RESUMO
PURPOSE: To identify microRNAs (miRNAs) directly regulating the proto-oncogene Bmi-1 expression in the development of hepatocellular carcinoma (HCC) and to explore the underlying molecular mechanisms. METHODS: Four HCC cell lines, including HepG2, Bel7404, Huh7, and PLC5, the normal hepatocellular cell line MIHA, and 30 HCC biopsies were included in this study. Potential miRNAs, which interact with Bmi-1 and are involved in the development of HCC were identified through bioinformatic analyses. The expression of miRNA and Bmi-1 in HCC cell lines and HCC tissues was analyzed using fluorescence protein analysis, real-time quantitative PCR (RT-qPCR), and Western blotting. RESULTS: Bioinformatic analysis suggested that miR-218 is a potential miRNA regulating Bmi-1 expression. Fluorescence protein analysis, RT-qPCR, and Western blotting confirmed the direct interaction between miR-218 and Bmi-1. In addition, increased expression of Bmi-1 was detected in HCC cell lines and HCC tissues. In most HCC tissues, the expression of miR-218 was decreased and was associated with increased expression of Bmi-1. CONCLUSION: miR-p218 downregulates the expression of the proto-oncogene Bmi-1 in HCC, and it may be an effective target for the treatment of this disease.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , MicroRNAs/genética , Proteína Quinase 7 Ativada por Mitógeno/biossíntese , Proteína Quinase 7 Ativada por Mitógeno/genética , Proto-Oncogene Mas , Proto-Oncogenes , TransfecçãoRESUMO
BACKGROUND: Testosterone is critical for maintaining spermatogenesis and male fertility. The accomplishment of these processes requires the synergistic actions of the classical and non-classical signaling pathways of androgens. METHODS: A murine testicular Sertoli cell line, TM4 cell was used to examine androgen actions in Sertoli cells. Western blot analysis and immunofluorescence assay were employed to study the testosterone-induced Androgen receptor (AR) translocation. Protein phosphorylation antibody array was applied to identify the phosphorylation sites under testosterone treatment, and these findings were verified by Western blot analysis. RESULTS: We found that a physiological dose of testosterone induced fast membrane association of AR. By using a phosphorylation antibody array, several phosphorylation sites, such as MEK1/2 (Ser217/221), Akt (Ser473), and Erk1/2 (Thr202/Tyr204) were rapidly phosphorylated within 5 min of testosterone treatment. Inhibition of the MEK and Akt signaling pathways prevented AR trafficking. Blocking of AR by flutamide eliminated the stimulation effect of testosterone on kinase phosphorylation. Testosterone induced kinase Src phosphorylation, and inhibition of Src restricted AR translocation to the membrane and the nucleus. CONCLUSION: Findings suggested that the membrane association of AR was mediated by the MEK and Akt phosphorylation signaling pathways, which resulted in Src activation and was initiated by testosterone binding to the membrane-localized AR. This study provides new insights into the testosterone signaling pathway in Sertoli cells, which mediate spermatogenesis. In addition, the study can be used in the diagnosis and treatment of male infertility caused by disorders in spermatogenesis.
