RESUMO
OBJECTIVE: Protein disulfide isomerase A3 (PDIA3) promotes the correct folding of newly synthesized glycoproteins in the endoplasmic reticulum. PDIA3 is overexpressed in most tumors, and it may become a biomarker of cancer prognosis and immunotherapy. Our study aims to detect the expression level of PDIA3 in gastric cancer (GC) and its association with GC development as wells as the underlying mechanisms. METHODS: GC cell lines with PDIA3 knockdown by siRNA, CRISPR-cas9 sgRNAs or a pharmacological inhibitor of LOC14 were prepared and used. PDIA3 knockout GC cells were established by CRISPR-cas9-PDIA3 system. The proliferation, migration, invasion and cell cycle of GC cells were analyzed by cell counting kit-8 assay, wound healing assay, transwell assay and flow cytometry, respectively. Immunodeficient nude mice was used to evaluate the role of PDIA3 in tumor formation. Quantitative PCR and western blot were used for examining gene and protein expressions. RNA sequencing was performed to see the altered gene expression. RESULTS: The expressions of PDIA3 in GC tissues and cells were increased significantly, and its expression was negatively correlated with the three-year survival rate of GC patients. Down-regulation of PDIA3 by siRNA, LOC14 or CRISPR-cas9 significantly inhibited proliferation, invasion and migration of GC cells TMK1 and AGS, with cell cycle arrested at G2/M phase. Meanwhile, decreased PDIA3 significantly inhibited growth of tumor xenograft in vivo. It was found that cyclin G1 (encoded by CCNG1 gene) expression was decreased by downregulation of PDIA3 in GC cells both in vitro and in vivo. In addition, protein levels of other cell cycle related factors including cyclin D1, CDK2, and CDK6 were also significantly decreased. Further study showed that STAT3 was associated with PDIA3-mediated cyclin G1 regulation. CONCLUSION: PDIA3 plays an oncogenic role in GC. Our findings unfolded the functional role of PDIA3 in GC development and highlighted a novel target for cancer therapeutic strategy.
Assuntos
Benzotiazóis , Neoplasias Gástricas , Animais , Camundongos , Humanos , Neoplasias Gástricas/patologia , Regulação para Baixo/genética , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Camundongos Nus , Ciclina G1/genética , RNA Guia de Sistemas CRISPR-Cas , Proliferação de Células/genética , Linhagem Celular Tumoral , Ciclo Celular/genética , RNA Interferente Pequeno/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Movimento Celular/genéticaRESUMO
Women with HR+HER2+ early-stage breast cancer are disadvantaged by the lack of clinical trials focused on women ≥70 years of age. In the past years, there has been increasing controversy on the use of toxic chemotherapy as standard of care treatment for early- stage HR+ HER2+ breast carcinoma in older women. With precision medicine coming of age, molecular profiling of tumors and circulating tumor DNA has identified target oncogenes that could be used in designing an optimal treatment for this group of women. This article reviews the current treatment of early-stage triple receptor positive breast cancer, the risks of chemotherapy in older women, and CCNG1, a novel biomarker in development for the use of DeltaRex-G, a CCNG1 inhibitor. Further, future perspectives for DeltaRex-G in older women with early stage CCNG1+ HR+ HER2+ breast cancer are discussed.
