CircRNA SEPT9 contributes to malignant behaviors of glioma cells via miR-432-5p-mediated regulation of LASP1.

BACKGROUND: Circular RNA (circRNA) septin 9 (circSEPT9; hsa_circ_0005320) has been reported to be abnormally up-regulated in glioma. However, the exact role and working mechanism of circSEPT9 in glioma progression are barely known. METHODS: RNA and protein levels were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay, respectively. Cell proliferation was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay and flow cytometry. Cell apoptosis was evaluated by flow cytometry. Cell motility was analyzed by transwell assays. Cell glycolytic metabolism was analyzed using commercial kits. Dual-luciferase reporter assay, RNA-pull down assay and RNA immunoprecipitation (RIP) assay were conducted to verify the intermolecular interactions. Xenograft mice model was utilized to assess the role of circSEPT9 in vivo. RESULTS: CircSEPT9 was highly expressed in glioma tissues and cell lines. CircSEPT9 interference inhibited the proliferation, migration, invasion and glycolytic metabolism and triggered the apoptosis of glioma cells. MicroRNA-432-5p (miR-432-5p) was a target of circSEPT9, and circSEPT9 silencing-mediated effects in glioma cells were largely alleviated by the addition of anti-miR-432-5p. MiR-432-5p bound to the 3' untranslated region (3'UTR) of LIM and SH3 protein 1 (LASP1), and LASP1 overexpression largely overturned miR-432-5p-induced effects in glioma cells. CircSEPT9 up-regulated LASP1 expression by acting as miR-432-5p sponge. CircSEPT9 silencing suppressed xenograft tumor growth in vivo. CONCLUSION: CircSEPT9 exerted an oncogenic role to enhance the malignant behaviors of glioma cells by binding to miR-432-5p to induce LASP1 expression.

miR-185 and SEPT5 Genes May Contribute to Parkinson's Disease Pathophysiology.

There are still unknown mechanisms involved in the development of Parkinson's disease (PD), which elucidating them can assist in developing efficient therapies. Recently, studies showed that genes located on the human chromosomal location 22q11.2 might be involved in the development of PD. Therefore, the present study was designed to evaluate the role of two genes located on the chromosomal location (miR-185 and SEPT5), which were the most probable candidates based on our bibliography. In vivo and in vitro models of PD were developed using male Wistar rats and SHSY-5Y cell line, respectively. The expression levels of miR-185, SEPT5, LRRK2, and PARK2 genes were measured at a mRNA level in dopaminergic areas of rats' brains and SHSY-5Y cells using the SYBR Green Real-Time PCR Method. Additionally, the effect of inhibition on the genes or their products on cell viability and gene expression pattern in SHSY-5Y cells was investigated. The level of miR-185 gene expression was significantly decreased in the substantia nigra (SN) and striatum (ST) of the rotenone-treated group (control group) compared to the healthy normal group (P < 0.05). In addition, there was a significant difference in the expression of SEPT5 gene (P < 0.05) in the substantia nigra between two studied groups. The results of an in vitro study showed no significant change in the expression of the genes; however, the inhibition on miR-185 gene expression led to the increase in LRRK2 gene expression in SHSY-5Y cells. The inhibition on LRRK2 protein also decreased the cellular toxicity effect of rotenone on SHSY-5Y cells. The results suggested the protective role of miR-185 gene in preventing the development of PD.

Role of septins in microbial infection.

Septins are widely recognized as a component of the cytoskeleton that is essential for cell division, and new work has shown that septins can recognise cell shape by assembling into filaments on membrane regions that display micrometer-scale curvature (e.g. at the cytokinetic furrow). Moreover, infection biology studies have illuminated important roles for septins in mediating the outcome of host-microbe interactions. In this Review, we discuss a selection of mechanistic insights recently gained from studying three infection paradigms: the rice blast fungus Magnaporthe oryzae, the poxvirus family member vaccinia virus and the Gram-negative bacterium Shigella flexneri These studies have respectively discovered that higher-order septin assemblies enable fungal invasion into plant cells, entrap viral particles at the plasma membrane and recognize dividing bacterial cells for delivery to lysosomes. Collectively, these insights illustrate how studying septin biology during microbial infection can provide fundamental advances in both cell and infection biology, and suggest new concepts underlying infection control.

Septin filament coalignment with microtubules depends on SEPT9_i1 and tubulin polyglutamylation, and is an early feature of acquired cell resistance to paclitaxel.