Assuntos
Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Testosterona/farmacologia , Antagonistas de Receptores de Andrógenos/farmacologia , Animais , Linhagem Celular , Flutamida/farmacologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Camundongos , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/química , Espermatogênese/efeitos dos fármacos , Quinases da Família src/metabolismoRESUMO
Phenotype-driven mutagenesis is an unbiased method to identify novel genes involved in spermatogenesis and other reproductive processes. Male repro29/repro29 mice generated by the Reproductive Genomics Program at the Jackson Laboratory were infertile with deformed sperm and poor motility. Using selected exonic capture and massively parallel sequencing technologies, we identified a nonsense mutation in the exon 6 of coiled-coil domain-containing 62 gene (Ccdc62), which results in a formation of a premature stop codon and a truncated protein. Among the tissues examined, CCDC62 was found to be expressed at the highest level in mouse testis by reverse transcriptase-PCR (RT-PCR) and Western blot analysis. With immunofluorescent staining, we demonstrated that CCDC62 was expressed in the cytoplasm and the developing acrosome in the spematids of mouse testis, and was specifically localized at the acrosome in mature sperm. The complementation analysis by mating repro29/+ mice with Ccdc62 -/- mice (generated by CRISPR-Cas9 strategy) further provided genetic proof that the infertility of repro29/repro29 mice was caused by Ccdc62 mutation. Finally, it was found that intracellular colocalization and interaction of CCDC62 and Golgi-associated PDZ and coiled-coil motif-containing protein may be important for acrosome formation. Taken together, this study identified a nonsense mutation in Ccdc62, which directly results in male infertility in repro29/repro29 mice.
Assuntos
Infertilidade Masculina/genética , Espermatogênese/genética , Fatores de Transcrição/genética , Acrossomo/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Proteínas de Transporte/metabolismo , Códon sem Sentido , Etilnitrosoureia , Feminino , Proteínas da Matriz do Complexo de Golgi , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência de DNA , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
STUDY QUESTION: What are the features of FAM71D (Family with sequence similarity 71, member D) expression and is there an association between FAM71D expression and sperm motility? SUMMARY ANSWER: FAM71D, a novel protein exclusively expressed in the testis, is located in sperm flagella and is functionally involved in sperm motility. WHAT IS KNOWN ALREADY: Some testis-specific proteins have been reported as potential diagnostic biomarkers to evaluate the spermatogenesis process and sperm quality. We have identified a novel testis-specific protein, FAM71D, through microarray data analysis, yet little is known about its expression and function. STUDY DESIGN, SIZE, DURATION: FAM71D mRNA and protein expression was quantified during mouse testis development. Its localization in germ cells was detected by dual-labeled immunostaining in testis sections and sperm smears. The clinical significance was assessed by comparing FAM71D expression in spermatozoa from normozoospermic controls and asthenozoospermic patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testes were dissected from C57BL/6 J male mice at postnatal ages of 1, 2, 3, 4, 6, 8 weeks and 6 months, and sperm was collected from cauda epididymides of adult mice by the swim-up method. Human spermatozoa were isolated from 100 human semen samples by density gradient Percoll centrifugation. RT-qPCR and western blot were performed to semi-quantify the expression of FAM71D in mouse testis, and in the ejaculated spermatozoa of normozoospermic controls and asthenozoospermic patients. Immunofluorescence staining was used to detect the localization of FAM71D. Co-immunoprecipitation assay was performed to evaluate the interaction between FAM71D and calmodulin. An antibody blocking assay was employed to assess the role of FAM71D in sperm motility. MAIN RESULTS AND THE ROLE OF CHANCE: Our results showed that FAM71D was exclusively expressed in the testis in an age-dependent manner. FAM71D expression exhibited dynamic change in the cytoplasm of spermatids during spermiogenesis and was finally retained in sperm flagella. FAM71D could interact with calmodulin. Use of anti-FAM71D antibody on sperm significantly decreased sperm motility. Expression level of FAM71D was markedly reduced in the ejaculated spermataozoa of asthenozoospermic patients (P < 0.05), and this was correlated with sperm progressive motility (r = 0.7435, P < 0.0001). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The sample size was limited and it is necessary to verify the correlation of FAM71D expression with sperm motility in larger cohorts. Furthermore, our results were descriptive and follow-up studies would be needed to elucidate the detailed role of FAM71D in sperm motility. WIDER IMPLICATIONS OF THE FINDINGS: This is the first systematic study to document the expression of endogenous FAM71D and a function for FAM71D in sperm motility. It provides new insights into our understanding of sperm motility regulation and causes of male infertility. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the National Natural Science Foundation of China, Guangdong Natural Science Foundation and the Shenzhen Project of Science and Technology. The authors have no competing interests.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Animais , Calmodulina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Análise do Sêmen , Cauda do Espermatozoide/metabolismo , Testículo/metabolismoRESUMO
