SPAG4L在人类睾丸中的表达研究

目的:观察人类睾丸新基因SPAG4L在人类不同发育阶段睾丸及隐睾中的表达,为了解该基因在精子发生中的功能奠定基础;方法:收集流产胎儿、成年人、老年人及隐睾患者的睾丸组织,应用RT-PCR和组织原位杂交技术检测SPAG4L mRNA的表达;结果:RT-PCR和组织原位杂交技术检测结果发现,SPAG4L在胎儿睾丸中几乎检测不到,在成年及老年男性睾丸中均有高表达,主要在精母细胞和圆形精子细胞中表达;在隐睾患者的睾丸中,精母细胞大量凋亡,SPAG4L表达明显下调;结论:SPAG4L主要在精子发生减数分裂阶段表达,受生长发育调控,提示该基因可能在精子发生减数分裂阶段发挥着重要的生理功能.     

DNA甲基化对癌睾丸抗原基因表达的调控及其意义

癌睾丸抗原所具有的表达特性和免疫原性使其有可能成为肿瘤免疫治疗的靶抗原,通过DNA去甲基化提高癌睾丸抗原基因的表达,是提高癌睾丸抗原疫苗疗效的途径之一。     

精子相关抗原4的功能及其在疾病中的作用

精子相关抗原4(sperm associated antigen 4,SPAG4)是SUN(Sad-1,UNC-84)蛋白家族成员。近年来的研究发现,SPAG4可以维持精子头部结构的完整性,参与精子尾部发育,其功能异常可导致雄性不育。此外,SPAG4 mRNA在多种肿瘤细胞中表达,是一种潜在的肿瘤标志物。对肾透明细胞癌的最新研究发现,SPAG4表达增高将促进肿瘤细胞的增殖和侵袭,其表达受缺氧诱导因子1(hypoxia inducible factor 1,HIF-1)调控。对SPAG4功能的深入研究,将有助于阐明男性不育的分子机制并为研究肿瘤的发生发展提供新的候选靶标。     

肿瘤-睾丸-脑抗原家族新成员PNMA5的克隆和表达谱分析

综合运用几种生物信息学数据库 ,分析了人类X染色体上可能的未知基因 ,筛选获得一个新的肿瘤抗原基因并克隆鉴定了它的全长cDNA .该基因全长为 3194bp ,编码一个 4 48个氨基酸残基的蛋白质 ,它与肿瘤 睾丸 脑抗原家族成员PNMA1、PNMA2及PNMA3有很高的同源性 ,命名为PNMA5 .在 16种人正常组织中 ,PNMA5仅在睾丸与脑中表达 ;而在所检测的肝癌、胃癌、肠癌、肺癌、乳腺癌与头颈部肿瘤 ,PNMA5在肠癌中表达 .PNMA5是肿瘤 睾丸 脑抗原家族的一个新成员 .认识这些肿瘤 睾丸 脑抗原的意义在于它们在肿瘤组织的表达可能是肿瘤破坏机体免疫耐受的一种机制 ,其相关神经副肿瘤综合症可能是一些隐匿肿瘤的早期表现 .     

肿瘤特异性抗原(MAGE)在肿瘤免疫治疗中的作用

目前,已经发现肿瘤特异性抗原Mage基因家族有55个成员,其中MageA、B和C三个亚家族在各种组织来源的恶性肿瘤中特异性表达,正常组织(睾丸除外)均不表达。所以,Mage抗原作为肿瘤的特异性标志,在肿瘤的检测和免疫治疗方面具有很大的应用潜力。本文介绍了Mage家族的基因定位,Mage蛋白的特性,Mage基因的作用。同时,总结了近年来Mage基因家族在临床方面的应用研究结果。     

胎盘特异性基因PLAC1与肿瘤

胎盘特异性基因PLAC1(placenta-specific 1 gene)具有癌-睾丸(cancer-testis antigen,CT)抗原的特点,即能在多种肿瘤组织中表达,而在正常组织(除胎盘和睾丸)几乎不表达。PLAC1编码一种膜蛋白,该蛋白质具有一定的免疫原性,能在肿瘤患者体内引起体液免疫和细胞免疫。目前的研究结果显示PLAC1代表一类新的肿瘤相关抗原,可能成为肿瘤免疫治疗的一个靶点。     

癌-睾丸抗原OY—TES-1蛋白在人肾癌中的表达

OY—TES-1属于癌一睾丸抗原（cancer testis antigen,CTA）,是一种在多种肿瘤组织中表达,在睾丸以外的正常组织几乎不表达的抗原。CTA既可引起体内免疫反应,亦可引起细胞免疫反应,已有部分CTA疫苗用于肿瘤临床治疗。为了解OY-TES-1在肾癌组织中的表达情况,我们运用免疫组织化学方法,检测了肾透明细胞癌、乳头状肾细胞癌、肾盂移行细胞癌等三种共40例肾癌标本。     