Cancer cell resistance to taxanes is a complex, multifactorial process, which results from the combination of several molecular and cellular changes. In breast cancer cells adapted to long-term paclitaxel treatment, we previously identified a new adaptive mechanism that contributes to resistance and involves high levels of tubulin tyrosination and long-chain polyglutamylation coupled with high levels of septin expression, especially that of SEPT9_i1. This in turn led to higher CLIP-170 and MCAK recruitment to microtubules to enhance microtubule dynamics and therefore counteract the stabilizing effects of taxanes. Here, we explored to which extent this new mechanism alone could trigger taxane resistance. We show that coupling septins (including SEPT9_i1) overexpression together with long-chain tubulin polyglutamylation induce significant paclitaxel resistance in several naive (taxane-sensitive) cell lines and accordingly stimulate the binding of CLIP-170 and MCAK to microtubules. Strikingly, such resistance was paralleled by a systematic relocalization of septin filaments from actin fibers to microtubules. We further show that this relocalization resulted from the overexpression of septins in a context of enhanced tubulin polyglutamylation and reveal that it could also be promoted by an acute treatment with paclitaxel of sensitve cell displaying a high basal level of SEPT9_i1. These findings point out the functional importance and the complex cellular dynamics of septins in the onset of cell resistance to death caused by microtubule-targeting antimitotic drugs of the taxane family.

A three-gene methylation marker panel for the nodal metastatic risk assessment of muscle-invasive bladder cancer.

PURPOSE: In this study, we aimed to identify a DNA methylation pattern suitable for prognosis assessment of muscle-invasive bladder cancer and to investigate metastasis-associated processes regulated by DNA methylation. METHODS: Genome-wide methylation analysis was performed on 23 muscle-invasive bladder tumors by microarray analysis. Validation was performed by the qAMP technique in two different patient cohorts (n = 32 and n = 100). mRNA expression was analyzed in 12 samples. Protein expression was determined using tissue microarrays of 291 patients. Bladder cancer cell lines T24 and 253JB-V were used for functional analyses. RESULTS: Microarray analyses revealed KISS1R, SEPT9 and CSAD as putative biomarkers with hypermethylation in node-positive tumors. The combination of the three genes predicted the metastatic risk with sensitivity of 73% and specificity of 71% in cohort 1, and sensitivity of 82% and specificity of 54% in cohort 2. mRNA expression differences were detected for KISS1R (p = 0.04). Protein expression of KISS1R was significantly reduced (p < 0.001). Knockdown of SEPT9v3 resulted in increased cell migration by 28% (p = 0.04) and increased invasion by 22% (p = 0.004). KISS1R overexpression resulted in decreased cell migration (25%, p = 0.1). CONCLUSIONS: We identified a methylation marker panel suitable to differentiate between patients with positive and negative lymph nodes at time of cystectomy. This enables a risk assessment for patients who potentially benefit from extended lymph node resection as well as from neoadjuvant chemotherapy and could improve the survival rates. Furthermore, we examined the impact of putative markers on tumor behavior. Hence, KISS1R and SEPT9 could represent a starting point for the development of novel therapy approaches.

iTRAQ-based proteomic analysis of DMH-induced colorectal cancer in mice reveals the expressions of ß-catenin, decorin, septin-7, and S100A10 expression in 53 cases of human hereditary polyposis colorectal cancer.

PURPOSE: The aim of this study is to explore the roles of ß-catenin, decorin, septin-7, and S100A10 expression in colorectal cancer development. METHODS: Twenty-five BALB/c mice were divided into five groups; four groups were administrated N,N-dimethylhydrazine for 0, 10, 15, and 20 weeks, and one group was administrated normal saline for 20 weeks. The colons were collected for histopathological analysis. Protein samples prepared from the frozen colon tissues of mice treated with N,N-dimethylhydrazine for the different time points were evaluated using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique coupled with the 2D liquid chromatography-tandem mass spectrometry analysis. Based on the proteomic analysis results, immunohistochemical staining of ß-catenin, decorin, septin-7, and S100A10 was performed in paraffin-embedded mice colorectal tissue, and 53 cases of human hereditary polyposis colorectal cancer samples. RESULTS: Colorectal cancer was observed in mice treated with N,N-dimethylhydrazine for 20 weeks, and adenomas were observed in mice subjected to the 10-, and 15-week treatments. Seventy-two differentially expressed proteins were involved in the development of cancer as per the iTRAQ and spectrometry analysis. In normal epithelium, adenoma, and cancer from human hereditary polyposis colorectal cancer, S100A10 expression (c2 = 100.989, P = 0.000) was highest in cancer, whereas decorin (c2 = 12.852, P = 0.002) and septin-7 (c2 = 66.519, P = 0.002) expressions were highest in the normal epithelium, which was confirmed via immunohistochemical staining. CONCLUSIONS: The subcellular localization of ß-catenin and decorin, septin-7, and S100A10 expressions are associated with the development of colorectal cancer in mice after N,N-dimethylhydrazine treatment and in human hereditary polyposis colorectal cancers.

