Application suite v4.12 program

The effects of miR-877-3p silencing on CC cell invasion were also determined. To do this, C-33A, SiHa and HeLa cells were transfected with NC and anti-miR-877-3p, as described above, for 4 days. Cells were FBS-starved overnight, trypsinized and 1.25 × 10⁵ cells were re-seeded in FBS-free DMEM on a 40-µL Matrigel^® layer (BD, Franklin Lakes, NJ, USA), which had been previously allowed to gel for 15–30 min at 37 °C in Transwell^® inserts with an 8-µm-pore membrane (Sarstedt, Nümbrecht, Germany). Inserts were placed in wells with 10% FBS-containing medium, and maintained for three more days. Finally, the gel layer was removed with a cotton-tipped swab, and invading cells that had reached the membrane were fixed and stained with a paraformaldehyde-containing crystal violet solution. Images were captured under a Leica DMi1 microscope with the Leica Application Suite v4.12 program (Leica, Wetzlar, Germany) at 50× magnification. The area occupied by invading cells was measured with the NIS Elements program (Nikon Instruments, Amsterdam, Netherlands).

To evaluate the proliferative ability of CC cell lines, C-33A, SiHa and HeLa cells were seeded (1 × 10⁴ cells/well) into 400 µL of medium in an E-plate L8 device (iCELLigence system, ACEA Biosciences, San Diego, CA, USA), after measuring the background in 100 µL of medium. Two replicates for each condition were analyzed. Cell attachment, spreading and proliferation were monitored by real-time cell analysis for 7 days, on the basis of changes in cell-sensor impedance, as previously described [47 (link)]. To assess the invasive capability of CC cell lines, cells were starved overnight. Next day, extracellular matrix layer was prepared by adding 40 µL of Matrigel^® (BD Biosciences, Franklin Lake, NJ, USA) into Transwell^® inserts with 8-μm-pore membranes (Sarstedt, Nümbrecht, Germany). After gelling for 15–30 min at 37 °C, starved cells were seeded (1.25 × 10⁵ cells/insert) on the gel layer in FBS-free medium, and the insert was placed in a well with FBS-containing medium. Cells were allowed to digest and penetrate the Matrigel^® layer as far as the membrane for 72 h. Then, non-invading cells and the gel layer were removed, and membranes were fixed and stained with a crystal violet solution containing paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA). Images were taken with a Leica DMi1 microscope and the Leica Application Suite v4.12 program (Leica, Wetzlar, Germany).