RESUMO
Co-immunoprecipitation (coIP) is an experimental technique to study protein-protein interactions (PPIs). However, single-step coIP can only be used to identify the interaction between two proteins and does not solve the interaction testing of ternary complexes. Here, we present a protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step coIP approach. We describe steps for cell culture and transfection, elution of target proteins, and two-step coIP including western blot analyses. For complete details on the use and execution of this protocol, please refer to Li et al.1.
Assuntos
Imunoprecipitação , Imunoprecipitação/métodos , Humanos , Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Western Blotting/métodos , Transfecção , Animais , Ligação Proteica , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/química , Células HEK293RESUMO
Embryonic stem cells (ESCs), with abilities of infinite proliferation (self-renewal) and to differentiate into distinct cell types (pluripotency), show attenuated inflammatory response against cytokines or pathogens, which is recognized as a unique characteristic of ESCs compared with somatic cells. However, the underlying molecular mechanisms remain unclear, and whether the attenuated inflammatory state is involved in ESC differentiation is completely unknown. Our recent study demonstrated that macroautophagy/autophagy-related protein ATG5 inhibits the inflammatory response of mouse ESCs (MmESCs) by promoting the degradation of BTRC/ß-TrCP1 and further the downregulation of NFKB/NF-κB signaling. In addition, maintenance of an attenuated inflammation status in MmESCs is required for their differentiation. In conclusion, ATG5 is a key regulator for the regulation of inflammatory response and differentiation of MmESCs.
Assuntos
Proteína 5 Relacionada à Autofagia , Autofagia , Diferenciação Celular , Inflamação , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Inflamação/patologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Transdução de Sinais , NF-kappa B/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is a highly aggressive solid malignancy in the word. Aberrant microRNA (miRNA) expression is involved in human diseases including cancer. In the current study, we explore the function of miR-98 in HCC cell proliferation. We found that expression level of miR-98 was significantly decreased in HCC tissues and cells lines compared with adjacent non-tumor issues and human hepatic cell line LO2. Increased expression of miR-98 suppressed HCC cell proliferation and arrested HCC cell cycle in G0/G1 phase. While, suppressed expression of miR-98 showed the opposite effect. Bioinformatics analysis revealed EZH2, a putative tumor promoter as a potential target of miR-98. Additionally, luciferase reporter assay revealed that miR-98 directly binds to the 3'-untranslated region (3'-UTR) of EZH2 mRNA. Furthermore, we demonstrated that miR-98 could reduce the Wnt/ß-catenin signal pathway by suppressing EZH2 directly. Moreover, inhibition of EZH2 abrogated the effect of miR-98 inhibitor on HCC cell proliferation. Taken together, these results suggested that miR-98 functioned as a potential tumor suppressor by regulating Wnt/ß-catenin signal pathway through direct suppression of EZH2 expression and might sever as a potential therapeutic target for HCC patients.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Regulação da Expressão Gênica/fisiologia , Humanos , MicroRNAs/genética , Transdução de Sinais/fisiologia , Proteínas Wnt/genética , beta Catenina/genéticaRESUMO
MicroRNAs (miRNAs) are emerging as critical regulators in carcinogenesis and tumor progression. miR-98 has previously been verified to be important in tumor progression, however, its function in hepatocellular carcinoma (HCC) remains to be elucidated. The expression levels of miR-98 in HCC tissues and cell lines were determined by reverse transcription quantitative polymerase chain reaction. Subsequently, the effect of miR98 on cell proliferation, migration and invasion was evaluated by MTT assay, transwell migration assay and transwell invasion assay. Furthermore, a luciferase reporter assay was conducted to confirm the action of miR98 on downstream target genes, including collagen triple helix repeat containing 1 (CTHRC1). In the present study, it was confirmed that miR98 was significantly downregulated in HCC tissues and cell lines. Overexpression of miR98 inhibited HCC cell proliferation, migration and invasion in vitro. In addition, at the molecular level, the tumor oncogene CTHRC1 was identified to be the direct target of miR-98. Our findings suggested that miR98 was significantly downregulated in HCC and suppressed HCC cell proliferation, migration and invasion partially via the downregulation of CTHRC1. Thus, these data demonstrated that miR-98 could be a potential therapeutic target in HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Neoplasias Hepáticas/metabolismo , MicroRNAs/fisiologia , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Proteínas da Matriz Extracelular/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/tratamento farmacológico , MicroRNAs/genética , MicroRNAs/uso terapêutico , Invasividade NeoplásicaRESUMO
