Three-dimensional organotypic culture using reconstituted basement membrane matrix Matrigel (rBM 3-D)
Three-dimensional organotypic culture using reconstituted basement membrane matrix Matrigel (rBM 3-D) is usually an indispensable tool to characterize morphogenesis of mammary epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix (ECM). cells in rBM 3-Deb culture. Overexpression of the differentially expressed miR-200 family member miR429 in MDA-MB231 cells attenuated their invasive stellate morphogenesis in rBM 3-Deb culture. In summary, CASP3 we provide the first miRNA signatures of morphogenesis of human breast malignancy cells in rBM 3-Deb culture and warrant further utilization of rBM 3-Deb culture in investigation of miRNAs in breast malignancy. properties of mammary epithelial cells (4-7). Genome wide manifestation profiling of breast malignancy in rBM 3-Deb culture has established gene manifestation signatures associated with unique morphogenesis of breast malignancy cell lines with diverse invasive and metastatic properties (8). The clinical significance of the gene manifestation information produced from rBM 3-Deb culture is usually confirmed in that the gene manifestation signature from rBM 3-Deb culture of breast malignancy cells holds prognostic values for patients with breast malignancy (9). microRNAs (miRNAs) are small non-coding RNAs that inhibit gene manifestation often via complementarity with its target sequences within the 3′ untranslated region (3′-UTR) of mRNA (10). Profiling miRNAs in human malignancy specimens and cell lines reveals a growing number of tumorigenic and tumor suppressive miRNAs (11). Among the tumor suppressive miRNAs, the let-7 family and miR-200 family are frequently silenced in malignancy (12). The let-7 family suppresses tumor growth via targeting cell cycle regulators (CDC25A and CDK6), promoters of growth (RAS and c-myc), and early embryonic genes (HMGA2) (13-15). The miR-200 family inhibits epithelial to mesenchymal transition (EMT) via targeting two EMT mediators, E-box binding transcription factors ZEB1 and ZEB2, and thereby suppresses attack and metastasis (16,17). Despite the importance of rBM 3-Deb culture and miRNAs in the research of breast malignancy, miRNAs have not been characterized in rBM 3-Deb culture of breast malignancy cells. The present study is usually targeted to elucidate the biology of miRNAs in morphogenesis of breast malignancy cells with diverse invasive and metastatic potentials in rBM 3-Deb culture. Materials and methods Reagents and plasmids Matrigel was purchased from BD Biosciences (Rockville, MD). Cell culture grade type I collagen was purchased from Sigma (St. Louis MO). A human miR-429 manifestation retroviral vector was generated by inserting the human pre-miR-429 into the pMSCV-puro-GFP-miR spine vector as we previously explained (18). Alexa CGP60474 CGP60474 594 conjugated filamentous actin (F-actin) binding phalloidin CGP60474 was purchased from Invitrogen (Carlsbad, CA). Cell culture and retroviral transduction Two human breast malignancy cell lines, MCF-7 cells (N variant) and MDA-MB231 cells were cultured in DMEM CGP60474 (Sigma) as previously explained (19,20). Stable ectopical manifestation of miR-429 in MDA-MB231 cells was accomplished by retroviral transduction as we previously explained (21). Briefly miR-429 conveying and its spine control retroviral vectors were produced using 293T cells. MDA-MB231 cells were then infected with the retroviruses and the stable transductants were selected and managed using puromycin made up of culture medium. rBM three dimensional organotypic culture Overlay rBM 3-Deb culture was carried out as explained (4). Briefly, MCF-7 and MDA-MB231 cells were seeded at 2105 cells/well in a 6-well cell culture plate that was coated with Matrigel. DMEM culture medium was supplemented with 4% of Matrigel and new medium was fed every two days. In the selected rBM 3-Deb culture of MCF-7 cells, Col-1 (2 (4,22). The cell morphogenesis was also visualized by staining for filamentous actin using Alexa 594 conjugated phalloidin followed by confocal fluorescent microscopy analysis on a Bio-Rad Radiance 2100 system (Hercules, CA), which is usually an established method to monitor morphogenesis of mammary epithelial cells (8). RNA extraction and analysis of mRNA and miRNA manifestation Total cell RNA was extracted using TRIzol (Invitrogen) from 2-Deb culture when the cells reached ~80% confluence and from rBM 3-Deb cultures on day 12 after the cells were seeded. Quantitative RT-PCR (qRT-PCR) was carried out to determine the manifestation.