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Neoplasias da Mama , Receptor ErbB-2 , Feminino , Humanos , Idoso , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Trastuzumab , Ciclina G1RESUMO
A significant decrease in LINC00467 expression in testicular germ cell tumors (TGCTs) was found in our previous study in comparison to adjacent tissue. Interestingly, the expression of LINC00467 correlated with the pathological grade of the tumor in TGCT patients. The higher the expression of LINC00467 was, the worse the prognosis of the patients with TGCT was. Despite these findings, the exact role of LINC00467 in the development of TGCTs requires further investigation. LINC00467 expression was downregulated in the NCCIT and TCam-2 cell lines via small interfering RNA (siRNA) silencing. The levels of gene expression were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Cell proliferation was evaluated by the MTT and Cell Counting Kit-8 (CCK8) assays, whereas flow cytometry was used to assess the effects on the cell cycle. Western blotting analysis was used to detect expression levels of protein. Additionally, RNA-sequencing and bioinformatics methods were used to investigate the mechanism of action of LINC00467 in TGCTs. The suppression of LINC00467 expression resulted in decreased cell proliferation and induced S-phase arrest. Furthermore, the suppression of LINC00467 downregulated proliferating cell nuclear antigen (PCNA), a protein related to cell cycle regulation, while it upregulated p21 expression. In other studies involving dihydrotestosterone (DHT) stimulation, it was observed that DHT could upregulate LINC00467 expression. In addition, silencing of the LINC00467 reversed the effect of testosterone on cell proliferation. The Gene Set Enrichment Analysis (GSEA) revealed that LINC00467 regulated the p53 pathway by modulating the expression of CCNG1. Our study found that LINC00467 regulates cell proliferation by inducing S-phase arrest through the cell cycle-related proteins PCNA and p21. These findings contribute to our understanding of non-coding RNAs mechanisms involved in the development of TGCTs.
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Neoplasias Embrionárias de Células Germinativas , Neoplasias Testiculares , Masculino , Humanos , Antígeno Nuclear de Célula em Proliferação , Neoplasias Testiculares/genética , Proliferação de Células/genética , RNA Interferente Pequeno/genética , Ciclina G1RESUMO
BACKGROUND: Protein arginine methyltransferase 6 (PRMT6) is a type I arginine methyltransferase that asymmetrically dimethylates histone H3 arginine 2 (H3R2me2a). However, the biological roles and underlying molecular mechanisms of PRMT6 in colorectal cancer (CRC) remain unclear. METHODS: PRMT6 expression in CRC tissue was examined using immunohistochemistry. The effect of PRMT6 on CRC cells was investigated in vitro and in vivo. Mass spectrometry, co-immunoprecipitation and GST pulldown assays were performed to identify interaction partners of PRMT6. RNA-seq, chromatin immunoprecipitation, Western blot and qRT-PCR assays were used to investigate the mechanism of PRMT6 in gene regulation. RESULTS: PRMT6 is significantly upregulated in CRC tissues and facilitates cell proliferation of CRC cells in vitro and in vivo. Through RNA-seq analysis, CDKN2B (p15INK4b) and CCNG1 were identified as new transcriptional targets of PRMT6. PRMT6-dependent H3R2me2a mark was predominantly deposited at the promoters of CDKN2B and CCNG1 in CRC cells. Furthermore, PRMT5 was firstly characterized as an interaction partner of PRMT6. Notably, H3R2me2a coincides with PRMT5-mediated H4R3me2s and H3R8me2s marks at the promoters of CDKN2B and CCNG1 genes, thus leading to transcriptional repression of these genes. CONCLUSIONS: PRMT6 functionally associates with PRMT5 to promote CRC progression through epigenetically repressing the expression of CDKN2B and CCNG1. These insights raise the possibility that combinational intervention of PRMT6 and PRMT5 may be a promising strategy for CRC therapy.
