RESUMO
The peritoneum, especially the omentum, is a common site for gastric cancer (GC) metastasis. Our aim was to expound the role and mechanisms of Piezo1 on GC omentum metastasis. A series of functional assays were performed to examine cell proliferation, clone formation, apoptosis, Ca2+ influx, mitochondrial membrane potential (MMP) and migration after overexpression or knockdown of Piezo1. A GC peritoneal implantation and metastasis model was conducted. After infection by si-Piezo1, the number and growth of tumours were observed in abdominal cavity. Fibre and angiogenesis were tested in metastatic tumour tissues. Piezo1 had higher expression in GC tissues with omentum metastasis and metastatic lymph node tissues than in GC tissues among 110 patients. High Piezo1 expression was associated with lymph metastasis, TNM and distant metastasis. Overexpressed Piezo1 facilitated cell proliferation and suppressed cell apoptosis in GC cells. Moreover, Ca2+ influx was elevated after up-regulation of Piezo1. Piezo1 promoted cell migration and Calpain1/2 expression via up-regulation of HIF-1α in GC cells. In vivo, Piezo1 knockdown significantly inhibited peritoneal metastasis of GC cells and blocked EMT process and angiogenesis. Our findings suggested that Piezo1 is a key component during GC omentum metastasis, which could be related to up-regulation of HIF-1α.
Assuntos
Regulação Neoplásica da Expressão Gênica , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mecanotransdução Celular , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Adulto , Idoso , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Imunofenotipagem , Masculino , Mecanotransdução Celular/genética , Potencial da Membrana Mitocondrial , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Omento/patologia , Neoplasias Gástricas/patologiaRESUMO
OBJECTIVES: To construct eukaryotic expressing recombinant vector of canine transferrin receptor gene (TfR ), then to transfect Chinese hamster ovary (CHO) cells with the vector for establishment of stable expression of TfR in CHO cell line. METHODS: The full-length TfR fragment was amplified by RT-PCR from the canine cells (walter reed dog cell, WRD) and then inserted into eukaryotic expression vector pCDNA3. After identification with enzyme digestion and sequencing, the recombinant vector was transfected into CHO cells by TransLipid Transfection Reagent. The stable transfected CHO cell line was then established by screening cultures with G418, and the expression of TfR was identified by RT-PCR, Western blot and immunofluorescence, respectively. RESULTS: The eukaryotic expression vector pCDNA3-TfR was constructed successfully by checking with enzyme digestion and sequencing, and the highly expressed canine TfR was observed in CHO cells transfected with pCDNA3-TfR by using RT-PCR, Western blot and immunofluorescence, respectively. The stable CHO cell line with canine TfR expression was established. CONCLUSIONS: The construction of the eukaryotic expression vector pCDNA3-TfR and the establishment of stable CHO cell line with TfR expression provide solid foundation for further experimental studies on the function of TfR.
Assuntos
Células CHO , Receptores da Transferrina/genética , Transfecção , Animais , Cricetinae , Cricetulus , Cães , Expressão Gênica , Vetores GenéticosRESUMO
Objective To investigate the expressions of silent mating type information regulation 2 homolog-1 (SIRT1), matrix metalloproteinase-9 (MMP-9) and hypoxia inducible factor 1α (HIF-1α) in hypoxic pulmonary vascular smooth muscle cells (PVSMCs) treated with Lycium barbarum polysaccharides (LBP). Methods PVSMCs were divided into 10 groups: normal oxygen (200 mL/L oxygen) cells, 2 µmol/mL LBP-treated normal oxygen cells, DMSO-treated normal oxygen cells, DMSO-treated hypoxic (100 mL/L oxygen) cells, (0.5, 1, 2) µmol/mL LBP-treated hypoxic cells; SIRT1 agonist (resveratrol or SRT-1720)-treated hypoxic cells, SIRT1 inhibitor EX-527-treated hypoxic cells. After 6, 12, 24 hours, the mRNA and protein expressions of SIRT1, MMP-9 and HIF-1α were measured by real-time quantitative PCR and Western blotting, respectively. In LBP-treated groups, the expressions of SIRT1, MMP-9 and HIF-1α mRNA and protein were detected 12 hours after LBP treatment. Results Under the condition of hypoxia, the expression levels of SIRT1 mRNA and protein in PVSMCs decreased, while MMP-9 and HIF-1α mRNA increased. Under hypoxia, SIRT1 expression was raised and MMP-9, HIF-1α were reduced by LBP treatment in a dose-dependent manner. Morever, resveratrol could inhibit the expression of MMP-9. Conclusion LBP can enhance the expression of SIRT1 and decrease the expression of MMP-9 and HIF-1α in hypoxic PVSMCs.