Identification of SEPTIN12 as a novel target of the androgen and estrogen receptors in human testicular cells.

SEPTIN12 (SEPT12) is a testis-enriched gene that is downregulated in the testis of infertile men with severe spermatogenic defects. While SEPT12 is involved in spermatogenic failure and sperm motility disorder, SEPT12 transcriptional regulation is still unknown. Here we report the promoter region of SEPT12 as a 245 bp segment upstream of the transcription start site. One androgen receptor (AR) and two estrogen receptor α (ERα) binding sites in this region were initially identified by bioinformatics prediction and confirmed by chromatin immunoprecipitation assay. Truncated ERα or AR binding sites decreased the promoter activity, which indicated that the ERα and AR are essential for the SEPT12 promoter. On the other hand, the promoter activity was enhanced by the treatment with 17ß-estradiol (E2) and 5α-dihydrotestosterone (5α-DHT). Thus, one androgen and two estrogen hormone responsive elements located in the promoter of SEPT12 gene can regulate SEPT12 expression. Two single nucleotide polymorphisms (SNPs), rs759992 T > C and rs3827527 C > T, were observed in the SEPT12 gene promoter region and were able to decrease the promoter activity. In conclusion, the current work identified the promoter of the human SEPT12 gene and provided key evidence about its transcriptional regulation via E2 and 5α-DHT. Since SEPT12 has an important role in spermatogenesis, SEPT12 expression analysis can be developed as a potential tool for the assessment of environmental or food pollution by hormones or for the evaluation of the risk of endocrine-disrupting chemicals (EDCs) in general.

Analysis of protein dynamics during cytokinesis in budding yeast.

Cytokinesis is essential for development and survival of all organisms by increasing cell number and diversity. It is a highly regulated process that requires spatiotemporal coordination of hundreds of proteins functioning in the assembly, constriction, and disassembly of a contractile actomyosin ring, targeted vesicle fusion, and localized extracellular matrix remodeling. Cytokinesis has been studied in multiple systems with a wide range of technologies to learn the common principles. In this chapter, we describe the analysis of protein dynamics during cytokinesis in the budding yeast Saccharomyces cerevisiae by several live-cell imaging methods. This, in combination with the power of yeast genetics, has yielded novel insights into the mechanism of cytokinesis. Similar approaches are increasingly used to study this fundamental process in more complex systems.

Transcript levels of the Aspergillus fumigatus Cdc42 module, polarisome, and septin genes show little change from dormancy to polarity establishment.

Aspergillus fumigatus is the most common airborne pathogen causing fatal mycoses in immunocompromised patients. During the first 8 hours of development A. fumigatus conidia break dormancy, expand isotopically, establish an axis of polarity, and begin to extend germ tubes in a polar manner. The transition from isotropic to polar growth is critical for tissue invasion and pathogenesis. In the current work, we used two-color microarrays to examine the A. fumigatus transcriptome during early development, focusing on the isotropic to polar switch. The most highly regulated transcripts in the isotropic to polar switch did not include known polarity genes. Transcripts encoding the Cdc42 module, polarisome components, and septins, known to be critical players in polarity, showed relatively steady levels during the isotropic to polar switch. Indeed, these transcripts were present in dormant conidia, and their levels changed little from dormancy through germ tube emergence. Not only did the isotropic to polar switch show little change in the expression of key polarity genes of the Cdc42 module, polarisome, and septins, it also showed the lowest overall levels of both up- and downregulation in early development.

Adenovirus-mediated over-expression of Septin4 ameliorates hepatic fibrosis in mouse livers infected with Schistosoma japonicum.