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Neoplasias Colorretais , Repressão Epigenética , Proteínas Nucleares , Proteína-Arginina N-Metiltransferases , Humanos , Arginina/química , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Ciclina G1/genética , Ciclina G1/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Repressão Epigenética/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismoRESUMO
Acute kidney injury (AKI) occurs in approximately 13% of hospitalized patients and predisposes patients to chronic kidney disease (CKD) through the AKI-to-CKD transition. Studies from our laboratory and others have demonstrated that maladaptive repair of proximal tubule cells (PTCs), including induction of dedifferentiation, G2/M cell cycle arrest, senescence, and profibrotic cytokine secretion, is a key process promoting AKI-to-CKD transition, kidney fibrosis, and CKD progression. The molecular mechanisms governing maladaptive repair and the relative contribution of dedifferentiation, G2/M arrest, and senescence to CKD remain to be resolved. We identified cyclin G1 (CG1) as a factor upregulated in chronically injured and maladaptively repaired PTCs. We demonstrated that global deletion of CG1 inhibits G2/M arrest and fibrosis. Pharmacological induction of G2/M arrest in CG1-knockout mice, however, did not fully reverse the antifibrotic phenotype. Knockout of CG1 did not alter dedifferentiation and proliferation in the adaptive repair response following AKI. Instead, CG1 specifically promoted the prolonged dedifferentiation of kidney tubule epithelial cells observed in CKD. Mechanistically, CG1 promotes dedifferentiation through activation of cyclin-dependent kinase 5 (CDK5). Deletion of CDK5 in kidney tubule cells did not prevent G2/M arrest but did inhibit dedifferentiation and fibrosis. Thus, CG1 and CDK5 represent a unique pathway that regulates maladaptive, but not adaptive, dedifferentiation, suggesting they could be therapeutic targets for CKD.
Assuntos
Injúria Renal Aguda , Insuficiência Renal Crônica , Camundongos , Animais , Camundongos Knockout , Ciclina G1 , Desdiferenciação Celular/genética , Quinase 5 Dependente de Ciclina/genética , Apoptose , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular , Injúria Renal Aguda/genética , Insuficiência Renal Crônica/genética , FibroseRESUMO
OBJECTIVE: To investigate the protective effects and regulatory mechanism of miR-488-3p on doxorubicin-induced cardiotoxicity. METHODS: The C57BL/6 mice and primary cardiomyocytes were used to construct doxorubicin-induced cardiomyocyte injury models in vivo and in vitro. The levels of miR-488-3p and its downstream target genes were analyzed by quantitative real-time PCR. Mouse cardiac function, cell survival, cellular injury-related proteins, and the apoptosis level of cardiomyocytes were analyzed by echocardiography, MTT analysis, Western blotting, and DNA laddering separately. RESULTS: Cardiomyocyte injury caused by a variety of stimuli can lead to the reduction of miR-488-3p level, especially when stimulated with doxorubicin. Doxorubicin led to significant decrease in cardiac function, cell autophagic flux blockage, and apoptosis in vivo and in vitro. The expression of miR-488-3p's target gene, CyclinG1, increased remarkably in the doxorubicin-treated neonatal mouse cardiomyocytes. Overexpression of miR-488-3p inhibited CyclinG1 expression, increased cardiomyocyte viability, and attenuated doxorubicin-induced cardiomyocyte autophagic flux blockage and apoptosis. CONCLUSIONS: miR-488-3p is one of the important protective miRNAs in doxorubicin-induced cardiotoxicity by inhibiting the expression of CyclinG1, which provides insight into the possible clinical application of miR-488-3p/CyclinG1 as therapeutic targets in doxorubicin-induced cardiovascular diseases.
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Antibióticos Antineoplásicos/efeitos adversos , Cardiotoxicidade/etiologia , Ciclina G1/antagonistas & inibidores , Doxorrubicina/efeitos adversos , MicroRNAs/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Animais , Humanos , Masculino , Camundongos , RatosRESUMO
Osteosarcoma (OS), a prevalent aggressive malignancy in the bone, has limited therapeutic targets and diagnostic biomarkers. In the current investigation, RT-qPCR showed that CDKN2B-AS1 was enhanced in OS samples and cells. This research was set to examine the modulation of CDKN2B-AS1 in OS. The expression of CDKN2B-AS1 and downstream molecules was analyzed by RT-qPCR method. CCK8, EdU staining along with Transwell assays were applied to evaluate cell proliferation and invasion. Those in vitro investigations specified that silencing of CDKN2B-AS1 with shRNAs obviously impeded the proliferation and invasion of MG63 cells. To authenticate the relationships between CDKN2B-AS1 and microRNA-122-5p (miR-122-5p) or cyclin G1 (CCNG1) and miR-122-5p, we next employed luciferase reporter assay. We displayed that CDKN2B-AS1 repressed miR-122-5p to restore CCNG1 expression. All in all, our findings substantiated the indispensable function of CDKN2B-AS1 in OS progression and the possible molecular mechanism.
