UCA1及其剪接变异体与膀胱癌

尿路上皮癌胚抗原1 (urothelial carcinoma antigen 1,UCA1) 在人膀胱移行细胞癌细胞系 BLZ-211 中包含3个剪接变异体：UCA1、UCA1a 和 UCA1b. 我们以往的研究表明, UCA1、UCA1a 均属于长非编码 RNA (long non-coding RNA,lncRNA),它们之间具有一段长 1 265 bp 的共同序列. 组织表达谱分析表明,它们具有相似的组织表达模式,提示它们可能与胚胎发育和膀胱癌发生发展密切相关. 异位表达UCA1基因、UCA1a 基因均可以促进人膀胱癌细胞生长,增强细胞的恶性表型,使其体外增殖、迁移、侵袭、抗凋亡能力明显增加,裸鼠致瘤能力明显增强,表明它们在膀胱癌的发生发展中均起到了重要的促进作用. 本文将从基因结构、组织表达谱及机制功能等不同角度系统地阐述UCA1基因及其剪接变异体在膀胱癌中的研究现状与进展.     

PNRC1剪接变异体的鉴定及其在辅激活核受体介导基因转录功能上的比较

为分析富含脯氨酸核受体辅调节蛋白1(PNRC1)选择性剪接, 及比较PNRC1剪接变异体在辅激活核受体介导基因转录功能上的差异,在生物信息学方法分析PNRC1剪接变异体的基础上,设计一定的特异性引物,采用RT-PCR结合克隆测序的方法对这些剪接变异体进行验证. 利用酵母双杂交和荧光素酶报告系统实验,分析它们与核受体的相互作用及比较它们在辅激活核受体介导基因转录功能上的差异.结果显示,生物信息学预测的几个剪接变异体真实存在于人的组织和细胞系中,这些剪接变异体在与雌激素受体α(ERα)、类固醇衍生因子1(SF1)等核受体的相互作用的强度及辅激活核受体介导基因转录功能上存在较大的差异. 研究提示,PNRC1这些剪接变异体在体内可能发挥不同的功能.     

人受精促进肽受体基因（TCP11b）的剪接、克隆和差别表达

运用同源比较和PCR法 ,从人睾丸组织中分离了人受精促进肽受体TCP11基因的一个新的剪切体TCP11b ,它编码 5 0 3个氨基酸的蛋白质 ,与TCP11a相比 ,在基因组的 5′端存在复杂的外显子剪接现象。运用荧光原位杂交 (FISH)方法 ,显示该基因定位到人染色体 6p2 1。Northern杂交及多组织RT PCR的结果显示该转录本在正常睾丸中表达 ,而其他组织、无精症患者及胎儿睾丸组织中未见该基因的表达。该结果结合mTcp 11功能的提示 ,TCP11b这种转录本对精子发生和人受精过程可能起重要作用。     

绵羊皮肤中GHR、IGF-1和IGF-1R基因表达的发育性变化及品种特点

采用相对定量反转录多聚酶链式反应 (RT-PCR)方法, 以18S rRNA作内标, 研究了罗米丽(Romilly Hillys)×中国美利奴(新疆军垦型)杂交一代优质细毛羊和哈萨克粗毛羊皮肤中生长激素受体(GHR)、胰岛素样生长因子1(IGF-1)和胰岛素样生长因子1受体(IGF-1R) mRNA发育性变化并进行了品种间比较。分别于30、60、90、135、180和255日龄称重、采毛样, 并于30、90、135和255日龄采皮样。结果表明: 粗毛羊和细毛羊体重、羊毛生长的发育模式没有明显的差异, 30~135日龄体重迅速增加, 135~255日龄增重十分缓慢; 30~135日龄羊毛日增长逐渐增加, 135~180日龄羊毛生长十分缓慢, 而180~255日龄又上升到较高水平。粗毛羊皮肤中GHR mRNA在30~90日龄显著增加 (P<0.05), 90日龄达到高峰, 此后显著下降(P<0.05); 细毛羊在135日龄时GHR mRNA极显著地升高(P<0.01), 此后又极显著地下降。粗毛羊皮肤中IGF-1、IGF-1R mRNA 30~90日龄上升, 90日龄之后极显著下降(P<0.01); 细毛羊皮肤中IGF-1、IGF-1R mRNA出生时较高, 然后逐渐下降。品种之间比较, 细毛羊GHR mRNA出现高峰晚于粗毛羊, 135日龄高峰时显著地高于粗毛羊; 粗毛羊IGF-1、IGF-1R mRNA在90日龄出现高峰, 并极显著或显著地高于细毛羊; 粗毛羊90日龄前GHR、IGF-1和IGF-1R mRNA高于细毛羊, 之后低于细毛羊。结果提示: 绵羊皮肤中GHR、IGF-1和IGF-1R基因表达有特定的发育模式, 并存在品种差异。     

