MicroRNA-338-3p and microRNA-451 contribute to the formation of basolateral polarity in epithelial cells

MicroRNAs are small noncoding RNA species, some of which are playing important roles in cell differentiation. However, the level of participations of microRNAs in epithelial cell differentiation is largely unknown. Here, utilizing an epithelial differentiation model with T84 cells, we demonstrate that miR-338-3p and miR-451 contribute to the formation of epithelial basolateral polarity by facilitating translocalization of β1 integrin to the basolateral membrane. Among 250 microRNAs screened in this study, the expression levels of four microRNAs (miR-33a, 210, 338-3p and 451) were significantly elevated in the differentiated stage of T84 cells, when epithelial cell polarity was established. To investigate the involvement of these microRNAs in terms of epithelial cell polarity, we executed loss-of- and gain-of-function analyses of these microRNAs. The blockade of endogenous miR-338-3p or miR-451 via each microRNA-specific antisense oligonucleotides inhibited the translocalization of β1 integrin to the basolateral membrane, whereas inhibition of miR-210 or miR-33a had no effect on it. On the other hand, simultaneous transfection of synthetic miR-338-3p and miR-451 accelerated the translocalization of β1 integrin to the basolateral membrane, although the introduction of individual synthetic microRNAs exhibited no effect. Therefore, we concluded that both miR-338-3p and miR-451 are necessary for the development of epithelial cell polarity

Immortalization of Fetal Bovine Colon Epithelial Cells by Expression of Human Cyclin D1, Mutant Cyclin Dependent Kinase 4, and Telomerase Reverse Transcriptase: An In Vitro Model for Bacterial Infection.

Cattle are the economically important animals in human society. They are essential for the production of livestock products such as milk and meats. The production efficiency of livestock products is negatively impacted by infection with zoonotic pathogens. To prevent and control infectious diseases, it is important to understand the interaction between cattle tissue and pathogenic bacteria. In this study, we established an in vitro infection model of an immortalized bovine colon-derived epithelial cell line by transducing the cells with lentiviral vectors containing genes encoding cell cycle regulators cyclin D1, mutant cyclin dependent kinase 4 (CDK4), and human telomerase reverse transcriptase (TERT). The established cell line showed continuous cell proliferation, expression of epithelial markers, and an intact karyotype, indicating that the cells maintained their original nature as colon-derived epithelium. Furthermore, we exposed the established cell line to two strains of Salmonella enterica and EHEC. Interestingly, S. Typhimurium showed higher affinity for the established cell line and invaded the cytoplasm than S. Enteritidis. Quantitative RT-PCR revealed that gene expression of Toll-like receptor 1 (TLR1), TLR 2 and TLR 3, whereas TLR 4, 5 and 6 were not detectable in established cells. Our established immortalized colon-derived epithelial cell should be a useful tool for studies evaluating the molecular mechanisms underlying bacterial infection

Fluorescence microscopic images of cells subjected to the adhesion and invasion assay.

<p><i>S</i>. Typhimurium (a-d: low-power fields and e-h; high-power fields), <i>S</i>. Enteritidis (i-l) after infection, and non-infected control (m-p)) in BFCE-K4DT cells (passage number = 15). Total bacteria were stained green (a, e, i, and m; black arrows), adherent bacteria were stained red (b, f, j, n; white arrows), nuclei of cells were stained blue, and merge images of extracellular bacteria (stars) and intracellular bacteria (black arrows) appear yellow and green respectively (d, h, l, p). Each staining was carried for 3 times and representative pictures were shown here.</p

Results of the immunoblotting analysis and TRAP assay.

<p>(A) Detection of CDK4 and cyclin D1 by immunoblotting. Lane 1, BFCE primary cells; Lanes 2 and 3, BFCE-K4DT cells. (B) Detection of telomerase activity by TRAP assay. Lane 1, 1 kb ladder marker; lane 2, CHAPS buffer as a negative control; lane 3, 293T cells as a positive control; lane 4, BFCE primary cells; lane 5 and 6, BFCE-K4DT cells. The 6-bp ladders were detectable in lanes of positive control and BFCE-K4DT cells. Each experiment was performed in duplicates after cloning (primary cells: 2 times passage, K4DT cells: 15 times passage) and representative data were shown here.</p

Toll-like receptors (TLRs) gene expression in established BFCE-K4DT cells.

<p>+: positive,</p><p>-: negative</p><p>Toll-like receptors (TLRs) gene expression in established BFCE-K4DT cells.</p

Karyotype analysis of established BFCE-K4DT cells.

<p>All mitotic chromosome spreads (50 of 50 examined) from BFCE-K4DT cells showed a 2n = 60XY pattern. This analysis was performed by using cloned cells after 15 times passage.</p