Assuntos
Neoplasias Ósseas , Ciclina G1 , MicroRNAs , Osteossarcoma , RNA Longo não Codificante , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Proliferação de Células , Ciclina G1/genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Osteossarcoma/genética , RNA Longo não Codificante/genéticaRESUMO
PURPOSE: Emerging evidence has shown that radiotherapy is an effective treatment for hepatocellular carcinoma (HCC), Micro(mi)RNAs are involved in regulating radiosensitivity in many cancers. MiR-122 accounts for approximately 70% of all cloned miRNAs in the liver, but there are few reports about whether it is involved in regulating of radiosensitivity in HCC cells. MATERIALS AND METHODS: HCC cells (HepG2 and Huh7) overexpressing miR-122 were constructed by transfecting them with lentiviral-miR-122. Then, their proliferation ability was analyzed by the MTT, and colony formation assays and a xenograft tumor model was used to detect their radiosensitivity. The expression of cyclin G1 mRNA and protein was detected by the quantitative real-time polymerase chain reaction and western blotting, respectively. RESULTS: Overexpression of miR-122 inhibited the proliferation of, and radiosensitized HCC cells. Cyclin G1 mRNA and protein level were suppressed in HepG2 tumors overexpression miR-122. CONCLUSION: MiR-122 may be useful as a potential radiosensitizer for HCC, and its mechanism is related to the regulation of cyclin G1.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/radioterapia , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina G1/genética , Ciclina G1/metabolismo , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/radioterapia , MicroRNAs/genética , MicroRNAs/metabolismo , RNA MensageiroRESUMO
Ovarian cancer (OC) brings about serious physical and psychological burden for female patients. LncRNA CASC9 has been reported to be intimately linked with the occurrence and development of several tumors. However, the biological role of lncRNA CASC9 in OC still lacks sufficient evidence. The expressions of CASC9 and miR-488-3p in OC cell lines and xenograft mice were detected by qRT-PCR assay. Cell Counting Kit-8 (CCK-8) assay was used to assess cell inhibition rate and cell proliferation in OVCAR-3 and OVCAR-3/DDP cells. Wound healing assay and transwell assay were performed to evaluate the capacity of migration and invasion, respectively. In addition, cell apoptosis was measured by TUNEL assay and cell cycle was assessed by flow cytometric analysis. Moreover, western blotting was carried out to detect the cyclinG1 (CCNG1)/TP53/MMP7 signaling and apoptosis-related proteins. Furthermore, luciferase reporter assay was performed to verify the combination of CASC9 with CCNG1 and miR-488-3p. The results of our study revealed that CASC9 expression was upregulated while miR-488-3p and CCNG1 expression was downregulated in OC cells with significant higher TP53 and MMP7 protein levels compared with normal ovarian surface epithelial cells. Additionally, luciferase reporter assay confirmed CASC9 bond to miR-488-3p/CCNG1. CASC9 silencing inhibited cell proliferation, migration, and invasion whereas promoted cell inhibition rate and apoptosis in vitro and in vivo. However, CASC9 overexpression showed the opposite effects. In summary, LncRNA CASC9 played a regulative role in ovarian carcinoma by cyclinG1/TP53/MMP7 signaling via binding to miR-488-3p in vivo and in vitro.