蚕豆细胞核仁和核质中剪接因子SC35的定位研究

本文以蚕豆(Vicia faba L.)根端分生组织细胞为材料,以抗SC35抗体为探针,在电镜下对SC35在高等植物细胞中的存在与否和分布特点进行了研究,发现经抗SC35抗体标记后,标明SC35位置的胶体金颗粒主要分布于核仁的致密纤维组分(DFC)、核质的染色质间颗粒(IGs)和染色质周边纤维处(PFs),而核仁的纤维中心(FC)、核仁液泡和集缩染色质团块中央部位的金颗粒很少。DFC, IGs和PFs处的金颗粒平均密度分别为65.89个/μm~2和36.28个/μm~2,远远高于集缩染色质团块中央部位以及FC和核仁液泡处的金颗粒平均密度(分别为5.90个/μm~2和6.26个/μm~2)。说明蚕豆细胞核仁的DFC,核质的IGs和PFs处富含剪接因子SC35。本文研究结果表明,SC35或SC35类蛋白在蚕豆细胞核质中的分布与其在哺乳动物细胞核质中的分布规律相似。同时本文首次报道了SC35或SC35类蛋白存在于核仁中。     

重组PAC1-EC1(N)对表达不同PAC1变体的细胞系细胞活性的影响

垂体腺苷酸环化酶激活多肽(pituitary adenylate cyclase-activating polypeptide,PACAP)特异受体PAC1(normal型)N端胞外域[简称PAC1-EC1(N)]具有调控PAC1活性的作用.为研究PAC1-EC1(N)对表达不同PAC1变体的细胞系活性的影响,利用基因...     

用于前体mRNA剪接机制研究的小基因模型的建立和研究应用

建立可用于选择性前体mRNA剪接分析的小基因模型. 以人或小鼠基因组DNA为模板, 通过PCR扩增获得GluR-B, FGF-2R和Zis小基因片段, 并将其克隆至真核表达载体中, 构建了小基因的质粒. 在此基础上, 将上述3个小基因模型和剪接因子Tra2β1或Zis2表达质粒共转染HeLa细胞, 并用RT-PCR进行了被剪接的小基因产物的半定量检测. 结果表明, 这些小基因可用于细胞水平的基因剪接分析, 利用该技术平台, 发现了Zis2亚型可以促进Zis小基因选择性剪接.     

杀伤血管内皮生长因子受体 1 阳性细胞的靶向毒素

白喉毒素 (diphtheria toxin DT) 是棒状白喉杆菌被β噬菌体感染后分泌的一种外毒素. 它可以阻断真核细胞的蛋白质合成,杀死细胞. 血管内皮生长因子 (VEGF) 的 R82A, K84A, H86A 突变体可以和肿瘤血管上高表达的 VEGF 受体 1 (VEGFR-1) 特异性结合. 首先从白喉杆菌中提取基因组 DNA,扩增出白喉毒素 C 区、 T 区基因. 并运用点突变技术,制成 VEGF 的 R82A, K84A, H86A 突变体. 利用这个可以和肿瘤血管上特异性受体相结合的 VEGF 的突变体,代替白喉毒素上的受体结合区,制成了针对 VEGFR-1 的靶向融合毒素——— DT391-mVEGF. 以去除了受体结合区的 DT391 为阴性对照,细胞实验表明,融合毒素对 VEGFR-1 阳性的肿瘤细胞有特异性杀伤作用.     