OBJECTIVE: To investigate the characteristics of the expression of the RIKEN cDNA 1700008O03 (1700008O03Rik) gene in the testis of the mouse from birth to sexual maturity and its potential role in regulating spermatogenesis. METHODS: Using mouse gene expression profile microarray, we screened the testis-specific gene 1700008O03Rik from the mouse. We studied the expression characteristics of the gene in the development of the mouse testis by reverse transcription PCR, quantitative real-time PCR, Western-blot, immunohistochemistry and immunofluorescence, and analyzed the structure of the 1700008O03Rik protein and its homology with other species using the bioinformatic software. RESULTS: 1700008O03Rik gene was highly expressed in the testis of the mouse, increasing in an age-dependent manner, and mainly in the endochylema of oblong spermatozoa. Bioinformatic analysis revealed a high homology of the 1700008O03Rik protein between human and mice, and phylogenetic tree analysis showed it to be highly conserved in mammalian evolution. CONCLUSIONS: 1700008O03Rik is a highly expressed gene in the mouse testis, mainly in the endochylema of oblong spermatozoa, which may be involved in the regulation of spermatogenesis in mice.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas/genética , Espermatogênese/genética , Espermatozoides/metabolismo , Testículo/metabolismo , Fatores Etários , Animais , Western Blotting , Biologia Computacional , DNA Complementar , Humanos , Masculino , CamundongosRESUMO
OBJECTIVE: To investigate the expression characteristic of the Daxx gene in the mouse testis and its role in spermatogenesis. METHODS: Real-time PCR, Western blot and immunofluorescence were used in examining the expression characteristics of DAXX in the testis tissue from wild-type, Sertoli cell-specific androgen receptor knockout (SCARKO) and androgen receptor knockout (ARKO) mice at different postnatal weeks . RESULTS: The Daxx gene was highly expressed in the testis tissue and mainly in the nuclei of the wild-type mice at 4 postnatal weeks. Compared with the wild-type, the ARKO mice showed a markedly decreased expression of DAXX (0.299±0.026), which displayed a polar distribution in the spermatogenic cells (0.853±0.058) and exhibited no significant difference in the SCARKO mice (1.000±0.015). CONCLUSIONS: The Daxx gene expression is the highest in the middle-stage development of the mouse testis, significantly decreased in ARKO mice as compared with the wild-type, and its location influenced by specific AR knockout in Sertoli cells. DAXX may be involved in the regulation of spermatogenesis in mice.
Assuntos
Proteínas de Transporte/genética , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Espermatogênese/genética , Testículo/metabolismo , Animais , Proteínas de Transporte/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Correpressoras , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos Knockout , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Receptores Androgênicos/genética , Células de SertoliRESUMO
Teratozoospermia is generally associated with clinical infertility. Despite numerous studies, the molecular mechanisms underlying male infertility are still poorly understood. In the present study, we demonstrated that deletion of Spata46, a gene encoding a novel protein of unknown function found in mouse testis, was responsible for male subfertility, and the cause of subfertility was characterized as abnormal sperm head shape and a failure of sperm-egg fusion. We also demonstrated that SPATA46 was expressed predominantly in condensed spermatids, with a highly specific localization restricted to the subacrosomal area; the protein is located at the nuclear membrane due to a transmembrane region in the N-terminus of the protein. At the subcellular level, SPATA46-deficient condensed spermatids displayed structural defects consisting of a discontinuous nuclear envelope and a cavity in the nucleus associated with an abnormal nuclear shape. Additionally, in vitro, we determined that the absence of SPATA46 led to accumulation of sperm around the perivitelline space of eggs, and the same phenomenon was also observed for natural sperm incubated with an anti-SPATA46 antibody, suggesting functional relevance of SPATA46 for sperm-egg fusion. Taken together, these results indicated that SPATA46 is a novel protein involved in reshaping of the sperm head and sperm-egg fusion.