Septin4 (Sept4) belongs to Septin family and may be involved in apoptosis, vesicle trafficking and other cell processes. In this study, we attempted to investigate the effect of Sept4 in hepatic fibrosis induced by Schistosoma japonicum. ICR mice infected with S. japonicum for 12weeks were treated with PBS, Ad-ctr and Ad-Sept4, respectively. All mice were killed at 2weeks after injection, and the changes in the fibrotic livers were detected via H&E staining, Sirius red staining, qRT-PCR, western blot and TUNEL analysis. In addition, pcDNA3.1-Sept4 plasmid was transfected into LX-2 cells to observe the effect of Sept4 on apoptosis of HSCs in vitro. Ad-Sept4 could ameliorate liver fibrosis, as detected by H&E staining and Sirius red staining. The number of TUNEL-positive cells was increased in the Ad-Sept4 treated group. The expression of Sept4 and cleaved-caspase-3 were all augmented, while the expression of α-SMA, Col1α1 and IL-13 were reduced in the Ad-Sept4 treated group, compared with that expressed in the Ad-ctr group. Over-expression of Sept4 in LX-2 cells could promote apoptosis of LX-2 cells in vitro. In conclusion, Ad-Sept4 can attenuate the development of liver fibrosis induced by S. japonicum through apoptosis.

Genetic deletion of SEPT7 reveals a cell type-specific role of septins in microtubule destabilization for the completion of cytokinesis.

Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.

Septin 7 immunoexpression in papillary thyroid carcinoma: a preliminary study.

Papillary thyroid carcinoma (PTC) is the most common type among thyroid cancers. The diagnosis of PTC may be challenging when follicular variant (FVPTC) of this disease is present due to the resemblance of nuclear properties of the classical type (CVPTC). However, making use of ancillary molecular markers in the diagnosis of PTC may help. In our study, we aimed to evaluate the SEPT7 protein expression in PTC. A total of 55 paraffin block tissue samples comprising encapsulated FVPTC (FVPTC(e), n=25), and CVPTC (n=15), and benign hyperfunctioning thyroid nodules (HypN, n=15) were used in this study. Nuclear, cytoplasmic, and overall (total) SEPT7 protein expression levels were determined by using immunohistochemistry. Nuclear, cytoplasmic, and overall SEPT7 expressions (p=0.02, p=0.001, p=0.002, respectively) were significantly lower in FVPTC(e) tissues when compared to HypN. In CVPTC group, nuclear expression was significantly lower (p=0.004) while overall and cytoplasmic expressions were not changed (p>0.05). In HypN group, highest nuclear (mean=2.73), cytoplasmic (mean=2.86), and overall (mean=2.86) expression scores were detected. Significantly lower SEPT7 expression in all expressional categories in FVPTC(e) group may be a sign of different molecular signature in this type of tissue.

Cell type-specific expression of SEPT3-homology subgroup members controls the subunit number of heteromeric septin complexes.

Septins are filament-forming proteins important for organizing the cortex of animal and fungal cells. In mammals, 13 septin paralogues were recently shown to assemble into core heterohexamer and heterooctamer complexes, which serve as building blocks for apolar filamentous structures that differ among cell types. To determine how tissue-specific septin paralogue expression may shape core heteromer repertoires and thereby modulate properties of septin filaments, we devised protocols to analyze native septin heteromers with distinct numbers of subunits. Our evidence based on genetically manipulated human cells supports and extends recent concepts of homology subgroup-restricted assembly into distinct categories of apolar heterohexamers and heterooctamers. We also identify a category of tetramers that have a subunit composition equivalent to an octameric building block. These atypical tetramers are prevalent in lymphocytes and neural tissues, in which octamers are abundant but hexamers are rare. Our results can be explained by tissue-specific expression of SEPT3 subgroup members: SEPT3, SEPT9, and SEPT12. These serve as cognate subunits in either heterooctamers or atypical tetramers but exhibit different preferences in various tissues. The identified tissue-specific repertoires of septin heteromers provide insights into how higher-order septin structures with differential properties and stabilities may form in diverse animal cell types.

Sept7b is essential for pronephric function and development of left-right asymmetry in zebrafish embryogenesis.

The conserved septin family of filamentous small GTPases plays important roles in mitosis, cell migration and cell morphogenesis by forming scaffolds and diffusion barriers. Recent studies in cultured cells in vitro indicate that a septin complex of septin 2, 7 and 9 is required for ciliogenesis and cilia function, but septin function in ciliogenesis in vertebrate organs in vivo is not understood. We show that sept7b is expressed in ciliated cells in different tissues during early zebrafish development. Knockdown of sept7b by using morpholino antisense oligonucleotides caused misorientation of basal bodies and cilia, reduction of apical actin and the shortening of motile cilia in Kupffer's vesicle and pronephric tubules. This resulted in pericardial and yolk sac edema, body axis curvature and hydrocephaly. Notably, in sept7b morphants we detected strong left-right asymmetry defects in the heart and lateral plate mesoderm (situs inversus), reduced fluid flow in the kidney, the formation of kidney cysts and loss of glomerular filtration barrier function. Thus, sept7b is essential during zebrafish development for pronephric function and ciliogenesis, and loss of expression of sept7b results in defects that resemble human ciliopathies.