促甲状腺激素释放激素受体-1(TRH-R1)蛋白在大鼠出生后睾丸不同发育阶段的表达和定位

目的研究促甲状腺激素释放激素受体-1(thyrotrophin-releasing hormone receptor type-1, TRH-R1)在大鼠睾丸出生后不同发育阶段的表达,探讨其在生殖发育调节中的作用.方法应用蛋白质免疫印迹杂交技术以及免疫组织化学ABC法检测TRH-R1在8d、15d、20d、35d、60d和90d大鼠睾丸中的表达和定位,并结合图像分析技术对免疫组化结果进行统计学分析观察其在发育过程中的变化.结果免疫印迹杂交发现TRH-R1蛋白表达于15d以后各阶段的大鼠睾丸;而运用免疫组化在第8d即检测到TRH-R1的表达,以后发育过程中的各个阶段均有阳性反应细胞, TRH-R1定位于大鼠睾丸的间质细胞;免疫反应阳性物均位于胞膜和胞质,胞核区为阴性;图像分析结果表明,随着大鼠睾丸的发育,TRH-R1表达量呈增多趋势,且具有统计学差异(P＜0.01).结论本实验证明TRH-R1在出生后8d大鼠的睾丸内即有表达,并持续表达于其后各个发育阶段;TRH-R1定位于睾丸的间质细胞,其表达量随着增龄变化呈增多趋势,即同发育过程相关.     

稳定表达“毒性”瞬时受体势A1通道细胞系的建立

瞬时受体势A1 (TRPA1) 是一种对低温敏感的离子通道,除响应温度外,也可被各种刺激性化合物激活,是许多感觉模型的转导通道．建立TRPA1异源表达系统将为药理分析及功能研究提供很大的便利,但是TRPA1的表达会引发细胞毒性,因此构建TRPA1稳定细胞系一直面临着挑战．在人胚肾细胞(HEK-293)中非调控的表达TRPA1稳定细胞系被成功建立．实验证实,培养至25代以上,该细胞系仍持续表达TRPA1,且细胞的功能检测也进一步验证了该重组TRPA1细胞系的稳定性及特异性．TRPA1-HEK细胞系不但是TRPA1功能性分析的便利工具,而且可应用于高通量药物筛选系统,鉴定TRPA1特异性调节剂．     

WITHDRAWN: Endosomal trafficking of EGFR regulated by hVps18 via interaction of MVB sorting machinery

Here, we report the cloning and characterization of RTVP-1b, a novel splice variant of human RTVP-1, which was isolated from the U87 glioma cell line. Sequence analysis revealed that RTVP-1b contains an additional 71 base exon between exons 2 and 3 that is missing in RTVP-1, leading to a frame-shift and a different putative protein. The deduced protein was 237 amino acids in length, sharing the N-terminal 141 amino acids with RTVP-1. RT-PCR analysis demonstrated that RTVP-1b was expressed in a wide range of tissues and that its expression was different from that of RTVP-1. In contrast, RTVP-1 and RTVP-1b showed similar patterns of expression in astrocytic tumors; highly expressed in glioblastomas as compared to normal brains, low-grade astrocytomas and anaplastic oligodendrogliomas. Overexpression of RTVP-1b increased glioma cell proliferation but did not affect cell migration. Our results suggest that RTVP-1b represents a potential prognostic marker and therapeutic target in gliomas.     

Molecular cloning and characterization of mouse aquaporin 6

In the rat kidney, aquaporin (AQP) 6 is localized in the intracellular vesicle membranes of type-A intercalated cells of the collecting duct; mouse AQP6 (mAQP6) has not been characterized. Although mAQP6 was originally cloned from cDNA in a mouse cerebellum library (GenBank NM 175087), we have independently cloned a cDNA encoding mAQP6 from an adult kidney cDNA library (C57BL/6J strain). We identified two different spliced variants of mAQP6: mAQP6a and mAQP6b. The mAQP6a isoform is almost identical to that of rat AQP6, whereas mAQP6b is identical to that reported in the mouse cerebellum library mentioned above. We found that the mRNA expression of these two spliced variants is regulated in a tissue-specific and age-dependent manner. Functional analyses of water and ion permeation revealed that mAQP6a functions like rat AQP6 and that mAQP6b does not function as either a water channel or an ion channel under our experimental conditions.     