Assuntos
Infertilidade Masculina/genética , Proteínas/genética , Espermátides/metabolismo , Espermatogênese/genética , Espermatozoides/metabolismo , Animais , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas/metabolismo , Cabeça do Espermatozoide/metabolismo , Interações Espermatozoide-Óvulo/genéticaRESUMO
OBJECTIVE: To investigate the expression characteristics of the gene of coiled-coil domain-containing protein 70 (Ccdc70) in the mouse testis and its potential role in spermatogenesis. METHODS: Using expression profile microarray, we screened the mouse testis-specific gene Ccdc70, studied its expression characteristics in the mouse testis by RT-PCR, real-time PCR, Western blot and immunohistochemistry, followed by bioinformatic analysis of the Ccdc70 protein. RESULTS: The Ccdc70 gene was expressed highly in the testis but lowly in the epididymis of the mice. The Ccdc70 protein was expressed mainly in the spermatocytes and round spermatids of the testis and in the epithelial cells of the epididymis. Bioinformatic analysis showed a structural domain in the Ccdc70 protein, which was highly conserved in mammalian evolution. CONCLUSION: The Ccdc70 gene is highly expressed in the mouse testis and mainly in the spermatocytes, round spermatids, and epididymal epithelial cells, which indicates that it is involved in the regulation of spermatogenesis and epididymal sperm maturation.
Assuntos
Proteínas/genética , Espermatogênese/genética , Testículo/metabolismo , Animais , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Masculino , CamundongosRESUMO
The acrosome is a specialized organelle that covers the anterior region of the sperm nucleus, and plays an essential role in mammalian fertilization. Although acrosome biogenesis is an important aspect of spermiogenesis, the molecular mechanism that regulates this event remains unknown. In the present study, we identified a novel gene, Fam170b (family with sequence similarity 170, member B), exclusively expressed in mouse testes. Fam170b expression first started at postnatal week 3, and increased in an age-dependent manner until plateauing in adulthood. Immunofluorescence staining revealed its enrichment in round spermatids, and redistribution to a perinuclear spot adjacent to the Golgi and the acrosome of elongating spermatids and spermatozoa; this localization was shared between mouse and human spermatozoa. Anti-FAM170B antibody was remarkably found to inhibit murine in vitro fertilization, specifically blocking the acrosome reaction. We further determined that FAM170B interacts with GOPC (Golgi-associated PDZ and coiled-coil motif containing protein) during acrosome formation, as verified by immunofluorescence and co-immunoprecipitation assays. Thus, we document the expression and function for the endogenous acrosomal protein FAM170B during spermiogenesis and fertilization.
