CD44 knockdown inhibits cell invasion and down-regulates MMP-9, MMP-14 and uPA.

<p>(A) The protein levels of CD44v, CD44s and pSTAT3 were markedly repressed in DCIS-shCD44 cells. (B) DCIS-shLuc or DCIS-shCD44 cells (2,000 cells/well) were incubated for 48 h and cell proliferation was determined by thymidine incorporation. Two separate experiments with triplicates were conducted (** p<0.01). (C) MCF10DCIS (DCIS), DCIS-shLuc or DCIS-shCD44 cells were incubated for 48 h in BME-coated invasion assay chambers. The number of cells that penetrated the BME layer was counted by Calcein-AM staining. Two separate experiments with triplicates were conducted (**<i>p</i><0.01). (D) DCIS-shLuc or DCIS-shCD44 cells were incubated for 48 h in Fluoroblok biocoat invasion assay chambers. Since both cells were labeled with green fluorescence, the cells that penetrated matrigel layer were detected as green pixels in the image. The green pixels were counted using Image-J program for quantitative evaluation. Two separate experiments with triplicates were conducted (*<i>p</i><0.05). (E) The mRNA expression levels of CD44 (20, the approximate qPCR cycle number of DCIS-shLuc cells at 24 h), MMP-2 (24), MMP-9 (29), MMP-14 (23) and uPA (21) in DCIS-shLuc and DCIS-shCD44 cells were determined after 24 h and 48 h of incubation. Three separate experiments with duplicates were conducted (**<i>p</i><0.01).