Septins of Platyhelminths: identification, phylogeny, expression and localization among developmental stages of Schistosoma mansoni.

Septins are a family of eukaryotic GTP binding proteins conserved from yeasts to humans. Originally identified in mutants of budding yeast, septins participate in diverse cellular functions including cytokinesis, organization of actin networks, cell polarity, vesicle trafficking and many others. Septins assemble into heteroligomers to form filaments and rings. Here, four septins of Schistosoma mansoni are described, which appear to be conserved within the phylum Platyhelminthes. These orthologues were related to the SEPT5, SEPT10 and SEPT7 septins of humans, and hence we have termed the schistosome septins SmSEPT5, SmSEPT10, SmSEPT7.1 and SmSEPT7.2. Septin transcripts were detected throughout the developmental cycle of the schistosome and a similar expression profile was observed for septins in the stages examined, consistent with concerted production of these proteins to form heterocomplexes. Immunolocalization analyses undertaken with antibodies specific for SmSEPT5 and SmSEPT10 revealed a broad tissue distribution of septins in the schistosomulum and colocalization of septin and actin in the longitudinal and circular muscles of the sporocyst. Ciliated epidermal plates of the miracidium were rich in septins. Expression levels for these septins were elevated in germ cells in the miracidium and sporocyst. Intriguingly, septins colocalize with the protonephridial system of the cercaria, which extends laterally along the length of this larval stage. Together, the findings revealed that schistosomes expressed several septins which likely form filaments within the cells, as in other eukaryotes. Identification and localization demonstrating a broad distribution of septins across organs and tissues of schistosome contributes towards the understanding of septins in schistosomes and other flatworms.

The role of Cdc42 and Gic1 in the regulation of septin filament formation and dissociation.

Septins are guanine nucleotide-binding proteins that polymerize into filamentous and higher-order structures. Cdc42 and its effector Gic1 are involved in septin recruitment, ring formation and dissociation. The regulatory mechanisms behind these processes are not well understood. Here, we have used electron microscopy and cryo electron tomography to elucidate the structural basis of the Gic1-septin and Gic1-Cdc42-septin interaction. We show that Gic1 acts as a scaffolding protein for septin filaments forming long and flexible filament cables. Cdc42 in its GTP-form binds to Gic1, which ultimately leads to the dissociation of Gic1 from the filament cables. Surprisingly, Cdc42-GDP is not inactive, but in the absence of Gic1 directly interacts with septin filaments resulting in their disassembly. We suggest that this unanticipated dual function of Cdc42 is crucial for the cell cycle. Based on our results we propose a novel regulatory mechanism for septin filament formation and dissociation. DOI: http://dx.doi.org/10.7554/eLife.01085.001.

Expression of Septin4 in human hepatic stellate cells LX-2 stimulated by LPS.

Septin4, a member of polymerizing GTP-binding proteins family, is reported to be involved in cytoskeletal organization in mitosis, apoptosis, fibrosis, and other cellular processes. Since various Septin4 expression patterns were reported in different diseases, this study aimed to investigate Septin4 expression in human LX-2 cell line stimulated by lipopolysaccharides (LPS) and attempted to clarify the relationship between Septin4 and hepatic inflammatory injury and fibrosis. In this subject, human stellate cell line LX-2 was stimulated by LPS. The expression of Septin4 was analyzed by Western blot and quantitative real-time PCR. To observe the relationship among Toll-like receptor 4 (TLR4), TGF-ß, and Septin4, proteins from the anti-TLR4 antibody blocked cells, as well as the TGF-ß-induced cells, were analyzed by the method of Western blot. As the results, LPS could induce the alteration of α-smooth muscle actin and Septin4 expression in LX-2 cells. Septin4 expression was regulated by LPS stimulation through TLR4 and TGF-ß pathway. These results therefore suggest that Septin4 may be involved in the process of activation of hepatic stellate cells by LPS stimulation. Further work would focus on the function of Septin4 in hepatic inflammatory injury and fibrosis.