Assuntos
Acrossomo/metabolismo , Fertilização , Proteínas de Plasma Seminal/genética , Reação Acrossômica , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Feminino , Fertilização in vitro , Proteínas da Matriz do Complexo de Golgi , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Plasma Seminal/metabolismo , Proteínas de Plasma Seminal/fisiologia , Espermatogênese , Testículo/metabolismoRESUMO
The serine/arginine-rich splicing actor 4 (SRSF4) is essential for pre-mRNA splicing and can influence alternative-splice-site choice. Little is known about the specific function of this gene in the reproductive system, although a recent study identified a SRSF4 polymorphism significantly associated with a decreased risk of non-obstructive azoospermia in Chinese men. We previously found that the expression of Srsf4 was up-regulated in the testes of Sertoli-cell-selective androgen receptor knockout (S-Ar(-/y)) mice compared to wild-type mice using digital gene expression analysis. In this study, we confirmed and extended the selective gene expression data: SRSF4 was mainly located in the nucleus of Sertoli cells, and Srsf4 expression in the Sertoli-cell-derived cell line TM4 is down-regulation by testosterone. Moreover, androgen receptor directly binds the androgen-responsive element of the Srsf4 promoter. Taken together, these results demonstrate that Srsf4 is a direct downstream target of the androgen receptor in mouse Sertoli cells.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas de Ligação a RNA/biossíntese , Receptores Androgênicos/metabolismo , Elementos de Resposta/fisiologia , Células de Sertoli/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Proteínas de Ligação a RNA/genética , Receptores Androgênicos/genética , Células de Sertoli/citologia , Testosterona/farmacologiaRESUMO
Androgens and the androgen receptor (AR) are of great importance to spermatogenesis and male fertility. AR knockout (ARKO) mice display a complete insensitivity to androgens and male infertility; however, the exact molecular mechanism for this effect remains unclear. In this study, we found that the expression levels of Prmt6 mRNA and protein were significantly up-regulated in the testes of ARKO mice compared to wild type (WT) mice. PRMT6 was principally localized to the nucleus of spermatogonia and spermatocytes by immunofluorescence staining. Furthermore, luciferase assay data showed that AR together with testosterone treatment suppressed Prmt6 transcription via binding to the androgen-responsive element (ARE) of the Prmt6 promoter. Moreover, knockdown of Prmt6 suppressed germ cells migration and promoted apoptosis. In addition, both of these cellular activities could not be enhanced by testosterone treatment. Taken together, these data indicate that PRMT6, which was down-regulated by AR and influenced cell migration and apoptosis of germ cells, could play a potentially important role in spermatogenesis.
Assuntos
Proteína-Arginina N-Metiltransferases/metabolismo , Receptores Androgênicos/fisiologia , Espermatogênese , Espermatozoides/fisiologia , Animais , Apoptose , Células COS , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Chlorocebus aethiops , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transporte Proteico , Proteína-Arginina N-Metiltransferases/genética , Transdução de Sinais , Testículo/enzimologia , Testosterona/fisiologiaRESUMO
OBJECTIVE: To identify the expression characteristics of the 1700001022RIK (RIKEN cDNA 1700001022) gene in mice and explore its function by bioinformatic analysis. METHODS: Using the expression profile of gene microarray, we detected the expression of a new testis-specific gene, 1700001022RIK, in mice. We analyzed its expression characteristics in the testis tissue and their changes in different developmental stages of the testis by RT-PCR, real-time RT-PCR, Western blot, and immunohistochemistry. We performed bioinformatic analysis using a bioinformatic software. RESULTS: The 1700001022RIK gene was specifically expressed in the mouse testis in an age-dependent manner, most highly in the adult mice. The 1700001022RIK protein was mainly expressed in the spermatogonia, spermatocytes, and round spermatids of the adult mice. Bioinformatic analysis showed that the 1700001022RIK protein amino acid sequence had a high similarity in human and mice, which indicated that this gene was highly conserved in mammals. CONCLUSION: 1700001022RIK is a testis-specific gene mainly expressed in the spermatogonia, spermatocytes, and round spermatids of seminiferous tubules, which might be involved in the regulation of spermatogenesis.
Assuntos
Expressão Gênica , Genômica , Chaperonas Moleculares/genética , Testículo , Fatores Etários , Animais , Western Blotting , Biologia Computacional , DNA Complementar , Masculino , Camundongos , Túbulos Seminíferos , Espermátides , Espermatócitos , Espermatogênese/genética , EspermatogôniasRESUMO
MicroRNAs (miRNAs) are known to mainly target protein-coding genes at post-transcriptional level, resulting in mRNA destabilization and/or translational repression. Long non-coding RNAs (lncRNAs) are emerging as a novel set of targets for miRNAs. Here, we report that downregulated hsa-miR-1 and upregulated lncRNA urothelial cancer associated 1 (UCA1) were inversely expressed in bladder cancer. Hsa-miR-1 decreased the expression of UCA1 in bladder cancer cells in an Ago2-slicer-dependent manner. The binding site between UCA1 and hsa-miR-1 was confirmed. Overexpression of hsa-miR-1 inhibited bladder cancer cell growth, induced apoptosis, and decreased cell motility. Knockdown of UCA1 expression phenocopied the effects of upregulation of hsa-miR-1. Transfection of UCA1 expression vector partly reversed the changes caused by transfection of pre-miR-1 plasmids. This study provides evidence for hsa-miR-1 to play tumor suppressive roles via downregulating lncRNA UCA1 in bladder cancer, which may have potential therapeutic significance.
