RESUMEN
Macrophage autophagy plays an important role in the development of atherosclerosis, but the precise mechanism mediating this process is unclear. The potential role of the X-box binding protein 1 (XBP1), a crucial transduction factor that is involved in endoplasmic reticulum stress and the unfolded protein response, in bone marrow-derived macrophage autophagy is unknown. This study mainly explores the roles of XBP1 mRNA splicing in bone marrow-derived macrophage autophagy. The present study shows that the transient overexpression of spliced XBP1 via adenovirus-mediated gene transfer induces autophagy and promotes proliferation in bone marrow-derived macrophages via the down-regulation of Beclin-1, but that the sustained overexpression of spliced XBP1 leads to apoptosis. When XBP1 is down-regulated in bone marrow-derived macrophages using siRNA, rapamycin-induced autophagosome formation is ablated. Furthermore, we have detected the overexpression of XBP1 in areas of atherosclerotic plaques in the arteries of ApoE-/- mice. These results demonstrate that XBP1 mRNA splicing plays an important role in maintaining the function of bone marrow-derived macrophages and provide new insight into the study and treatment of atherosclerosis.
Asunto(s)
Autofagia/genética , Supervivencia Celular/genética , Proteínas de Unión al ADN/fisiología , Macrófagos/citología , Empalme del ARN , Factores de Transcripción/fisiología , Animales , Aterosclerosis/fisiopatología , Secuencia de Bases , Proliferación Celular , Células Cultivadas , Cartilla de ADN , Proteínas de Unión al ADN/genética , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Factores de Transcripción del Factor Regulador X , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Proteína 1 de Unión a la X-BoxRESUMEN
OBJECTIVE: To construct a replication-incompetent adenovirus vector targeting cancer stem cells by modified touchdown PCR and overlap extension PCR and investigate its infection efficiency in CD133(+) SW480 cells in vitro. METHODS: The two portions of the fiber gene encoding the Ad5 fiber knob domain with the HI loop deleted were amplified using two pairs of designed primers and then linked by overlap extension PCR. The product obtained was identified by sequencing and inserted into prokaryotic expression vector pEGFP-N1. The product, pEGFP-N1 KNOBδHI, contained a unique EcoRV restriction site in the deleted portion of the sequence encoding the HI loop. The gene sequences of the adenovirus fiber were amplified using both common PCR and overlap extension PCR, then identified by sequencing and inserted into pNEB193, resulting in pNEB-F5. CD133(+) SW480 cells were infected with the generated adenovirus vectors Ad5-GFP and Ad5FHI-GFP to investigate the infection efficiency using fluorescent microscope. RESULTS: The target fragments of expected sizes were amplified by touchdown PCR and overlap extension PCR, but not by common PCR. Ad5FHI-GFP showed a higher infection efficiency than Ad5-GFP in CD133(+) SW480 cells. CONCLUSION: Compared with common PCR, touchdown PCR and overlap extension PCR can significantly improve the specificity and efficiency of the PCR products for constructing CD133(+) cancer stem cell-selective adenovirus type 5 vector, which provides carriers for tumor-targeted gene therapy.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos , Células Madre Neoplásicas/citología , Reacción en Cadena de la Polimerasa/métodos , Antígeno AC133 , Antígenos CD , Línea Celular Tumoral , Glicoproteínas , Humanos , Células Madre Neoplásicas/virología , Péptidos , Plásmidos/genéticaRESUMEN
OBJECTIVE: To select the peptides that specifically bind human cancer stem cell surface marker CD133 from the Ph.D.-7>(TM) phage peptide library. METHODS: With a biotinylated extracellular fragment of human cancer stem cell surface marker CD133 as the target protein, the CD133 high-affinity peptides were screened from the phage peptide library by liquid phase panning. The clones with high-binding force with human CD133 were then identified by sandwich ELISA and their single-stranded DNA was extracted to test the specificity by competitive ELISA. The amino acid sequences of the selected peptides derived from the phage DNA sequences were synthesized after sequence alignment analysis, and their capacity of binding with colorectal carcinoma cells was assessed by immunofluorescence technique. RESULTS: After 4 rounds of liquid phase selection, the phages capable of specific binding with human CD133 were effectively enriched, with an enrichment ratio of 388 times compared to that at the fourth and first rounds. Thirteen out of the 20 clones from the fourth round of panning were identified as positive clones, among which 11 had identical amino acid sequence of TISWPPR, and 2 had the sequence of STTKLAL, and the former sequence showed a stronger binding specificity to CD133. CONCLUSION: We have successfully obtained a peptide that specifically binds human CD133 from the Ph.D.-7(TM) phage peptide library, demonstrating the feasibility of screening small molecule high-affinity polypeptides from phage peptide library by liquid-phase panning.