Assuntos
MicroRNAs/genética , Neoplasias Ovarianas/patologia , RNA Longo não Codificante/genética , Transdução de Sinais , Animais , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ciclina G1/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinase 7 da Matriz/genética , Camundongos , Transplante de Neoplasias , Neoplasias Ovarianas/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Species differences in the metabolism of xenobiotics by cytochrome P450 are critical in evaluating the use of experimental animals in studying toxic compounds relevant to human diseases. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is produced by high-temperature cooking of fish and meat, is activated to become a carcinogen by cytochrome P4501A2 (CYP1A2) through N2 -hydroxylation in humans, but is detoxified by Cyp1a2 through 4'-hydroxylation in mice. CYP1A-humanized (hCYP1A) mice, in which mouse Cyp1a is replaced with human CYP1A, show constitutive human xenobiotic metabolism by hCYP1A, thereby serving as a suitable model for studying PhIP. Previous studies have demonstrated that oral administration of PhIP induces colon tumors in hCYP1A mice; however, these studies used a super-high dose, raising concerns regarding the relevance of the mechanism to human cancer. Herein, we systematically investigated PhIP-induced colon carcinogenesis in hCYP1A mice treated with lower doses. We found that a dose 2000 times lower than that used previously, which is comparable to human daily intake levels, could induce colon tumors, albeit at a lower incidence rate. We further investigated the transcriptome changes in the colon of hCYP1A mice treated with PhIP and identified that PhIP treatment increased the expression of Bax, Btg2, Ccng1, Cdkn1a, and Trp53inp1 and decreased the expression of Igf1 and Ccnd1. Since these genes are key components of the p53-dependent DNA damage response, the altered expression patterns indicated PhIP-induced DNA damage in hCYP1A mice. Together, these results prove that hCYP1A mice are suitable for studying PhIP-induced carcinogenesis and show that PhIP is an important colorectal cancer carcinogen in human diet.
Assuntos
Carcinógenos/toxicidade , Neoplasias do Colo/genética , Citocromo P-450 CYP1A2/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imidazóis/toxicidade , Proteína Supressora de Tumor p53/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Culinária/métodos , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina G1/genética , Ciclina G1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Dano ao DNA , Feminino , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Inativação Metabólica/genética , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Transgenes , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Long noncoding RNA (LncRNA) ophthalaldehyde-interacting protein 5 antisense transcript 1 (OIP5AS1) serves major roles in the progression of various types of cancer. The present study investigated its biological function in ovarian cancer (OC) and its mechanisms. The levels of OIP5AS1, microRNA1283p (miR1283p) and cyclin G1 (CCNG1) were examined by reverse transcriptionquantitative PCR. Cell viability, apoptosis, migration and invasion were detected to analyze cellular progression. Glycolytic metabolism was assessed by detecting the levels of glucose consumption and lactate production. CCNG1 and hexokinase 2 protein levels were measured by western blotting. Dualluciferase reporter assay, RNA immunoprecipitation and RNA pulldown assays were performed to affirm the interaction between two molecules. OIP5AS1 was found to be upregulated in OC tissues and cells. Knockdown of OIP5AS1 suppressed cell viability, migration, invasion and glycolysis while promoting apoptosis in OC cells. OIP5AS1 interacted with miR1283p and functioned as an oncogene by sequestering miR1283p. In addition, CCNG1 was a target gene for miR1283p and miR1283p regulated the CCNG1induced effects on OC cells by downregulating CCNG1. OIP5AS1 upregulated the expression of CCNG1 via targeting miR1283p. OIP5AS1 knockdown also inhibited tumor growth of OC in vivo by modulating the expression of miR1283p and CCNG1. Collectively, these data illustrated that the oncogenic role of OIP5AS1 in OC was associated with the miR1283p/CCNG1 axis at least in part. OIP5AS1 might be a probable diagnostic and therapeutic biomarker for the treatment of OC patients.
