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Background: MicroRNAs are noncoding RNA molecules of ~ 22 nucleotides with diagnostic and therapeutic action [Curr Drug Targets, 2015. 16(12): p. 1381-403], affecting the expression of mRNAs involved in invasion, migration, and development [Oncotarget, 2015. 6(9): p. 6472-98, Cancer Manag Res, 2014. 6: p. 205-16]. miR-200c is part of the miR-200c/141 cluster on chromosome 12p13. Its mechanism of action when encapsulated is critical in lung cancer when patients express changes in miRNAs. miR-200c be a potential biomarkers for various lung diseases. As a potential therapy, miR-200c can impacts lives as target lung cancer is a leading cause of death with about 234,000 cases annually, high heterogeneity, complex screening, and a 5-year survival rate of 16% [CA Cancer J Clin, 2016.66(1): p. 7-30]. Encapsulated miR-200c efficiently enhances bioavailability, pharmacokinetics of therapeutics and targeting to cells, improves efficacy and provides potential cure., Methods: The functions of miR-200c were determined in non-metastatic KW-634 and metastatic 821-T4 and 821-LN mouse lung cancer cell lines after various Nano vehicle treatments. Viability and cytotoxicity were determined by cell cycle and quantitative real-time PCR analyses were used to quantify levels of miR-200c and its target genes. In situ hybridization was used to visualize patterns of expression of miR-200c and others in the lung and many organs. Next-generation sequencing accession number GSE125000, invasion and migration assays using transwell chambers, and ActivSignal were used to elucidate the activation and inhibition profiles and perform direct expression measurements and modification of cellular components., Results: Due to their effectiveness as intracellular vesicles transporting miR-200c into, out, and between parts of the cells, miR-200c is encapsulated with cholesterol, an integral part of the biological membranes with very important physical properties of the vehicle. Nano miR-200c showed efficient cellular uptake in KW-634, 821-T4, and 821-LN cells with important changes in gene expression and new isoforms. In KW-634, when treated with encapsulated miR-200c and compare to the non-encapsulated control; miR-29b increased by 5261-fold, and in 821-T4/LN, miR-1247 increased by 150-fold. Conversely, miR-1247 and miR-675 decreased by 348 and 1029.5-fold, respectively. miR-189 decreased by 34-fold in treated 821-T4 cells. A reduction of growth was observed only after 48 h of treatment with Nano miR-200c. Moreover, labeling the vehicle with carboxy-fluorescein showed that the encapsulated particles enter the nucleus and mitochondria. Encapsulated miR-200c by entering the cells, the nucleus and mitochondria, trigger changes in cell cycle phases with 4 up to 12 fold percentage in G2 and S phase respectively compare to miR-200c. Endogenous expression of Nkx2.1, miR-200c, and their targets Myb, Nfib, Six4 and Six1 showed an inverse correlation, as observed in development., Conclusions: Little is known about miR-200c involvement in regulatory processes. Nano miR-200c affects invasion and migration mechanisms. The expression of encapsulated miR-200c contributes to the inhibition/activation of Kras, EMT, Hippo, regulatory pathways and blockers of metastasis. Delivery of miR-200c increases the expression of miR-29b, an EMY regulator, and miR-1247, an inhibitor of cancer genes, both tumor suppressors involved in lung metastasis. Encapsulated miR-200c act on different proteins that regulates cell cycle pathways. These findings represent a part of a regulatory network providing new insights towards improvement of therapy.

Background: The transcription factor NK2 homeobox 1 (Nkx2-1) plays essential roles in epithelial cell proliferation and differentiation in mouse and human lung development and tumorigenesis. A better understanding of genes and pathways downstream of Nkx2-1 will clarify the multiple roles of this critical lung factor. Nkx2-1 regulates directly or indirectly numerous protein-coding genes; however, there is a paucity of information about Nkx2-1-regulated microRNAs (miRNAs)., Methods and Results: By miRNA array analyses of mouse epithelial cell lines in which endogenous Nkx2-1 was knocked-down, we revealed that 29 miRNAs were negatively regulated including miR-200c, and 39 miRNAs were positively regulated by Nkx2-1 including miR-1195. Mouse lungs lacking functional phosphorylated Nkx2-1 showed increased expression of miR-200c and alterations in the expression of other top regulated miRNAs. Moreover, chromatin immunoprecipitation assays showed binding of NKX2-1 protein to regulatory regions of these miRNAs. Promoter reporter assays indicated that 1kb of the miR-200c 5' flanking region was transcriptionally active but did not mediate Nkx2-1- repression of miR-200c expression. 3'UTR reporter assays support a direct regulation of the predicted targets Nfib and Myb by miR-200c., Conclusions: These studies suggest that Nkx2-1 controls the expression of specific miRNAs in lung epithelial cells. In particular, we identified a regulatory link between Nkx2-1, the known tumor suppressor miR-200c, and the developmental and oncogenic transcription factors Nfib and Myb, adding new players to the regulatory mechanisms driven by Nkx2-1 in lung epithelial cells that may have implications in lung development and tumorigenesis.