Neuroblastoma is a brain malignancy of childhood and accounts for 7-10% of childhood cancers, leading to approximately 15% of pediatric cancer deaths. MicroRNAs (miRNAs) are a family of short (about 18-25 nucleotides), noncoding and single stranded endogenous RNAs, which complementarily bind to the 3' untranslated regions of their target genes. Recently, glutamine metabolism has been recognized as an important nutrition source for tumor cells, and hence targeting glutamine metabolism could benefit to development of anti-cancer agents. In this study, we investigate the roles of miR-513c in human neuroblastoma. We report miR-513c is significantly downregulated in human neuroblastoma tissues compared with their adjacent normal tissues. Moreover, miR-513c is significantly downregulated in neuroblastoma cell lines compared with normal neuroblast cells. Overexpression of miR-513c suppresses neuroblastoma cells' migration, invasion, and proliferation. We demonstrate the glutaminase (GLS) is a direct target of miR-513c in human neuroblastoma cells. In addition, we found restoration of GLS expression recovered the neuroblastoma cells' migration, invasion, and proliferation. In summary, this study illustrates a miR-513c mediated neuroblastoma cells suppression, providing a new aspect on the miRNA-based therapeutic approach for the treatments of neuroblastoma.

Insufficient angiogenesis is a common problem in bladder tissue engineering and is believed to be a major factor responsible for graft shrinkage. In this study, we investigated the use of bladder acellular matrix allografts (BAMAs) modified with vascular endothelial growth factor (VEGF)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) for the long-term sustained release of VEGF to enhance blood supply and inhibit graft shrinkage in a rabbit model of bladder reconstruction. Rabbits underwent partial bladder cystectomy using a 2 × 3 cm BAMA modified with VEGF-loaded PLGA NPs in the experimental group, while no modification was used in the control. Histology and immunohistochemical analyses showed that urothelium, smooth muscle fibers and blood vessels were formed in both groups at 4 and 12 weeks postoperatively. The microvessel density in the experiment group was significantly higher than that in control and the contracture rate declined to 27%. In vitro functional experiments indicated that the characteristics of regenerated bladders were similar to native bladders. The VEGF release from BAMA in vivo was almost 83% within 3 months. Our data demonstrated the effectiveness of VEGF-loaded PLGA NPs-modified BAMAs to enhance neovascularization and solve the problems of insufficient angiogenesis and graft shrinkage associated with bladder tissue engineering.

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Macroscopic evidence of contracture has been identified as a major issue during the regeneration process. We hypothesize that lack of angiogenesis is the primary cause of contracture and explore a nanomedicine approach to achieve sustained release of vascular endothelial growth factor (VEGF) to stimulate angiogenesis. We evaluate the efficacy of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) for long-term (3 months) sustained release of VEGF in bladder acellular matrix allografts (BAMA) in a swine model. We anticipate that the sustained release of VEGF could stimulate angiogenesis along the regeneration process and thereby inhibit contracture. Bladder was replaced with BAMA (5×5 cm), modified with PLGA NPs encapsulated with VEGF in a pig model. The time points chosen for sampling were 1, 2, 4, and 12 weeks. The regenerated areas were then measured to obtain the contracture rate, and the extent of revascularization was calculated using histological and morphological features. In the control group of animals, the bladder was replaced with only BAMA. The in vivo release of VEGF was evident for ∼3 months, achieving the goal of long-acting sustained release, and successfully promoted the regeneration of blood vessels and smooth muscle fibers. In addition, less collagen deposition was observed in the experimental group compared with control. Most importantly, the inhibition of contracture was highly significant, and the ultimate contracture rate decreased by ∼57% in the experimental group compared with control. In isolated strips analysis, there were no significant differences between BAMA-regenerated (either VEGF added or not) and autogenous bladder. BAMA modified with VEGF-loaded PLGA-NPs can sustainably release VEGF in vivo (>3 months) to stimulate angiogenesis leading to the inhibition of contracture. This is the first study to report a viable nanomedicine-based strategy to overcome contracture during bladder regeneration induced by BAMA. Furthermore, this study also confirms that insufficient angiogenesis plays a crucial role in the onset of contracture.