Assuntos
Ciclina G1/genética , MicroRNAs/genética , Neoplasias Ovarianas/genética , RNA Longo não Codificante/genética , Adolescente , Adulto , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Adulto JovemRESUMO
Radiotherapy plays an important role in the treatment of hepatocellular carcinoma (HCC). Cyclin G1 is a novel member of the cyclin family, and it is abnormally expressed in HCC. In this study we investigated the role of cyclin G1 in the radiotherapy of HCC cells. The expression of cyclin G1 was silenced by transfection of cyclin G1-siRNA into HepG2 cells and Huh7 cells, and the expression of cyclin G1 mRNA and protein was measured by qRT-PCR and Western blot analysis. The proliferation was analyzed using MTT assay, and the radiosensitivity of HCC cells was detected using colony formation assay and a xenograft tumor model. The expression of apoptosis-related proteins (Bcl-2 and Bax) was detected by Western blot analysis, and caspase-3 was detected using fluorimetry. The expression of cyclin G1 mRNA and protein in HepG2/Huh7-cyclin G1-siRNA cells was found to be significantly decreased compared to that in HepG2/Huh7 cells. Silencing the expression of cyclin G1 inhibited the proliferation of HCC cells and enhanced radiosensitivity in HCC cells in vitro and in vivo. Knockdown of cyclin G1 expression significantly decreased Bcl-2 expression, and increased Bax expression and caspase-3 activity in HCC cells. Silencing of cyclin G1 expression enhances the radiosensitivity of HCC cells in vitro and in vivo. The mechanism for this may be related to the regulation of apoptosis-related proteins.
Assuntos
Carcinoma Hepatocelular/radioterapia , Ciclina G1/genética , Neoplasias Hepáticas/radioterapia , Tolerância a Radiação/genética , Animais , Apoptose/efeitos da radiação , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Caspase 3/genética , Linhagem Celular Tumoral , Ciclina G1/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Xenoenxertos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
Shwachman-Diamond syndrome (SDS) is characterized by exocrine pancreatic insufficiency, neutropenia, and skeletal abnormalities. Biallelic mutations in SBDS, which encodes a ribosome maturation factor, are found in 90% of SDS cases. Sbds-/- mice are embryonic lethal. Using CRISPR/Cas9 editing, we created sbds-deficient zebrafish strains. Sbds protein levels progressively decreased and became undetectable at 10 days postfertilization (dpf). Polysome analysis revealed decreased 80S ribosomes. Homozygous mutant fish developed normally until 15 dpf. Mutant fish subsequently had stunted growth and showed signs of atrophy in pancreas, liver, and intestine. In addition, neutropenia occurred by 5 dpf. Upregulation of tp53 mRNA did not occur until 10 dpf, and inhibition of proliferation correlated with death by 21 dpf. Transcriptome analysis showed tp53 activation through upregulation of genes involved in cell cycle arrest, cdkn1a and ccng1, and apoptosis, puma and mdm2. However, elimination of Tp53 function did not prevent lethality. Because of growth retardation and atrophy of intestinal epithelia, we studied the effects of starvation on WT fish. Starved WT fish showed intestinal atrophy, zymogen granule loss, and tp53 upregulation - similar to the mutant phenotype. In addition, there was reduction in neutral lipid storage and ribosomal protein amount, similar to the mutant phenotype. Thus, loss of Sbds in zebrafish phenocopies much of the human disease and is associated with growth arrest and tissue atrophy, particularly of the gastrointestinal system, at the larval stage. A variety of stress responses, some associated with Tp53, contribute to pathophysiology of SDS.