人类生精相关基因TSARG4的cDNA克隆

为了探索精子生成的分子机制 ,从人精子外部致密纤维蛋白相关基因SPAG4(spermantigen 4)和小鼠精母细胞中表达的AK0 0 62 2 5基因出发 ,找到两个人类EST ,BG72 0 5 64和AI70 0 45 4,其中BG72 0 5 64在人睾丸中表达。运用“间隙填充法”填平这两个EST之间的间隙 ,从人睾丸文库中快速克隆了同源于SPAG4和AK0 0 62 2 5基因的人类TSARG4基因 (testisandspermatogenesisrelatedgene 4) (GenBank登录号为AF40 13 5 0 ) ,并用RT PCR对该基因阅读框进行验证。TSARG4基因全长 12 5 2bp ,开放阅读框为 94～ 12 3 3bp ,定位于 2 0q11.2 ,推定编码 3 79个氨基酸 ,预计分子量为 43 0 81.45 ,等电点为 8.61,该基因与小鼠精母细胞基因AK0 0 62 2 5编码的氨基酸序列同源性 74% ,与人类SPAG4基因编码的氨基酸序列同源性 45 %。RT PCR表明人类TSARG4基因在多个组织中均有表达 ,而同源的小鼠AK0 0 62 2 5基因仅在睾丸中表达     

带Myc、His标签的SPAG4L真核表达载体的构建与表达

为了获得带有His、Myc 双标签的SPAG4L蛋白,建立能够稳定表达该蛋白的细胞系,本研究利用PCR技术从人睾丸cDNA中扩增SPAG4L开放阅读框,构建到pcDNA3.1(+)myc-his真核表达载体中进行测序和双酶切验证。将pcDNA3.1/myc-His(-)A/SPAG4L质粒和空白载体分别转染HeLa细胞,G418筛选后建立稳定转染细胞系。用Western blotting和免疫荧光技术对新建立的稳定转染细胞系进行检测。结果表明,成功扩增了SPAG4L基因,构建到pcDNA3.1/myc-His(-)A真核表达载体后,经酶切和测序验证所插入的SPAG4L序列完全正确;pcDNA3.1/myc-His(-)A/SPAG4L转染HeLa细胞后,经G418筛选后建立了稳定转染细胞系。Western blotting检测后发现,新建立的细胞系能够正确表达SPAG4L及其标签蛋白,进一步的免疫荧光实验发现,SPAG4L能够和内质网标签蛋白PDI共定位。研究结果所提供的稳定转染细胞系将为下一步进行免疫共沉淀和pull-down实验提供了有力的工具。     

前列腺癌潜在的标志物PAGE4/CT16.7

前列腺癌相关基因4(prostate-associated gene 4,PAGE4)是癌-睾丸抗原家族成员之一,具有在生殖系统组织中表达,而在非生殖系统组织中几乎不表达的特性。目前已发现前列腺癌患者可引起PAGE4的细胞免疫反应,前列腺炎患者血清中可检测到PAGE4蛋白。因此PAGE4有望成为前列腺癌免疫治疗潜在靶点及前列腺疾病筛查的又一新的血清标志物。对PAGE4基因及蛋白质的结构特点、表达特性、抗原肽筛选和生物学功能等方面进行综述。     

Sperm Associated Antigen 9 Plays an Important Role in Bladder Transitional Cell Carcinoma

Background

Majority of bladder cancer deaths are caused due to transitional cell carcinoma (TCC) which is the most prevalent and chemoresistant malignancy of urinary bladder. Therefore, we analyzed the role of Sperm associated antigen 9 (SPAG9) in bladder TCC.

Methodology and Findings

We examined SPAG9 expression and humoral response in 125 bladder TCC patients. Four bladder cancer cell lines were assessed for SPAG9 expression. In addition, we investigated the effect of SPAG9 ablation on cellular proliferation, cell cycle, migration and invasion in UM-UC-3 bladder cancer cells by employing gene silencing approach. Our SPAG9 gene and protein expression analysis revealed SPAG9 expression in 81% of bladder TCC tissue specimens. High SPAG9 expression (>60% SPAG9 positive cells) was found to be significantly associated with superficial non-muscle invasive stage (P = 0.042) and low grade tumors (P = 0.002) suggesting SPAG9 putative role in early spread and tumorigenesis. Humoral response against SPAG9 was observed in 95% of patients found positive for SPAG9 expression. All four bladder cancer cell lines revealed SPAG9 expression. In addition, SPAG9 gene silencing in UM-UC-3 cells resulted in induction of G₀–G₁ arrest characterized by up-regulation of p16 and p21 and consequent down-regulation of cyclin E, cyclin D and cyclin B, CDK4 and CDK1. Further, SPAG9 gene silencing also resulted in reduction in cellular growth, and migration and invasion ability of cancer cells in vitro.

Conclusions

Collectively, our data in clinical specimens indicated that SPAG9 is potential biomarker and therapeutic target for bladder TCC.     

