Fig 1.
The effect of miR-335 on MT1-MMP expression and cell surface activation of proMMP-2.
(A) The expression of miR-335 in untreated HT-1080, BPH-1 and U87GM cells as measured by real-time RT-PCR. The base-line level of miR-335 was about 15 and 25 fold greater in BPH-1 and U87GM cells respectively, than in HT-1080 cells. (B) miR-335 stimulation of cell surface MT1-MMP activity. The addition of miR-335 in contrast to a control miR (miRNC) or no addition stimulated proMMP-2 activation in HT-1080 and BPH-1 cells, but not U87GM cells. The addition of ConA increased proMMP-2 activation in all three cell lines, and miR-335 stimulated proMMP-2 activation in HT-1080 and BPH-1, but not U87GM cells. (C). The effect of miR-335 on expression of mRNA for MT1-MMP and EMMPRIN in relation to control no addition or control miRNA (miR-NC) as determined by real-time RT-PCR in BPH-1 and HT-1080 cells. The messages for both MT1-MMP and EMMPRIN were increased by miR-335 in BPH-1 cells but were unchanged (MT1-MMP) or decreased (EMMPRIN) by miR-335 in HT-1080 cells. (D and E) The effect of miR-335 on the protein levels of MT1-MMP in BPH-1 and HT-1080 cells determined by immuno-blotting (D) and quantification of the level of MT1-MMP as the ratio of MT1-MMP/GAPDH (E). The level of MT1-MMP was increased in HT-1080 cells treated with miR-335 alone or with addition of ConA. No change in the level of MT1-MMP in BPH-1 cells was evident.
Fig 2.
The effect of miR-335 on cell surface localization of MT1-MMP as determined by confocal microscopy in HT-1080 cells.
The level of cell surface MT1-MMP observed in control miR treated cells was reduced by treatment with a miR-335 inhibitor. In contrast, cell surface localization of MT1-MMP was increased by miR-335. The bars indicate 20 microns.
Fig 3.
The effect of miR-335 on cell migration of HT-1080 and BPH-1 cells in an in vitro model of wound healing.
Treatment of cells with miR-335 increased the rate of cell migration observed at 4 and 8 hours after a scratch was made in a confluent layer of cells using a pipette tip. The data were quantified as Remaining Area of Wound (μ2).
Fig 4.
The effect of miR-335 on cell proliferation.
Cell proliferation was measured using the CCK-8 assay. Increased levels of proliferation in miR-335 treated cells were found at 72 and 96 hrs for HT-1080, 96 hrs for BPH-1, and 72 hrs for U87GM cells. There was no change in rate of proliferation in HCT116, and a decrease in proliferation of MCF7 and MDA-MB-231 cells.