RESUMO
EPB41L3 may play a role as a metastasis suppressor by supporting regular arrangements of actin stress fibres and alleviating the increase in cell motility associated with enhanced metastatic potential. Downregulation of epb41l3 has been observed in many cancers, but the role of this gene in esophageal squamous cell carcinoma (ESCC) remains unclear. Our study aimed to determine the effect of epb41l3 on ESCC cell migration and invasion. We investigated epb41l3 protein expression in tumour and non-tumour tissues by immunohistochemical staining. Expression in the non-neoplastic human esophageal cell line Het-1a and four ESCC cell lines - Kyse150, Kyse510, Kyse450 and Caes17 - was assessed by quantitative Polymerase Chain Reaction (qPCR) and Western blotting. Furthermore, an EPB41L3 overexpression plasmid and EPB41L3-specific small interfering RNA were used to upregulate EPB41L3 expression in Kyse150 cells and to downregulate EPB41L3 expression in Kyse450 cells, respectively. Cell migration and invasion were evaluated by wound healing and transwell assays, respectively. The expression levels of p-AKT, matrix metalloproteinase (MMP)2 and MMP9 were evaluated. Expression of epb41l3 was significantly lower in tumour tissues than in non-tumour tissues and in ESCC cell lines compared with the Het-1a cell line. Kyse450 and Caes17 cells exhibited higher expression of epb41l3 than Kyse150 and Kyse510 cells. Overexpressing epb41l3 decreased Kyse150 cell migration and invasion, whereas EPB41L3-specific small interfering RNA silencing increased these functions in Kyse450 cells. Furthermore, overexpressing epb41l3 led to downregulation of MMP2 and MMP9 in Kyse150 and Kyse510 cells. Our findings reveal that EPB41L3 suppresses tumour cell invasion and inhibits MMP2 and MMP9 expression in ESCC cells.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Proteínas dos Microfilamentos/metabolismo , Western Blotting , Carcinoma de Células Escamosas/genética , Movimento Celular , Regulação para Baixo , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Proteínas dos Microfilamentos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Rapid assessment and optimization of the incompatible metabolic modules remain a challenge. Here, we developed a systematic approach to characterize the module interactions and improve the problematic modules during the 6-deoxyerythronolide B (6dEB) biosynthesis in E. coli. Tremendous differences in the overall trends of flux changes of various metabolic modules were firstly uncovered based on in silico fluxome analysis and comparative transcriptome analysis. Potential targets for improving 6dEB biosynthesis were identified through analyzing these discrepancies. All 25 predicted targets at modules of PP pathway and nucleotide metabolism were firstly tested for improving the 6dEB production in E. coli via synthetic antisense RNAs. Down-regulation of 18 targets genes leads to more than 20% increase in 6dEB yield. Combinatorial repression of targets with greater than 60% increase in 6dEB titer, e.g., anti-guaB/anti-zwf led to a 296.2% increase in 6dEB production (210.4 mg/L in flask) compared to the control (53.1 mg/L). This is the highest yield yet reported for polyketide heterologous biosynthesis in E. coli. This study demonstrates a strategy to enhance the yield of heterologous products in the chassis cell and indicates the effectiveness of antisense RNA for use in metabolic engineering.
Assuntos
Eritromicina/análogos & derivados , Escherichia coli/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Engenharia Metabólica/métodos , RNA Antissenso/farmacologia , Antibacterianos/biossíntese , Simulação por Computador , Regulação para Baixo , Eritromicina/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Via de Pentose Fosfato/efeitos dos fármacosRESUMO
Expelling heterologous compounds out of hosts by transporters is a potential strategy to enhance product titers in microbial cell factories. In this work, to increase heterologous polyketide 6-deoxyerythronolide B (6dEB, erythromycin precursor) production, tripartite multidrug efflux pumps MacAB-TolC, AcrAB-TolC, MdtEF-TolC, and MexAB-OprM were modulated in a 6dEB production strain. Compared with the control, overexpression of a single component of efflux pumps (except oprM) repressed 6dEB production, but modulation of two components MacA and MacB or the complete pumps MacAB-TolC and MdtEF-TolC significantly improved 6dEB titer by 100 ± 11, 118 ± 54, and 98 ± 12 %, respectively. In addition, to avoid the challenging fine-tuning components of pumps, the transcriptional regulators of efflux pumps were modulated to improve the 6dEB production. Overexpression of RpoH (activator of MdtEF-TolC) and EvgA (activator of EmrKY-TolC and AcrAD-TolC) strongly increased 6dEB titer by 152 ± 54 and 142 ± 85 %, respectively. This is the first report of transporter engineering for improving heterologous polyketide production in Escherichia coli. Our results provide an effective strategy for improving the yield of the heterologous products in chassis cell.