Assuntos
Carcinoma de Células de Transição/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Western Blotting , Proliferação de Células , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Androgen signaling plays a crucial role in spermatogenesis, yet few downstream targets for this signaling pathway have been identified. In the current study, we found that the expression of heat-shock transcription factor 1 (Hsf1) was increased in the testes of Sertoli cell-selective androgen receptor knockout (S-AR(-/y) ) mice compared with wild-type mice by quantitative real-time PCR, and the expression of HSF1 in the S-AR(-/y) Sertoli cells was significantly increased, based on immunofluorescence analysis. In vitro cell-culture studies showed that testosterone repressed the expression of Hsf1 in TM4 cells, a mouse Sertoli cell line. Moreover, a luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay showed that testosterone repressed Hsf1 expression by facilitating the binding of androgen receptor to the Hsf1 promoter. Our experiments also demonstrated that testosterone-mediated inhibition of Hsf1 transcription down-regulated the expression of heat-shock proteins HSP105 and HSP60. Taken together, these results reveal that Hsf1 is a novel target of androgen receptor in mouse Sertoli cells, and testosterone and its receptor regulate the process of spermatogenesis partially by inhibiting Hsf1 expression.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Receptores Androgênicos/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Fatores de Transcrição/biossíntese , Animais , Linhagem Celular , Chaperonina 60/biossíntese , Chaperonina 60/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP110/biossíntese , Proteínas de Choque Térmico HSP110/genética , Fatores de Transcrição de Choque Térmico , Masculino , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Receptores Androgênicos/genética , Células de Sertoli/citologia , Testosterona/genética , Testosterona/metabolismo , Fatores de Transcrição/genéticaRESUMO
Idiopathic azoospermia (IA) is a severe form of male infertility due to unknown causes. The HSF2 gene, encoding the heat shock transcription factor 2, had been suggested to play a significant role in the spermatogenesis process since the Hsf2-knockout male mice showed spermatogenesis defects. To examine whether HSF2 is involved in the pathogenesis of IA in human, we sequenced all the exons of HSF2 in 766 patients diagnosed with IA and 521 proven fertile men. A number of coding mutations private to the patient group, which include three synonymous mutations and five missense mutations, were identified. Of the missense mutations, our functional assay demonstrated that one heterozygous mutation, R502H, caused a complete loss of HSF2 function and that the mutant suppressed the normal function of the wild-type (WT) allele through a dominant-negative effect, thus leading to the dominant penetrance of the mutant allele. These results support a role for HSF2 in the pathogenesis of IA and further implicate this transcription factor as a potential therapeutic target.
