MMP-9 participates in tumour growth, invasion, metastasis and vascularisation. Thus, inhibition of MMP-9 may be involved in the process of tumourigenesis. We have investigated the effect of RNAi-mediated MMP-9 silencing on inhibiting invasion and migration of mouse melanoma cell B16. A specific and optimised siRNA vector was used to target MMP-9 mRNA synthesis in B16 cells. Changes of invasion and migration capability of B16 cell were examined after transfection at different time, and a footpad tumour model was performed to measure the effect of MMP-9 siRNA on melanoma tumourigenesis in vivo. In vitro, down-regulation of MMP-9 expression significantly inhibited B16 cell invasion and migration. In vivo, intratumoural injection of plasmid DNA expressing MMP-9 siRNA every 3 days for three times remarkably inhibited melanoma growth and also suppressed tumour metastasis. The results indicate that RNA-mediated targeting of MMP-9 may have promising applications for the treatment of melanoma.
Protocells are believed to consist of a lipid membrane and encapsulated nucleic acid. As the lipid membrane is impermeable to macromolecules like nucleic acids, the processes by which nucleic acids become encapsulated inside lipid membrane compartments are still unknown. In this paper, a freeze-thaw method was modified and applied to giant unilamellar vesicles (GUVs) and deoxyribonucleic acid (DNA) in mixed solution resulting in the efficient encapsulation of 6.4 kb plasmid DNA and similar length linear DNA into GUVs. The mechanism of encapsulation was followed by observing the effect of freeze-thaw temperatures on GUV morphological change, DNA encapsulation and ice crystal formation, and analyzing their correlation. Following ice crystal formation, the shape of spherical GUVs was altered and membrane integrity was damaged and this was found to be a necessary condition for encapsulation. Heating alone had no effects on DNA encapsulation, but was helpful for restoring the spherical shape and membrane integrity of GUVs damaged during freezing. These results suggested that freeze-thaw could promote the encapsulation of DNA into GUVs by a mechanism: the vesicle membrane was breached by ice crystal formation during freezing, DNA entered into damaged GUVs through these membrane gaps and was encapsulated after the membrane was resealed during the thawing process. The process described herein therefore describes a simple way for the encapsulation of nucleic acids and potentially other macromolecules into lipid vesicles, a process by which early protocells might have formed.
Extracellular vesicles from highly metastatic tumor cells have been shown to mediate epithelial-mesenchymal transition (EMT)-related events in recipient cells. In endometrial epithelial cells, EMT processes are known to be involved in the development of adenomyosis. We aimed to investigate whether adenomyosis-derived extracellular vesicles (AMEVs) are able to induce an EMT process in endometrial epithelial cells. In this study, AMEVs were isolated from patients with adenomyosis and characterized by transmission electron microscopy, Western blot, and nanoparticle tracking. Primary endometrial epithelial cells (EECs) were derived from normal endometrium tissues from patients with leiomyoma and co-cultured with AMEVs
in vitro
. AMEV uptake was examined by fluorescence confocal microscopy. The invasion of EECs was confirmed by Transwell assay. Immunohistochemistry, Western blot, and qRT-PCR were performed on EECs to illustrate the expression levels of cytokeratin 19, E-cadherin, vimentin, and zinc finger E-box-binding homeobox 1 (ZEB1). The results indicated that the cellular fluorescence intensity gradually increased after 48 h of co-culture, but decreased after 72 h. After co-culturing with AMEVs for 72 h, EECs expressed significantly lower levels of cytokeratin 19 and E-cadherin, and significantly higher levels of vimentin and ZEB1. Together these results demonstrated that AMEVs induce an EMT process and enhance the invasion of EECs. These changes may contribute to the pathogenesis and progression of adenomyosis.
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