Identification of a novel splice variant of X-linked inhibitor of apoptosis-associated factor 1

XAF1 (XIAP-associated factor 1) binds to XIAP and blocks its anti-apoptotic activity. It has been reported that XAF1 is mainly expressed in normal tissues but is missing or present at low levels in most cancer cell lines, which implies a tumor-suppressing function. In the present study we describe the identification of a novel splice variant of human XAF1, designated XAF1C, which contains a cryptic exon. Incorporation of this exon (exon 4b) into the mRNA introduces an in-frame stop codon, resulting in a shortened open-reading frame (ORF) of 495 nucleotides. This ORF is predicted to encode a 164 amino acid (AA) protein lacking the C-terminal domain of the previously described XAF1(A), but containing a unique 24 AA carboxy terminus. Like XAF1(A), XAF1C mRNA expression was detected in a variety of human cancer cell lines and also in normal human tissues. The ratio of XAF1(A) and XAF1C mRNA expression differs amongst the cell lines tested, suggesting differential mRNA stabilities and/or the existence of tissue- or cell type-specific splicing regulation. In transfected cells, xaf1c encodes a truncated protein of 18kDa, which is distributed primarily in the nucleus.     

Discovery of potent transient receptor potential vanilloid 1 antagonists: Design and synthesis of phenoxyacetamide derivatives

We aimed to discover a novel type of transient receptor potential vanilloid 1 (TRPV1) antagonist because such antagonists are possible drug candidates for treating various disorders. We modified the structure of hit compound 7 (human TRPV1 IC₅₀ = 411 nM) and converted its pyrrolidino group to a (hydroxyethyl)methylamino group, which substantially improved inhibitory activity (15d; human TRPV1 IC₅₀ = 33 nM). In addition, 15d ameliorated bladder overactivity in rats in vivo.     

A truncated splice variant of KCNQ1 cloned from rat heart

KCNQ1 encodes a pore-forming subunit of potassium channels. Mutations in this gene cause inherited diseases, i.e., Romano-Ward syndrome and Jervell and Lange-Nielsen syndrome. A truncated isoform of KCNQ1 was reported to be expressed physiologically and to suppress a delayed rectifier potassium current dominant-negatively in human heart. However, it is not known whether this way of modulation occurs in other species. We cloned another truncated splice variant of KCNQ1 (tr-rKCNQ1) from rat heart. Judging from the deleted sequence of the tr-rKCNQ1, the genomic structure of rat in this portion might be different from those of human and mouse. Otherwise, an unknown exon might exist. RT-PCR analysis demonstrated that the tr-rKCNQ1 was expressed in fetal and neonatal hearts. When this gene was expressed along with a full-length KCNQ1, it suppressed potassium currents, whether a regulatory subunit minK was co-expressed or not.     

Characterization of the canine mda-7 gene,transcripts and expression patterns

Human melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) displays potent growth suppressing and cell killing activity against a wide variety of human and rodent cancer cells. In this study, we identified a canine ortholog of the human mda-7/IL-24 gene located within a cluster of IL-10 family members on chromosome 7. The full-length mRNA sequence of canine mda-7 was determined, which encodes a 186-amino acid protein that has 66% similarity to human MDA-7/IL-24. Canine MDA-7 is constitutively expressed in cultured normal canine epidermal keratinocytes (NCEKs), and its expression levels are increased after lipopolysaccharide stimulation. In cultured NCEKs, the canine mda-7 pre-mRNA is differentially spliced, via exon skipping and alternate 5′-splice donor sites, to yield five splice variants (canine mda-7sv1, canine mda-7sv2, canine mda-7sv3, canine mda-7sv4 and canine mda-7sv5) that encode four protein isoforms of the canine MDA-7 protein. These protein isoforms have a conserved N-terminus (signal peptide sequence) and are dissimilar in amino acid sequences at their C-terminus. Canine MDA-7 is not expressed in primary canine tumor samples, and most tumor derived cancer cell lines tested, like its human counterpart. Unlike human MDA-7/IL-24, canine mda-7 mRNA is not expressed in unstimulated or lipopolysaccharide (LPS), concanavalin A (ConA) or phytohemagglutinin (PHA) stimulated canine peripheral blood mononuclear cells (PBMCs). Furthermore, in-silico analysis revealed that canonical canine MDA-7 has a potential 28 amino acid signal peptide sequence that can target it for active secretion. This data suggests that canine mda-7 is indeed an ortholog of human mda-7/IL-24, its protein product has high amino acid similarity to human MDA-7/IL-24 protein and it may possess similar biological properties to human MDA-7/IL-24, but its expression pattern is more restricted than its human ortholog.