Assuntos
Azoospermia/genética , Genes Dominantes , Proteínas de Choque Térmico/genética , Mutação , Fatores de Transcrição/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Many genes are regulated by androgen and its receptor (AR), but the direct target genes of AR, especially those involved in spermatogenesis and male infertility, remain unclear. Here, we identified ubiquitin-conjugating enzyme E2B (Ube2b) as a critical target gene of AR. The expression of UBE2B was decreased in the testes of Sertoli cell AR knockout (S-AR(-/y)) mice analyzed by quantitative RT-PCR (qRT-PCR) and immunofluorescence. The upregulation of Ube2b gene by testosterone was further demonstrated by Western blot and qRT-PCR in TM4 cells, a mouse Sertoli cell line. Moreover, luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay validated that the ligand-bound AR activated Ube2b transcription via direct binding to the androgen-responsive element of the Ube2b promoter. In vitro analyses showed that testosterone increased UBE2B expression and activated H2A ubiquitylation, while downregulation of UBE2B blocked the testosterone-induced H2A ubiquitylation. The ubiquitylation of H2A was markedly decreased in the testes of S-AR(-/y) mice by immunohistochemistry. Digital gene expression analysis showed that 113 genes were significantly downregulated and 71 were upregulated by UBE2B in TM4 cells. These results suggest that Ube2b, as a direct AR transcriptional target in Sertoli cells, mediates the function of AR in spermatogenesis by promoting H2A ubiquitylation.
Assuntos
Receptores Androgênicos/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Animais , Células Cultivadas , Regulação para Baixo/fisiologia , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Receptores Androgênicos/genética , Transdução de Sinais/fisiologia , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinação/fisiologia , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: The molecular mechanisms involved in the development and progression of clear cell renal cell carcinomas (ccRCCs) are poorly understood. The objective of this study was to analyze the expression of dual-specificity phosphatase 9 (DUSP-9) and determine its clinical significance in human ccRCCs. METHODS: The expression of DUSP-9 mRNA was determined in 46 paired samples of ccRCCs and adjacent normal tissues by using real-time qPCR. The expression of the DUSP-9 was determined in 211 samples of ccRCCs and 107 paired samples of adjacent normal tissues by immunohistochemical analysis. Statistical analysis was performed to define the relationship between the expression of DUSP-9 and the clinical features of ccRCC. RESULTS: The mRNA level of DUSP-9, which was determined by real-time RT-PCR, was found to be significantly lower in tumorous tissues than in the adjacent non-tumorous tissues (p < 0.001). An immunohistochemical analysis of 107 paired tissue specimens showed that the DUSP-9 expression was lower in tumorous tissues than in the adjacent non-tumorous tissues (p < 0.001). Moreover, there was a significant correlation between the DUSP-9 expression in ccRCCs and gender (p = 0.031), tumor size (p = 0.001), pathologic stage (p = 0.001), Fuhrman grade (p = 0.002), T stage (p = 0.001), N classification (p = 0.012), metastasis (p = 0.005), and recurrence (p < 0.001). Patients with lower DUSP-9 expression had shorter overall survival time than those with higher DUSP-9 expression (p < 0.001). Multivariate analysis indicated that low expression of the DUSP-9 was an independent predictor for poor survival of ccRCC patients. CONCLUSION: To our knowledge, this is the first study that determines the relationship between DUSP-9 expression and prognosis in ccRCC. We found that decreased expression of DUSP-9 is associated with poor prognosis in ccRCC. DUSP-9 may represent a novel and useful prognostic marker for ccRCC.
