Carraro DM, Elias EV, Andrade VP. invasive properties. The ectopic expression of PLC\2 in non\transformed and DCIS\derived cells is, to some extent, dependent on the de\regulation of miR\146a, a tumor suppressor miRNA in invasive breast cancer. Interestingly, an inverse relationship between the two molecules, indicative of a role of miR\146a in targeting PLC\2, was not detected in primary DCIS from patients who developed a second invasive breast neoplasia. This suggests that alterations of the PLC\2/miR\146a relationship in DCIS may constitute a molecular risk factor for the appearance of new breast lesions. Since neither traditional classification systems nor molecular characterizations are able to predict the malignant potential of DCIS, as is possible for invasive ductal carcinoma (IDC), we propose that the assessment of the PLC\2/miR\146a levels at diagnosis could be beneficial for identifying whether DCIS patients may have either a low or high propensity for invasive recurrence. values <0.05 were considered statistically significant. 3.?RESULTS 3.1. PLC\2 is present in DCIS tissues and affects EMT markers, CD133 level, and Fmoc-Val-Cit-PAB-PNP invasion capability in DCIS\derived cells Fmoc-Val-Cit-PAB-PNP Immunohistochemical analysis performed on FFPE tissue sections from 70 pure DCIS (Cohort 1) with different histopathological features demonstrated that PLC\2 is expressed, to a variable extent (Figure ?(Figure1A),1A), in all breast tumor samples. The analysis of PLC\2 staining, arbitrarily quantified as weak, moderate or strong following a previously established criterion,6 showed that protein levels are significantly lower in DCIS with respect to unrelated IDC (Cohort 2), even though this PLC isozyme was expressed at moderate or high levels in 26% and 20% of DCIS samples, respectively (Figure ?(Figure11B). Open in a separate window Figure 1 PLC\2 is expressed in primary DCIS. In (A) immunohistochemical analysis of PLC\2 expression of FFPE sections from healthy breast tissue (a) and DCISs with different histological features (b\d) derived from Cohort 1. b: low\grade ductal non comedo; c: intermediate\grade ductal non comedo; d: high grade ductal comedo. Bar?=?100?m. In (B) graphical representation of levels of PLC\2 staining in primary DCIS (Cohort 1) and unrelated IDC (Cohort 2). [Color figure can be viewed at wileyonlinelibrary.com] The amount of PLC\2 found in DCIS did not significantly correlate with any of the main clinic\pathological factors and biological markers for breast tumors (Table ?(Table1).1). Conversely, a significant correlation was observed between PLC\2 staining and the age at diagnosis, since the majority of patients 50 developed primary DCIS with low levels of the protein and none of the tumors from patients over 65 showed strong PLC\2 staining (Table ?(Table11). Table 1 Correlation of PLC\2 staining with clinico\pathological factors and biological markers in DCIS (%)(%)(%)promoter by chromatin immunoprecipitation in MCF10DCIS cells Fmoc-Val-Cit-PAB-PNP transfected with miR\146a inhibitor or mimic. The Fmoc-Val-Cit-PAB-PNP bands correspond to PCR products obtained amplifying a Mouse monoclonal to OCT4 137?bp DNA fragment encompassing a consensus\binding site for NF\kB. Input: genomic DNA not subjected to immunoprecipitation (positive control); IgG: samples immunoprecipitated with a non\specific antibody (negative control). All experiments were performed in triplicate. In (F) percentage of luciferase activity in MCF10DCIS cells co\transfected for 24?h with 250?ng of PLC\2 3\UTR luciferase reporter vector and with different concentrations of miR\146a mimic. Values obtained from cells transfected with scramble miRNA.