Method: In this study, the potential targets of miR-145 were identified bio-informatically using different target prediction tools. The identified target genes were validated in vitro by dual luciferase assay. Wound healing and soft agar colony assay assessed cell proliferation and migration. miR-145 expression level was measured quantitatively by RT-PCR at different stages of breast tumor. Western blot was used to verify the role of miR-145 in EMT transition using key marker proteins.
Result: Wound healing and soft agar colony assays, using miR-145 over-expressing stably transfected MCF7 cells, unraveled its role as a pro-proliferation candidate in cancerous cells. The association between miR-145 over-expression and differential methylation patterns in representative target genes (DR5, BCL2, TP53, RNF8, TIP60, CHK2, and DCR2) supported the inference drawn. These in vitro observations were validated in a representative set of nodal positive tumors of stage 3 and 4 depicting higher miR-145 expression as compared to early stages. Further, the role of miR-145 in epithelial-mesenchymal (EMT) transition found support through the observation of two key markers, Vimentin and ALDL, where a positive correlation with Vimentin protein and a negative correlation with ALDL mRNA expression were observed.
Conclusion: Our results demonstrate miR-145 as a pro-cancerous candidate, evident from the phenotypes of aggressive cellular proliferation, epithelial to mesenchymal transition, hypermethylation of CpG sites in DDR and apoptotic genes and upregulation of miR-145 in later stages of tumor tissues.
MATERIALS AND METHODS: The antisolvent precipitation method was used for formulation of nanoparticles. Factorial design (32) was utilized as a tool to analyze the effect of Ch and TGP concentration on particle size and entrapment efficiency of nanoparticles.
RESULTS: Formulated nanoparticles showed high entrapment efficiency (67.19±0.42-83.36±0.23%) and small size (53.3-383.1 nm). The present investigation involved utilization of two biological membranes (egg and tomato) as biological barriers for drug release. The study revealed that drug release from tomato membranes was retarded (as compared to egg membranes) but the release pattern matched that of egg membranes. All formulations followed the Baker-Lansdale model of drug release irrespective of the two different biological barriers. Stability studies were carried out for 45 days and exhibited less variation in particle size as well as a reduction in entrapment efficiency. Simvastatin loaded PEC stabilized nanoparticles exhibited better control on growth of human breast cancer cell lines than simple simvastatin. An unusual anticancer effect of simvastatin nanoparticles is also supported by several other research studies.
CONCLUSION: The present study involves first-time synthesis of Ch-TGP polyelectrolyte complex stabilized nanoparticles of simvastatin against MCF-7 cells. It recommends that, in future, theoretical modeling and IVIVC should be carried out for perfect designing of delivery systems.