INTRODUCTION: long non-coding RNA ANRIL (lncRNA ANRIL) has been demonstrated to play a crucial role in cancer progression. However, its effects in hepatocellular carcinoma (HCC) have not been explored. The aim of this study was to investigate the clinical significance of lncRNA NRIL in human HCC. METHODS: In this study, we determined for the first time the expression of lncRNA ANRIL in human HCC by quantitative Real-time-PCR analysis. Kaplan-Meier curves and multivariate Cox proportional models were used to study the impact on clinical outcome. Small interfering RNA (siRNA) was used to silence lncRNA ANRIL and to explore the effects of reduced lncRNA ANRIL expression on cell growth and metastasis. RESULTS: lncRNA ANRIL expression in HCC tissues was significantly higher than in the adjacent non-tumor tissues (P<0.05). The expression of lncRNA ANRIL was remarkably associated with the histologic grade and TNM stage of HCC patients (P<0.05). In addition, HCC patients with higher lncRNA ANRIL expression had significantly poorer overall survival (P<0.05). Multivariate analysis suggested that high lncRNA ANRIL expression was an independent predictor of poor prognosis (P<0.05). Moreover, in vitro assays revealed that the decreased expression of lncRNA ANRIL could suppress the cell proliferation, migration and invasion HCC cells. CONCLUSIONS: Our results suggest that lncRNA ANRIL may serve as an efficient clinical biomarker and a therapeutic target for HCC patients.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , RNA Longo não Codificante/biossíntese , Adulto , Idoso , Carcinoma Hepatocelular/mortalidade , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , TransfecçãoRESUMO
INTRODUCTION: Dysregulation of long non-coding RNAs (lncRNAs) play important roles in tumor progression. The aim of our study was to explore the clinicopathologic and prognostic significance of lncRNA CCAT2 expression in human gastric cancer. METHODS: Expression levels of lncRNA CCAT2 in 85 pairs of gastric cancer and adjacent non-tumor tissues were detected by quantitative real-time PCR (qRT-PCR). In order to determine its prognostic value, overall survival and progression-free survival were evaluated using the Kaplan-Meier method, and multivariate analysis was performed using the Cox proportional hazard analysis. RESULTS: Expression levels of lncRNA CCAT2 in gastric cancer tissues were significantly higher than those in adjacent non-tumor tissues. By statistical analyses, high lncRNA CCAT2 expression was observed to be closely correlated with higher incidence of lymph node metastasis and distance metastasis. Moreover, patients with high lncRNA CCAT2 expression had shorter overall survival and progression-free survival compared with the low lncRNA CCAT2 group. Multivariate analyses indicated that high lncRNA CCAT2 expression was an independent poor prognostic factor for gastric cancer patients. CONCLUSIONS: Our results suggested that up-regulation of lncRNA CCAT2 was correlated with gastric cancer progression, and lncRNA CCAT2 might be a potential molecular biomarker for predicting the prognosis of patients.
Assuntos
Adenocarcinoma/patologia , Biomarcadores Tumorais/genética , Neoplasias Gástricas/patologia , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adulto , Idoso , Progressão da Doença , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , RNA Longo não Codificante , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Gástricas/genética , Neoplasias Gástricas/mortalidade , Regulação para CimaRESUMO
BACKGROUND: MicroRNAs (miRNA) have been documented playing a critical role in cancer progression. Although miR-338-3p has been implicated in several cancers, its role in gastric cancer is still unknown. The aim of our study was to investigate the role of miR-338-3p in gastric cancer progression. METHODS: Expression levels of miR-338-3p in gastric cancer cell lines and tissues were determined by quantitative real-time PCR (qRT-PCR). The effect of miR-338-3p on proliferation was evaluated by MTT assay, cell migration and invasion were evaluated by transwell migration and invasion assays. Furthermore, luciferase reporter assay was conducted to confirm the target gene of miR-338-3p, and the results were validated in gastric cancer cells. RESULTS: In the present study, we found that miR-338-3p was down-regulated in both gastric cancer cell lines and tissues. Enforced expression of miR-338-3p inhibited proliferation, migration and invasion of gastric cancer cells in vitro. Moreover, we identified A disintegrin and metalloproteinase 17 (ADAM17) gene as potential target of miR-338-3p. Importantly, ADAM17 rescued the miR-338-3p mediated inhibition of cell proliferation, migration and invasion. CONCLUSIONS: Our study suggested that miR-338-3p is significantly decreased in gastric cancer, and inhibits cell proliferation, migration and invasion partially via the downregulation of ADAM17. Thus, miR-338-3p may represent a potential therapeutic target for gastric cancer intervention.
Assuntos
Proteínas ADAM/biossíntese , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Neoplasias Gástricas/patologia , Proteínas ADAM/genética , Proteína ADAM17 , Western Blotting , Movimento Celular/genética , Proliferação de Células/genética , Humanos , MicroRNAs/metabolismo , Invasividade Neoplásica , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , TransfecçãoRESUMO
BACKGROUND: A large number of studies demonstrated that microRNAs play important roles in the progression and development of human cancers. However, the expression level of miR-107 and its biological function in hepatocellular carcinoma (HCC) remains unclear. METHOD: Quantitative real-time PCR (qRT-PCR) was used to evaluate the expression level of miR-107 in HCC tissues and cell lines. Then, we explored the function of miR-107 to determine its potential roles on HCC cell proliferation in vitro. Luciferase reporter assay was used to confirm the target gene of miR-107, and the results were validated in cell lines. RESULTS: miR-107 was significantly up-regulated in HCC tissues and cell lines. The enforced expression of miR-107 was able to promote cell proliferation in HepG2 cells. At the molecular level, our results suggested that expression of Axin2 was negatively regulated by miR-107. CONCLUSION: Our observations suggested that miR-107 could promote HCC cells proliferation via targeting Axin2 and might represent a potential therapeutic target for HCC.