Molecular cloning and characterization of the macaque sperm associated antigen 9 (SPAG9): an orthologue of human SPAG9 gene

The present study was conducted to isolate macaque proteomic homologue of human SPAG9 (EMBL nomenclature human sperm associated antigen 9: hSPAG9; Shankar et al., 1998: Biochem Biophys Res Commun 243:561-565) in order to find out whether the macaque can provide a suitable model for examining its immunocontraception effects. Macaque SPAG9 was cloned and sequenced from the macaque testis cDNA library. The macaque cDNA contained open reading frame encoding 712 amino acids. A 84.9% and 94% homology between macaque and human SPAG9 was found at protein and DNA levels. Northern analysis and RNA in situ hybridization experiments revealed testis- and stage-specific expressions of macaque SPAG9 mRNA, mainly confined to round spermatid suggesting haploid germ cell expression. Anti-human SPAG9 antibodies recognized native SPAG9 in macaque sperm extract in Western blotting and the acrosomal compartment region of macaque sperm in indirect immunofluorescence. Flow cytometry analysis further revealed surface localization of macaque SPAG9 in live macaque sperm. The amino acid sequence data for nonhuman primate SPAG9 suggest that antibodies generated by vaccinating macaque with hSPAG9 will recognize nonhuman primate SPAG9, supporting the testing of SPAG9 contraceptive vaccine based on hSPAG9 in the nonhuman primate model.     

Sperm-associated antigen 9 overexpression correlates with poor prognosis and insensitive to Taxol treatment in breast cancer

Sperm-associated antigen 9 (SPAG9) has been reported to express in several cancers and have clinical significance. Using immunohistochemistry, we found that there was a strong association among SPAG9 expression and tumor size, TNM stage, histological grade, lymph node metastasis, and recurrence. It suggested that SPAG9-elevated expression was an independently prognostic indicator for both OS and DFS. Furthermore, the selected treatment of chemotherapy with Taxol/non-Taxol significantly affects OS and DFS. To sum up, SPAG9-elevated expression contributes to malignant behavior and poor prognosis of breast cancer and may support a potential indicator in treatment selection.     

Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein

Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.     

The expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in peripheral blood of patients with HCC and lung cancer

There are different views of how the immune system participates in the reaction to cancer. Here, we evaluated expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in patients with hepatocellular carcinoma (HCC) and lung cancer to explore tumor immunity. Our analysis showed that levels of HSP70 and SPAG9 antibody were significantly higher in the serum of lung cancer and HCC patients than in the serum of healthy subjects (P < 0.001), but there were no differences in levels of HSP70 antibody in patients and controls. Levels of serum SPAG9 antibody in newly diagnosed lung cancer patients were significantly higher than in treated lung cancer patients (P < 0.05), but there were no differences in levels of HSP70 or HSP70 antibody. Levels of serum HSP70 and SPAG9 antibody, but not HSP70 antibody, were also higher in hepatitis/cirrhosis patients than in healthy subjects (P = 0.005, P < 0.001). Levels of serum SPAG9 antibody were significantly higher in HCC patients than in hepatitis/cirrhosis patients, but there were no differences in HSP70 or HSP70 antibody levels. Finally, levels of serum HSP70 and SPAG9 antibody were significantly higher in HCC patients than in lung cancer patients (P < 0.05, P < 0.001). These results indicate that cancer-testis antigen SPAG9 induces a strong humoral immune response in cancer patients but HSP70 does not. These results show that SPAG9 has potential as a tumor-specific biomarker.     

Accelerated mortality from hydrocephalus and pneumonia in mice with a combined deficiency of SPAG6 and SPAG16L reveals a functional interrelationship between the two central apparatus proteins

SPAG6 and SPAG16L are proteins localized to the "9+2" axoneme central apparatus. Both are essential for sperm motility and male fertility. These two proteins are also expressed in other tissues containing ciliated cells, such as brain and lung. To study the effects of combined deficiency of these two proteins, a double mutant mouse model was created. The double mutant mice displayed a more profound phenotype of growth retardation and hydrocephalus compared to mice nullizygous for SPAG6 and SPAG16L alone. The double mutant mice died younger, and mortality was significantly higher than in single mutant mice. In addition, the double mutant mice demonstrated pneumonia and its complications, including hemorrhage, edema, and atelectasis, phenotypes not observed in mice nullizygous for mutations in the individual genes. No other cilia-related phenotypic change was detected in double mutant mice including lateralization defects. The ultrastructure of cilia in both the brain and lung of the double mutant mice appeared normal. This model of combined SPAG6 and SPAG16L deficiency provides a new platform to study primary ciliary dyskinesia. The findings also demonstrate that SPAG6 and SPAG16L have related roles in controlling the function of cilia in the brain and lung.     

Dissecting the axoneme interactome: the mammalian orthologue of Chlamydomonas PF6 interacts with sperm-associated antigen 6, the mammalian orthologue of Chlamydomonas PF16

The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat.