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
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.
Assuntos
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
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
Disruption of extravillous trophoblast (EVT) migration and invasion is considered to be responsible for pathological placentation in preeclampsia (PE). Cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression. However, its biological function and underlying molecular mechanism in PE are poorly understood. In this study, clinical data demonstrated that CCNG2 was significantly upregulated in PE placenta and associated with invasive EVT dysfunction. Additionally, Ccng2 knockout led to an attenuation of PE-like symptoms in the PE mouse model produced via treatment with NG-nitro-L-arginine methyl ester (L-NAME). In vitro, CCNG2 inhibited the migration, invasion, and endothelial-like network formation of human trophoblast cell line HTR8/SVneo. Mechanically, CCNG2 suppressed JNK-dependent Wnt/PCP signaling and its downstream indicators including epithelial-to-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs) via promoting the polyubiquitination degradation of dishevelled 2 (Dvl2) protein in HTR8/SVneo cells. We also discovered that the E3 ligase Ring finger protein 123 (RNF123), as a novel CCNG2 target among HTR8/SVneo cells, interacted with Dvl2 and participated in CCNG2-induced polyubiquitination degradation of Dvl2. Moreover, we verified that the treatment of HTR8/SVneo cells with RNF123-specific siRNA improved polyubiquitination-induced degradation of Dvl2 and the activity of Wnt/PCP-JNK signaling mediated by CCNG2. Taken together, our results reveal that the CCNG2/RNF123/Dvl2/JNK axis may be involved in the pathogenesis and progression of PE through trophoblastic cell function modulation, thus probably providing us with new therapeutic strategies for PE treatment.
Assuntos
Movimento Celular/genética , Ciclina G1/metabolismo , Ciclina G2/metabolismo , Proteínas Desgrenhadas/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Pré-Eclâmpsia/metabolismo , Trofoblastos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima/genética , Adulto , Animais , Linhagem Celular , Ciclina G1/genética , Ciclina G2/genética , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Gravidez , Transfecção , Ubiquitina-Proteína Ligases/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
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
In eukaryotes, the cell cycle is driven by the actions of several cyclin dependent kinases (CDKs) and an array of regulatory proteins called cyclins, due to the cyclical expression patterns of the latter. In yeast, the accepted pattern of cyclin waves is based on qualitative studies performed by different laboratories using different strain backgrounds, different growing conditions and media, and different kinds of genetic manipulation. Additionally, only the subset of cyclins regulating Cdc28 was included, while the Pho85 cyclins were excluded. We describe a comprehensive, quantitative and accurate blueprint of G1 cyclins in the yeast Saccharomyces cerevisiae that, in addition to validating previous conclusions, yields new findings and establishes an accurate G1 cyclin blueprint. For the purposes of this research, we produced a collection of strains with all G1 cyclins identically tagged using the same and most respectful procedure possible. We report the contribution of each G1 cyclin for a broad array of growing and stress conditions, describe an unknown role for Pcl2 in heat-stress conditions and demonstrate the importance of maintaining the 3'UTR sequence of cyclins untouched during the tagging process.
Assuntos
Ciclina G1/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ciclo Celular , Ciclina G1/classificação , Ciclina G1/metabolismo , Genótipo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse FisiológicoRESUMO
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.
Assuntos
Ciclina G1/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Técnicas de Silenciamento de Genes , Humanos , RNA Interferente Pequeno/metabolismo , Câncer Papilífero da Tireoide/patologia , Câncer Papilífero da Tireoide/cirurgia , Glândula Tireoide/patologia , Glândula Tireoide/cirurgia , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/cirurgia , TireoidectomiaRESUMO
TP53 mutation is considerably common in advanced high-grade serous ovarian cancer (HGSOC) and significantly associated with a poor prognosis. In this study, we investigated the role of Cyclin G1 (CCNG1), a target gene of wild-type TP53 (P53wt), in HGSOC and the possible regulatory mechanism between TP53 mutant (P53mt) and CCNG1 in the progression of HGSOC. High expression level of CCNG1 was found in 61.3% of HGSOC tissues and only 18.2% in fimbriae of fallopian tubes. Additionally, overexpression of CCNG1 was significantly associated with a shorter overall survival (P < 0.0001) and progression-free survival (P < 0.0004) in HGSOC patients. In vitro, CCNG1 promoted both tumor cell motility by inducing epithelial-mesenchymal transition (EMT) and resistance to cisplatin (CDDP). In vivo, knockdown expression of CCNG1 inhibited cancer metastasis. Furthermore, P53mt increased the expression of CCNG1 by regulating Notch3 expression, and a positive correlation between CCNG1 and Notch3 protein expression was observed by Immunohistochemistry (IHC) (r = 0.39, P: 0.01528). In conclusion, the activation of P53mt-Notch3-CCNG1 pathway was responsible for tumor progression to advanced disease with correlation with worse prognosis in patients with HGSOC. These data suggest a possible molecular mechanism of disease and highlights CCNG1's potential role as a therapeutic target in HGSOC.
Assuntos
Ciclina G1/genética , Neoplasias Ovarianas/genética , Receptor Notch3/genética , Proteína Supressora de Tumor p53/genética , Antineoplásicos/uso terapêutico , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Cisplatino/uso terapêutico , Ciclina G1/metabolismo , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Neoplasias Ovarianas/patologia , Prognóstico , Receptor Notch3/metabolismoRESUMO
The acquisition of a castration-resistant prostate cancer phenotype by prostate cancer cells is the alteration that has the worst prognosis for patients. The aim of this study was to evaluate the role of the microRNAs-23b/-27b as well as the possible CCNG1 target gene in tissue samples from patients with localized prostate cancer that progressed to castration-resistant prostate cancer and in a castration-resistant prostate cancer cell line (PC-3). The microRNAs and target gene expression levels of the surgical specimens were analyzed by quantitative real-time polymerase chain reaction. The prostate cancer cell line, PC-3, was transfected with pre-miR-23b, pre-miR-27b, and their respective controls using Lipofectamine RNAiMAX and exposed or not to flutamide. After transfections, expression levels of both the microRNAs and the gene, CCNG1, were analyzed by quantitative real-time polymerase chain reaction. The apoptosis and cell cycle assays were performed on the mini MUSE cytometer. MicroRNAs-23b/-27b were underexpressed in surgical specimens of prostate cancer; however, their target gene, CCNG1, was overexpressed in 69% of the cases. After transfection with the microRNAs-23b/-27b and flutamide, we observed a reduction in gene expression compared with cells that were treated only with microRNAs or only with flutamide. In the apoptosis assay, we demonstrated cell sensitization following transfection with microRNAs-23b/-27b and potentiation when co-administered with flutamide. The number of cells in apoptosis was almost three times higher with the simultaneous treatments (miR + flutamide) compared with the control (p < 0.05). In the cell cycle assay, only flutamide treatment showed better results; a higher number of cells were found in the G0-G1 phase, and a lower percentage of cells completed the final phase of the cycle (p < 0.05). We conclude that microRNAs-23b/-27b are downexpressed in prostate cancer, and their target gene, CCNG1, is overexpressed. We postulated that microRNAs-23b/-27b sensitize the PC-3 cell line and that after the addition of flutamide in the apoptosis assay, we would observe synergism in the treatments between miR and flutamide. In the cell cycle assay, the use of flutamide was sufficient to decrease the number of cells in mitosis. Therefore, we postulate that microRNAs, along with other drugs, may become very useful therapeutic tools in the treatment of castration-resistant prostate cancer.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclina G1/genética , Flutamida/metabolismo , MicroRNAs/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Linhagem Celular Tumoral , Fase G1/efeitos dos fármacos , Fase G1/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mitose/efeitos dos fármacos , Mitose/genética , Próstata/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/genética , Transfecção/métodosRESUMO
BACKGROUND: MicroRNA-122 (miR-122), a pivotal liver-specific miRNA, is frequently repressed in hepatocellular carcinoma (HCC) and associated with poor prognosis. Long non-coding RNA (lncRNA) HOTAIR has been proved to function as an oncogene in multiple cancers including HCC. However, the relationship between HOTAIR and miR-122 in HCC remains largely unknown. METHODS: We investigated the function of HOTAIR and miR-122 in HCC cell models and a xenograft mouse model. The regulatory network between HOTAIR and miR-122 was further detected following overexpression or knockdown of HOTAIR. DNA methylation status of miR-122 promoter region, as well as expression levels of DNMTs, EZH2 and Cyclin G1 were analyzed. FINDINGS: In this study, we found that HOTAIR was highly expressed whereas miR-122 was suppressed in HCC, and HOTAIR negatively regulated miR-122 expression in HCC cells. Furthermore, knockdown of HOTAIR dramatically inhibited HCC cell proliferation and induced cell cycle arrest in vitro and suppressed tumorigenicity in vivo by upregulating miR-122 expression. Mechanistically, a CpG island was located in the miR-122 promoter region. HOTAIR epigenetically suppressed miR-122 expression via DNMTs-mediated DNA methylation. Moreover, HOTAIR upregulated DNMTs expression via EZH2. In addition, suppression of miR-122 induced by HOTAIR directly reactivated oncogene Cyclin G1 expression. Collectively, our results suggest that HOTAIR epigenetically suppresses miR-122 expression via DNA methylation, leading to activation of Cyclin G1 and promotion of tumorigenicity in HCC, which provide new insight into the mechanism of HOTAIR-mediated hepatocarcinogenesis via suppressing miR-122.
Assuntos
Carcinoma Hepatocelular/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , MicroRNAs/genética , Interferência de RNA , RNA Longo não Codificante/genética , Animais , Sequência de Bases , Carcinoma Hepatocelular/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Ciclina G1/genética , Ciclina G1/metabolismo , Modelos Animais de Doenças , Epigênese Genética , Feminino , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: miR-516b, as a tumor suppressor in several tumors, its regulatory role in esophageal squamous cell carcinoma (ESCC) hasn't been previously reported. OBJECTIVE: This study was to investigate the potential role of miR-516b in ESCC. METHODS: miR-516b expression was measured in ESCC tumor specimens and matched adjacent non-cancerous tissues from 80 ESCC patients. The association between miR-516b and clinicopathological features of these patients was analyzed. The effect of miR-516b was evaluated by cell proliferation, migration, invasion and apoptosis assays in ESCC cell line EC9706 and TE-9. The role of miR-516b in vivo was further studied by constructing ESCC xenograft mice model. The direct target of miR-516b was predicted by public miRNA database and confirmed by luciferase reporter assay. The regulation of miR-516b on the target gene was further confirmed in vitro and in vivo. The expressions of proteins related to cell cycle and apoptosis were analyzed by western blot analysis, and cell migration and invasion were assessed by transwell assays. RESULTS: miR-516b expression was reduced in ESCC tissues and cells, and correlated with advanced TNM stage, depth of invasion, lymphatic metastasis and poorer overall survival in ESCC patients. miR-516b was upregulated by miR-516b mimics repressing cell proliferation, and inducing G1 cell cycle arrest and apoptosis. miR-516b upregulation also suppressed the growth of ESCC xenograft tumor in nude mice and the invasion of ESCC cells via regulating the epithelial-mesenchymal transition pathway. CCNG1 was identified as a direct downstream target of miR-516b. CONCLUSION: The results demonstrated miR-516b functions as a tumor suppressor by directly modulating CCNG1 expression in ESCC cells, and may be a novel therapeutic and prognostic biomarker for ESCC.
Assuntos
Ciclina G1/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Genes Supressores de Tumor , MicroRNAs/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ciclina G1/genética , Regulação para Baixo , Transição Epitelial-Mesenquimal , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Transdução de Sinais , Fatores de Tempo , Carga TumoralRESUMO
Budding yeast cells produce a finite number of daughter cells before they die. Why old yeast cells stop dividing and die is unclear. We found that age-induced accumulation of the G1/S-phase inhibitor Whi5 and defects in G1/S cyclin transcription cause cell cycle delays and genomic instability that result in cell death. We further identified extrachromosomal rDNA (ribosomal DNA) circles (ERCs) to cause the G1/S cyclin expression defect in old cells. Spontaneous segregation of Whi5 and ERCs into daughter cells rejuvenates old mothers, but daughters that inherit these aging factors die rapidly. Our results identify deregulation of the G1/S-phase transition as the proximal cause of age-induced proliferation decline and cell death in budding yeast.
Assuntos
Pontos de Checagem da Fase G1 do Ciclo Celular , Aneuploidia , Divisão Celular , Ciclina G1/genética , Ciclina G1/metabolismo , Dano ao DNA , DNA Ribossômico/química , Proteínas Fúngicas/metabolismo , Expressão Gênica , Saccharomycetales/citologia , Saccharomycetales/genética , Saccharomycetales/metabolismo , Transcrição GênicaRESUMO
For triage purposes following a nuclear accident, blood-based gene expression biomarkers can provide rapid dose estimates for a large number of individuals. Ionizing-radiation-responsive genes are regulated through the DNA damage-response pathway, which includes activation of multiple transcription factors. Modulators of this pathway could potentially affect the response of these biomarkers and consequently compromise accurate dose estimation calculations. In the present study, four potential confounding factors were selected: cancer condition, sex, simulated bacterial infection (lipopolysaccharide), and curcumin, an anti-inflammatory/antioxidant agent. Their potential influence on the transcriptional response to radiation of the genes CCNG1 and PHPT1, two biomarkers of radiation exposure ex vivo, was assessed. First, both CCNG1 and PHPT1 were detected in vivo in blood samples from radiotherapy patients and as such were validated as biomarkers of exposure. Importantly, their basal expression level was slightly but significantly affected in vivo by patients' cancer condition. Moreover, lipopolysaccharide stimulation of blood irradiated ex vivo led to a significant modification of CCNG1 and PHPT1 transcriptional response in a dose- and time-dependent manner with opposite regulatory effects. Curcumin also affected CCNG1 and PHPT1 transcriptional response counteracting some of the radiation induction. No differences were observed based on sex. Dose estimations calculated using linear regression were affected by lipopolysaccharide and curcumin. In conclusion, several confounding factors tested in this study can indeed modulate the transcriptional response of CCNG1 and PHPT1 and consequently can affect radiation exposure dose estimations but not to a level which should prevent the biomarkers' use for triage purposes.
Assuntos
Biomarcadores/sangue , Ciclina G1/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Neoplasias/sangue , Monoéster Fosfórico Hidrolases/genética , Dosagem Radioterapêutica/normas , Radioterapia de Intensidade Modulada , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Estudos de Casos e Controles , Curcumina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/radioterapiaRESUMO
BACKGROUND: This investigation aimed to evaluate the role of methylation in the regulation of microRNA (miR)-122, miR-125b and miR-106b gene expression and the expression of their target genes during isoniazid (INH)-induced liver injury. METHODS: Rats were given INH 50 mg kg- 1·d- 1 once per day for 3, 7, 10, 14, 21 and 28 days and were sacrificed. Samples of blood and liver were obtained. RESULTS: We analysed the methylation and expression levels of miR-122, miR-125b and miR-106b and their potential gene targets in livers. Liver tissue pathologies, histological scores and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities changed, indicating the occurrence of liver injury. Relative expression levels of miR-122, miR-125b and miR-106b genes in the liver decreased after INH administration and correlated with the scores of liver pathology and serum AST and ALT activities, suggesting that miR-122, miR-125b and miR-106b are associated with INH-induced liver injury. The amount of methylated miR-122, miR-125b and miR-106b in the liver increased after INH administration and correlated with their expression levels, suggesting the role of methylation in regulating miRNA gene expression. Two miR-122 gene targets, cell cycle protein G1 (Cyclin G1) and cationic amino acid transporter-1 (CAT-1), also increased at the mRNA and protein levels, which suggests that lower levels of miR-122 contribute to the upregulation of Cyclin G1 and CAT-1 and might play a role in INH-induced liver injury. Signal transducer and activator of transcription 3 (STAT3) was a common target gene of miR-125b and miR-106b, and its expression levels of mRNA and protein increased after INH administration. The protein expression of phosphorylated (p)-STAT3 and the mRNA expression of RAR-related orphan receptor gamma (RORγt) regulated by p-STAT3 also increased. Meanwhile, the mRNA and protein expression of interleukin (IL)-17 regulated by RORγt, and the mRNA and protein expression of CXCL1 and MIP-2 regulated by IL-17 increased after INH administration. These results demonstrate that lower levels of hepatic miR-125b and miR-106b contribute to the upregulation of STAT3 in stimulating the secretion of inflammatory factors during INH-induced liver injury. CONCLUSIONS: Our results suggested that DNA methylation probably regulates the expression of miRNA genes (miR-122, miR-125b, and miR-106b), affecting the expression of their gene targets (Cyclin G1, CAT-1, and STAT3) and participating in the process of INH-induced liver injury.