Assuntos
Proteína Axina/metabolismo , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Proteína Axina/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Luciferases/genética , Luciferases/metabolismo , MicroRNAs/genética , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: Laparoscope-assisted gastrectomy in treating patients with gastric cancers developed with a background of highly invasive traditional surgery and is being increasingly performed in the Asian Pacific area. This study systemically investigated the technique and clinical results for comparison with traditional radical subtotal gastrectomy for gastric cancers. METHODS: Clinical studies evaluating the effectiveness and side effects of laparoscope-assisted gastrectomy in treating patients with gastric cancers were identified using a predefined search strategy. Summary rates of effectiveness and side effects of laparoscope-assisted gastrectomy were calculated. RESULTS: Thirteen clinical studies which including 1,412 patients with gastric cancer treated by laparoscope-assisted gastrectomy were considered eligible for inclusion. Systemic analysis showed that, for all patients, the pooled resection rate was 100%. Major adverse effects were anastomotic stenosis, abdominal abscess, abdominal bleeding, postoperative ileus. Treatment related death occurred in 0. 71% (10/1412). CONCLUSION: This systemic analysis suggests that laparoscope-assisted gastrectomy in treating patients with gastric cancers is associated with good curative rate and acceptable complications.
Assuntos
Gastrectomia/métodos , Laparoscopia/métodos , Neoplasias Gástricas/cirurgia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Gastrectomia/efeitos adversos , Humanos , Laparoscopia/efeitos adversos , Pessoa de Meia-Idade , Complicações Pós-Operatórias/cirurgia , Adulto JovemRESUMO
INTRODUCTION: Previous studies have shown that the dysregulation of miRNAs are frequently associated with cancer progression. Deregulation of miR-211 has been observed in various types of human cancers. However, its biological function in gastric cancer (GC) is still unknown. METHODS: The expression of miR-211 in GC was detected by using quantitative real-time PCR (qRT-PCR). The miR-211 mimics and inhibitor were designed and transfected into BGC-823 cells. Then, we explore the probable biological function of miR-211 in gastric cancer cell proliferation and invasion in vitro. A luciferase reporter assay and western blot were performed to confirm the target gene of miR-211. RESULTS: MiR-211 was significantly down-regulated in GC. Over-expression of miR-211 inhibited gastric cancer cell proliferation and invasion in vitro, conversely, down-regulated expression of miR-211 promoted gastric cancer cell proliferation and invasion. In addition, the sex-determining region Y-related high mobility group box 4 (SOX4) is identified as a target of miR-211 in GC cells, and SOX4 expression levels was inversely correlated with miR-211. Furthermore, knockdown of Sox4 inhibited the proliferation and invasion in GC cells. CONCLUSION: miR-211 could inhibit GC cell proliferation and invasion partially by down-regulating SOX4. MiR-211 might be a potential therapeutic target for GC treatment in the future.
Assuntos
Movimento Celular , Proliferação de Células , MicroRNAs/metabolismo , Fatores de Transcrição SOXC/metabolismo , Neoplasias Gástricas/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Invasividade Neoplásica , Fatores de Transcrição SOXC/genética , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Fatores de Tempo , TransfecçãoRESUMO
OBJECTIVE: To induce the differentiation of human bone marrow mesenchymal stem cells (HMSCs) into hepatocyte-like cells with hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4) in vitro. METHODS: HMSCs were induced to differentiate into hepatocyte-like cells by HGF (group B), FGF-4 (group C) and HGF+FGF-4 (group D) in vitro. Undifferentiated HMSCs and L-02 cells were used as the negative (group A) and positive (group E) controls, respectively. The changes of cell morphology were observed microscopically. The expressions of hepatic markers, alpha fetoprotein (AFP) and CK-18, were detected by immunocytochemical staining at different times after induction, and the differentiation ratios of the various groups of HMSCs were calculated on the basis of image analysis. The expressions of AFP and ALB were detected by immunofluorescence assay in each group at different times after induction, and the expressions of AFP and ALB mRNA by RT-PCR. RESULTS: HMSCs gradually transformed into spindle-shaped, round, polygonal or irregular cells after induction. Immunocytochemical staining revealed positive AFP and CK18 expressions in groups B, C, and D after induction as well as in group E. The positive units (PU) of AFP and CK18 in group D calculated according to image analysis were significantly higher than that of groups A, B, and C. The expressions of AFP and ALB detected by immunofluorescence were both positive after induction in all groups except group A, similar to the findings of the expressions of AFP and ALB mRNA by RT-PCR. CONCLUSION: HMSCs can be induced to differentiate into hepatocyte-like cells by HGF, FGF-4 and their combination at certain concentrations, and the hepatocyte-like cells can express some hepatic markers such as AFP, ALB, CK18, etc. HGF+FGF-4 may achieve more effective induction of HMSC differentiation into hepatocyte-like cells, and the efficiency of HGF is greater than that of FGF-4.