Assuntos
Neutropenia/genética , Proteínas Nucleares/genética , Síndrome de Shwachman-Diamond/genética , Proteínas de Peixe-Zebra/genética , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Atrofia , Ciclina G1/genética , Ciclina G1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fígado/metabolismo , Fígado/patologia , Neutropenia/metabolismo , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Pâncreas/metabolismo , Pâncreas/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ribossomos/metabolismo , Síndrome de Shwachman-Diamond/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/metabolismoRESUMO
OBJECTIVE: The roles of microRNAs (miRNAs) have been widely exploited in cancer. MiRNAs have become a potential breakthrough in cancer diagnosis and treatment. Here, the regulatory mechanism of microRNA-488 (miR-488) was investigated in ovarian cancer (OC). PATIENTS AND METHODS: The expression levels of miR-488 and CCNG1 (Cyclin G1) were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot assays. Transwell assay and epithelial-mesenchymal transition (EMT) markers were used to clarify the effect of miR-488 on cell metastasis. The dual-luciferase reporter assay was used to verify the relation between miR-488 and CCNG1. RESULTS: The expression of miR-488 was reduced in OC, which was associated with poor clinical outcomes and prognosis in OC patients. MiR-488 inhibited cell metastasis in OC by blocking EMT and promoting tumor suppressor p53 expression. In addition, CCNG1 was confirmed as a direct target of miR-488. Upregulation of CCNG1 impaired the inhibitory effect of miR-488 in OC. CONCLUSIONS: MiR-488 serves as a tumor inhibitor in OC by suppressing cell metastasis, indicating that miR-488 has a great potential in the diagnosis and treatment of OC.
Assuntos
Ciclina G1/metabolismo , MicroRNAs/metabolismo , Neoplasias Ovarianas/metabolismo , Proteína Supressora de Tumor p53/genética , Células Cultivadas , Feminino , Humanos , MicroRNAs/genética , Neoplasias Ovarianas/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
The master tumor suppressor p53 controls transcription of a wide-ranging gene network involved in apoptosis, cell cycle arrest, DNA damage repair, and senescence. Recent studies revealed pervasive binding of p53 to cis-regulatory elements (CREs), which are non-coding segments of DNA that spatially and temporally control transcription through the combinatorial binding of local transcription factors. Although the role of p53 as a strong trans-activator of gene expression is well known, the co-regulatory factors and local sequences acting at p53-bound CREs are comparatively understudied. We designed and executed a massively parallel reporter assay (MPRA) to investigate the effect of transcription factor binding motifs and local sequence context on p53-bound CRE activity. Our data indicate that p53-bound CREs are both positively and negatively affected by alterations in local sequence context and changes to co-regulatory TF motifs. Our data suggest p53 has the flexibility to cooperate with a variety of transcription factors in order to regulate CRE activity. By utilizing different sets of co-factors across CREs, we hypothesize that global p53 activity is guarded against loss of any one regulatory partner, allowing for dynamic and redundant control of p53-mediated transcription.
Assuntos
Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Ciclina G1/genética , Fator 15 de Diferenciação de Crescimento/genética , Humanos , Imidazóis/farmacologia , Camundongos , Motivos de Nucleotídeos , Piperazinas/farmacologia , Transcrição GênicaRESUMO
BACKGROUND: The downstream targets of farnesoid X receptor (FXR) such as miRNAs have a potent effect on the progression of many types of cancer. We aim to study the effects of FXR on osteosarcoma (OS) development and the potential role of microRNA-23b-3p. METHODS: The expressions of FXR and miR-23b-3p in normal osteoblasts and five osteosarcoma cell lines were measured. Their correlations were analyzed by Pearson's test and verified by the introduction of FXR agonist, GW4064. TargetScan predicted that cyclin G1 (CCNG1) was a target for miR-23b-3p. The transfection of FXR siRNA was performed to confirm the correlation between FXR and miR-23b-3p. We further transfected miR-23b-3p inhibitor into MG-63 cells, and the transfected cells were treated with 5 µM GW4064 for 48 h. Quantitative PCR (qPCR) and Western blot were performed for expression analysis. Cell proliferation, cell apoptosis rate, and cell cycle distribution were assessed by clone formation assay and flow cytometry. RESULTS: Scatter plot showed a positive correlation between FXR and miR-23b-3p (Pearson's coefficient test R2 = 1.00, P = 0.0028). As CCNG1 is a target for miR-23b-3p, the treatment of GW4064 induced the downregulation of CCNG1 through upregulating miR-23b-3p. The inhibition of miR-23b-3p obviously promoted cell viability, proliferation, and cell cycle progression but reduced apoptosis rate of MG-63 cells; however, the treatment of GW4064 could partially reverse the effects of the inhibition of miR-23b-3p on OS cells. CONCLUSIONS: Upregulated FXR by GW4064 can obviously suppress OS cell development, and the suppressive effects may rely on miR-23b-3p/CCNG1 pathway.