Structures of septin filaments prepared from rat brain and expressed in bacteria.

Septin forms a conserved family of cytoskeletal GTP-binding proteins that have diverse roles in protein scaffolding, vesicle trafficking and cytokinesis. There are 14 mammalian septin isoforms and these isoforms assemble into hetero-oligomeric rod-shaped complexes and these short filaments are the basal units to construct higher-order structures such as longer filaments, rings, gauzes or hourglasses. Septin expressed in a eukaryotic expression system forms various structures such as bundles, sheets, helixes, and rings. Septin expressed in bacteria formed hexameric short filaments and single or parallel long filaments, but no such higher order structures were observed so far. In a previous study, we showed maturation-dependent localization of septin isoforms to the lipid raft fraction of rat brain. In this study, we attempted further purification of raft-localized septin isoforms. Repeated cycles of extraction with high MgCl(2) solution and precipitation under low ionic solution were combined with several column procedures. The obtained fraction contained several septin isoforms and showed rings of bundled filaments with a diameter of ~0.4µm. Several non-septin proteins were also detected in the fraction. We also attempted expression of septin isoforms in bacteria and found that the expressed septin complexes formed bundles of filaments. In addition to linear and curled filaments, circular bundles of thin filaments with a diameter of ~0.6µm were also observed. These results suggest that the curvature of the bundles of septin filaments may be regulated by the regulatory factor(s) in the lipid raft.

Identification of endometriosis-related genes by representational difference analysis of cDNA.

BACKGROUND: Accumulated evidence reveals that abnormally expressed genes in eutopic endometrium of endometriosis play a critical role in the pathogenesis of endometriosis. AIMS: Identification of endometriosis-related genes for further revealing the pathogenesis of endometriosis and offering the basis for developing the molecular-targeted diagnosis and therapy of endometriosis. METHODS: Forty women with endometriosis and forty control women without endometriosis during their secretory phase were selected for this study. cDNA representational difference analysis (cDNA-RDA) was performed to screen the up-regulated genes in eutopic endometrium samples of endometriosis (n = 10) compared with the controls (n = 10). To validate the results, MAT2A, the most abundantly expressed gene, was selected to detect mRNA and protein levels between eutopic endometrium of endometriosis (n = 40) and controls (n = 40) using immunohistochemistry, real-time fluorescent quantitative PCR and Western blotting. RESULTS: Ten up-regulated genes were identified in eutopic endometria of endometriosis compared with controls. Among these genes, COX-2, BRAF, NRAS and CFL1 have already been reported to be associated with the endometriosis in previous studies. MAT2A, SEPT9, ATAD3A and CADM2 have been reported to be involved in other diseases but not in endometriosis. NAA15 and CCDC21 have not reported in any diseases. Further study showed that MAT2A protein was localised in both endometrial glandular and stromal cells. Compared with controls, the mRNA and protein levels of MAT2A were significantly higher in eutopic endometrium of endometriosis (P < 0.05). CONCLUSIONS: cDNA-RDA can be used to effectively identify the endometriosis-related genes, which can provide novel experimental data and further understand the pathogenesis of endometriosis.

Expression and function of neuronal growth-associated proteins (nGAPs) in PC12 cells.

The growth cone plays crucial roles in neural wiring, synapse formation, and axonal regeneration. Continuous rearrangement of cytoskeletal elements and targeting of transported vesicles to the plasma membrane are essential to growth cone motility; however, the proteins directly involved in these processes and their specific functions are not well established. We recently identified 17 proteins as functional marker proteins of the mammalian growth cone and as neuronal growth-associated proteins in rat cortical neurons (nGAPs; Nozumi et al., 2009). To determine whether these 17 proteins are growth cone markers in other neuronal cell types, we examined their expression and function in PC12D cells. We found that all 17 nGAPs were highly concentrated in the growth cones of PC12D cells, and that knockdown of all of them by RNAi reduced or inhibited neurite outgrowth, indicating that all of the 17 nGAPs may be general growth cone markers. Among them, eight proteins were shown to regulate the amount of F-actin in PC12D growth cones. Two of these nGAP that are cytoskeletal proteins, Cap1 and Sept2, increased the mean growth cone area and the mean neurite length by regulating the amount of F-actin; Sept2 also induced filopodial growth. Taken together, our data suggested that some of the nGAPs were generalized markers of the growth cone in multiple neuronal cell types and some of them, such as Cap1 and Sept2, regulated growth cone morphology through rearrangement of F-actin and thereby controlled neurite outgrowth.