RESUMO
Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is an emerging method for the analysis of metal nanoparticles (NPs) in single cells. However, two main obstacles, low analytical throughput and lack of commercial reference materials, need to be overcome. In this work, we demonstrated the principles of a new approach termed "single-cell isotope dilution analysis" (SCIDA) to remove the two obstacles. For a proof of concept, macrophage cells were chosen as a model to study the uptake of silver NPs (AgNPs) at a single-cell level. Single cells exposed to AgNPs were placed in an array by a microfluidic technique; each cell in the array was precisely dispensed with a known picoliter droplet of an enriched isotope solution with a commercial inkjet printer; accurate quantification of AgNPs in single cells was done by using isotope dilution LA-ICP-MS. The average Ag mass of 1100 single cells, 396 ± 219 fg Ag per cell, was in good accord with the average of the population of cells determined by solution ICP-MS analysis. The detection limit was 0.2 fg Ag per cell. The SCIDA approach is expected to be widely applied for the study of cell-NP interactions and biological effects of NPs at the single-cell level.
Assuntos
Espectrometria de Massas , Nanopartículas Metálicas , Prata/química , Prata/metabolismo , Análise de Célula Única/métodos , Animais , Transporte Biológico , Isótopos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Células RAW 264.7RESUMO
Purpose: Various studies have identified miR-202 critically participated in the development of different cancers. However, the potential mechanisms underlying the carcinogenesis of pancreatic cancer (PC) still remains elusive. Methods: In the study, cell proliferation assay, colony formation assay, EdU incorporation assay, Luciferase reporter assay, lactate production, glucose consumption assay, real-time PCR and western blot were used to investigate the mechanism of hexokinase 2 (HK2) regulated by miR-202 in pancreatic cancer in vitro and in vivo. Results: Here we found that miR-202 was decreased in the PC tissues, and its low expression was correlated with a poor prognosis of PC patients. Overexpression of miR-202 in PC cells reduced cell proliferation and tumorigenesis by impairing glycolysis, while downregulation of miR-202 promoted the cells proliferative capacity. Mechanically, we demonstrated that HK2, an enzyme that catalyzes the irreversible rate-limiting step of glycolysis, as the direct target of miR-202. Overexpression of miR-202 suppressed both the mRNA and protein levels of HK2, whereas re-introduction of HK2 abrogated miR-202-mediated glycolytic inhibition. In addition, the expression of miR-202 was negatively associated with HK2 level in a cohort of PC tissues. Conclusion: Our findings validate the mechanism that miR-202 reprograms the metabolic process to promote PC progression, thus providing potential prognostic predictors for PC patients.