的标本,在这16例杂合子标本中,有7例发生了基因组印记缺失;H19基因发生基因组缺失现象与其启动子区域的高度去甲基化正相关。结论H19基因的基因组印记缺失可能和宫颈癌的发生有着高度的相关性。     

Methylation-sensitive restriction enzyme nested real time PCR,a potential approach for sperm DNA identification

Mammal H19 gene is an imprinting gene in which the paternal allele is silenced. On H19 imprinting control region (ICR), one of the mechanisms regulating the paternal allelic specific silence is DNA methylation in somatic cells throughout the individual's whole life. Nevertheless, this pattern of DNA methylation is erased and re-established in germline. As results, in mature sperm H19 ICR shows biallelic methylation instead of paternal specific methylation in somatic cells. Although the data were mainly from experiments on mice the same mechanisms are believed existing in human germline. We designed an experiment to probe the sperm DNA by methylation sensitive restriction enzyme based nested qPCR (MSRE-nested-qPCR). The genomic DNA digested/undigested by HhaI was amplified by outer primers encompassing four HhaI sites on H19 ICR. These PCR products were used as templates for second round real-time PCR to quantify the DNA methylation level. The results showed that DNA methylation level at H19 ICR were 55.27 ± 8.36% in 32 blood samples and 101.94 ± 11.66% in 31 semen samples. Based on our data sperm DNA could be identified if H19 ICR methylation level is over 78.62%.     

辐射对小鼠精子发生过程中印记基因表达的影响

目的筛选在小鼠精子发生过程中受电离辐射影响表达发生改变的印记基因。方法选择雄性BALB／c小鼠8只，分为实验组和对照组X射线全身照射0．1Gy／d，连续13d，建立辐射干扰小鼠精子发生模型。提取实验组和对照组睾丸RNA，采用上海生物芯片中心小鼠寡核苷酸基因表达谱芯片筛选表达发生改变的印记基因，对感兴趣的基因经半定量RT—PCR验证。结果12个表达发生改变的印记基因，6个上调，6个下调，其中Igf2、Peg3基因Ratio值分别为3．859和0．397，经半定量RT—PCR验证与芯片结果相符。结论X射线全身照射可导致小鼠睾丸部分印记基因表达发生改变。     

长链非编码RNA在甲状腺癌中的研究进展

甲状腺癌是近年来发病率快速增长的内分泌肿瘤。人类恶性肿瘤与环境因素密切相关,环境改变可诱导机体内某些致病基因发生变化,从而促进了疾病的发生。在对人类的基因转录组的研究过程中,发现了一类长度超过200个核苷酸的非编码RNA,即长链非编码RNA（LncRNAs）,其通过调节基因表达参与细胞分化、增殖、凋亡、迁移和侵袭等肿瘤的发生发展。越来越多的研究发现LncRNAs与甲状腺癌关系密切,许多LncRNAs对甲状腺有致癌或抑癌作用,但其具体功能和作用机制尚不明确。笔者对LncRNAs在甲状腺癌中的最新研究进展进行综述,为探讨LncRNAs在甲状腺癌中的作用机制及其临床应用价值提供依据。     

Forensic STRs as potential disease markers: A study of VWA and von Willebrand's Disease

In recent years it has been established that non-coding variants may be in linkage disequilibrium (LD) with coding variants up to several thousand base pairs away forming haplotype blocks. These non-coding markers may be haplotype specific and, therefore, informative regarding the surrounding coding sequence. In this study, we chose to study the VWA short tandem repeat (STR) as it is targeted in all major commercial kits utilized in routine forensic DNA profiling and is located in the von Willebrand Factor (vWF) gene; a gene associated with von Willebrand's Disease (vWD). We examined the VWA STR together with single nucleotide polymorphisms (SNPs) located throughout the vWF gene to identify haplotype structures and the extent of LD between markers in the region. Several areas exhibiting LD were identified by population data analysis in the 178 kilobase (178kb) vWF gene, which was supported by family studies. However, there appeared to be no evidence of LD blocks surrounding the VWA STR and evidence for recombination within 3 kb of VWA, hence, it is unlikely that VWA STR alleles could be used to predict haplotypes within the vWF gene that are associated with different forms of vWD.     

Parentally imprinted allele typing at a short tandem repeat locus in intron 1a of imprinted gene KCNQ1

A short tandem repeat (STR) in the intron 1a of paternally imprinted gene, KCNQ1, is evaluated as a new probe for use in parentally imprinting allele (PIA) typing. This typing can determine the inheritance of one allele from father by the methylation difference. Allelic and genotypic frequencies of the STR were determined using samples from 175 unrelated Japanese and 170 unrelated Germans. The polymorphism information contents were 0.652 and 0.634 for the Japanese and the Germans, respectively, indicating usefulness in individual identification. This method was applied to five Japanese families consisting of 19 individuals. Genomic DNA was digested by methylation-sensitive restriction endonucleases, HhaI and HapII, followed by PCR amplification using two-step sandwich primer sets and the products were analyzed on polyacrylamide gel electrophoresis. For all of the families, each child's paternal allele given by PIA typing corresponded to one of the two alleles from father, not the two from mother, that were determined by the STR genotyping. The results demonstrate that this STR probe is feasible for use in PIA typing and that its typing method can contribute to paternity testing.