Assuntos
Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Fosfatases de Especificidade Dupla/metabolismo , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Adulto , Idoso , Carcinoma de Células Renais/genética , Fosfatases de Especificidade Dupla/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Renais/genética , Masculino , Pessoa de Meia-Idade , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/metabolismoRESUMO
A novel gene, TSG23/Tsg23, was identified by comparing the expression profiles of human adult and fetal testis using Affymetrix Genechips. RT-PCR analysis from multiple human and mouse tissues indicated TSG23/Tsg23 mRNA was mainly expressed in the testis. In situ hybridization revealed that TSG23/Tsg23 mRNA was located in spermatocytes and round spermatids of the seminiferous tubules in human and mouse testis. To further confirm the result from RT-PCR, the antibody for human TSG23 was generated against the protein encoded by the gene. Western blot analysis demonstrated that TSG23 was mainly expressed in human testis, with a molecular weight of about 23 kDa. Immunohistochemistry showed that TSG23 was predominantly located in spermatocytes and round spermatids, consistent with the results from in situ hybridization. In order to explore the function of TSG23 in spermatogenesis, the study compared the expression of TSG23 in the testis from fertile persons and from patients with azoospermia. The results showed that there was less expression in patients with obstructive azoospermia compared with fertile persons, and no detectable TSG23 at mRNA and protein levels in patients with non-obstructive azoospermia. The expression of Tsg23 mRNA was considerably decreased in a time-dependent manner in the testis of an azoospermic mouse model induced by Busulfan. These data suggest that TSG23/Tsg23 is involved in human and mouse spermatogenesis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Espermatogênese/genética , Testículo/fisiologia , Adulto , Sequência de Aminoácidos , Animais , Azoospermia/genética , Sequência de Bases , Feminino , Feto/anatomia & histologia , Feto/fisiologia , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Alinhamento de Sequência , Espermátides/fisiologia , Espermatócitos/fisiologia , Distribuição Tecidual , Adulto JovemRESUMO
microRNAs (miRs) have been investigated as a novel class of regulators of cellular processes, including proliferation, apoptosis and metabolism. In particular, miR30b has been demonstrated to be deregulated in certain types of cancer, including lung, colorectal and gastric cancer. Previous studies of miR30b in renal clear cell carcinoma demonstrated that the expression level of miR30b was associated with distant metastasis. However, the function of miR30b in renal cell carcinoma (RCC) remained to be elucidated. In the present study, the expression of miR30b in 31 paired RCC tissues from four cell lines (786O, 769P, ACHN and 293T) was detected by reverse transcriptionquantitative polymerase chain reaction. In addition, the effect of miR30b on cell proliferation in RCC cells was also determined using MTT and Cell Counting Kit8 assay analyses. Furthermore, the function of miR30b in cell migration and invasion was determined by wound scratch and Transwell assays. Flow cytometry was also performed to quantify the effect of miR30b on cell apoptosis. The results of the current study indicated that miR30b was upregulated in RCC tissues from affected cell lines when compared with adjacent normal tissues and a normal kidney cell line, which is different to the downregulation of miR30b as observed in other types of cancer. miR30b is associated with RCC cell proliferation, invasion, migration and apoptosis, which indicated that miR30b acts as an oncogene in RCC. To the best of our knowledge, the present study is the first to demonstrate the upregulation of miR30b in RCC tissues and describe miR30b as an oncogene in RCC in the regulation of cell proliferation, migration, invasion and apoptosis. Further studies will define the target gene of miR30b in RCC and investigate the potential role of miR30b as a biomarker for RCC.
Assuntos
Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Rim/patologia , MicroRNAs/genética , Adulto , Idoso , Apoptose , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Oncogenes , Regulação para CimaRESUMO
There is increasing evidence that the deregulation of microRNAs (miRNAs; miRs) contributes to tumorigenesis. Previous studies have shown that miR195 is downregulated in various types of cancer. The present study aimed to investigate the function and expression levels of miR125b. Results of qPCR revealed that miR1953p, the mature sequence of miR195, was upregulated in renal cell carcinoma (RCC) tissues and cell lines (786O, 769P and ACHN). This indicated that the function and role of miR1953p may differ in different types of tumor. To assess the function of miR1953p in RCC cell lines, cell proliferation was examined using MTT and CCK8 assays, mobility was assessed using a cell scratch assay, Transwell migration assay and invasion assay, and apoptosis was examined using flow cytometry. These assessments were also performed in cells with upregulated or downregulated miR1953p via transfection with synthesized miR1953p mimic or inhibitor. The results revealed that the overexpression of miR1953p promoted 786O and ACHN RCC cell proliferation, migration and invasion, and inhibited cell apoptosis, whereas the downregulation of miR1953p suppressed cell proliferation, migration and invasion, and induced cell apoptosis. These results indicated that miR1953p was associated with the tumorigenesis of RCC, with further investigations to focus on the pathway and use of miR1953p as a clinical biomarker for RCC.