*3.7. Antitumoral E*ff*ects*

The tumor suppressor miR200c inhibits the epithelial–mesenchymal transition, a process involved in metastasis by enhancing the motility and migration of tumor cells, by targeting ZEB1 and ZEB2. ZEB1 and ZEB2 are transcriptional repressors, which downregulate the marker E-cadherin [7,51]. Furthermore, Kopp et al. reported that one of the most prominent oncogenes KRAS is targeted by miR200c, which results in an altered cell cycle of the cancer cells [6]. Notably, they could show that miR200c inhibits cell cycle progression by decreasing the G1-population. We have performed direct investigations of these antitumoral e ffects through tumor cell migration and cell cycle analysis on two di fferent EGFR overexpressing cell lines, T24 bladder cancer and HeLa cervical cancer cells using miR200c loaded MSN-454-GE11 samples.

Cell migration was studied using a scratch assay, as shown in Figure 7. T24 cells and HeLa cells were incubated with MSN-454-GE11 for 4 h (5 μg miR200c/well), then the medium was changed. After additional 24 h, the cell layer was broken by a scratch using a 200 μL Eppendorf pipette tip. The closure of the scratch, which is an indicator for cell migration, was measured at indicated time points. In both cell lines, the scratch was almost completely closed after 48 h when MSN-454-GE11 was loaded with Ctrl-RNA (88% scratch closure for T24 and 82% closure for HeLa cells). In contrast, with MSN-454-GE11 particles loaded with miR200c, a scratch closure of only 48% (for T24) and 53% (for HeLa cells) was observed after this time. Hence, miR200c delivered by MSN-454-GE11 significantly hinders cell migration.

**Figure 7.** Inhibition of tumor cell migration. Images of a scratch assay of T24 and HeLa cells. Cells were treated with MSN160 nm-454-GE11 loaded with miR200c. The cell layer was broken after 24 h through a scratch and the closure was monitored for 48 h.

In addition, the effect of miR200c on the cell cycle was studied. Cells were transfected with MSN-454-GE11 loaded with miR200c or with the control RNA Ctrl and incubated for 4 h (Figure 8). A significant decrease in the number of cells in the G1 phase is observed when exposed to miR200C delivered by MSN, in combination with an increase in the number of cells in the S-phase. Thus, tumor cells transfected with MSN-454-GE11 loaded with miR200c showed the expected decreased migration and changes in the cell cycle.

**Figure 8.** Cell cycle analysis via flow cytometry of cell stages G1, S and G2 of T24 cells at 72 h after treatment. For statistical analysis, a two-tailed t-test was performed (*n* = 3, mean ± SD, \*\*\* *p* < 0.01)