Assuntos
MicroRNAs/metabolismo , Osteossarcoma/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Apoptose , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Ciclina G1/metabolismo , Humanos , Isoxazóis/farmacologia , Osteoblastos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Citoplasmáticos e Nucleares/fisiologia , Regulação para CimaRESUMO
Capsaicin (8-methyl-N-vanillyl-6-noneamide), which is an active component in red chili peppers, is used as a chemopreventive agent that shows favorable cytotoxicity against cancer cells. Accumulating evidence indicates that capsaicin preferentially inhibits a tumor-associated NADH oxidase (tNOX, ENOX2) that is ubiquitously expressed in cancer but not in non-transformed cells. This attenuates cancer cell growth by inducing apoptosis. The capsaicin-mediated inhibition of tNOX was recently shown to prolong the cell cycle. However, the molecular events underlying this regulation have not yet been investigated. In the present study, we used a cellular thermal shift assay (CETSA) to detect "target engagement" of capsaicin and its consequent impact on cell cycle progression. Our results indicated that capsaicin engaged with tNOX and triggered the proteasomal degradation of tNOX, which leads to the inhibition of NAD+-dependent SIRT1 deacetylase. Ultimately, the acetylation levels of c-Myc and p53 were enhanced, which suppressed the activation of G1 cyclin/Cyclin-dependent kinase complexes and triggered cell cycle arrest in cancer cells. The results obtained when tNOX was overexpressed in non-cancer cells validated its importance in cell cycle progression. These findings provide the first molecular insights into the regulatory role of tNOX and the anti-proliferative property of capsaicin in regulating the cell cycle of bladder cancer cells.
Assuntos
Capsaicina/farmacologia , Ciclina G1/metabolismo , NADH NADPH Oxirredutases/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
OBJECTIVE: The aim of this study was to investigate the regulatory mechanism of miR-23a on biological behaviors of papillary thyroid carcinoma (PTC) cells, such as cell proliferation, cell cycle and apoptosis. PATIENTS AND METHODS: The expression of miR-23a in 28 paired of PTC tissue samples and matched adjacent tissues was detected by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Meanwhile, miR-23a expression in PTC cell lines was also detected by qRT-PCR. Subsequently, miR-23a mimics and inhibitor were transfected into PTC cells. The effects of gain or loss of miR-23a on cell proliferation, cell cycle and apoptosis were analyzed. Bioinformatics analysis, Dual-Luciferase activity assay and Western blot were recruited to validate the potential target gene of miR-23a. RESULTS: The expression of miR-23a was significantly decreased in PTC tissue samples and cell lines. Upregulation of miR-23a in PTC cells markedly decreased cell proliferation, induced cell cycle arrest at G0/G1 phase and promoted cell apoptosis. However, decreased miR-23a exerted the opposite effects. Dual-Luciferase, qRT-PCR and Western blot showed that CCNG1 was a target gene of miR-23a. Furthermore, the silence of CCNG1 intensified the suppressive effect of miR-23a on cell growth. CONCLUSIONS: MiR-23a was involved in the development of PTC via targeting CCNG1, which might provide a new prospect for PTC diagnosis and therapy.