HCMV, HSV-1 and HSV-2 have been shown to induce persistence in C. trachomatis co-infections with similar features as those described here for HHV6

May 24, 2017

g adaptor molecules of the TLR/IL1 signaling cascade such as MAP3K7IP7/TAB2. However, 21825001 although TAB2 expression was found to be inhibited by miR-155 in our microarray experiments, we clearly showed that miR-155 specifically inhibited IL1-induced KGF production but did not affect the expression level of three other cytokines, indicating that in our model, miR-155 does not affect the TLR/IL1 signalling cascade. In conclusion, this study provides a global characterization of miR-155 targets in human lung fibroblasts. It leads to the identification of KGF/FGF7 as a mesenchymal-specific miR-155 target. To date, this is the first report relating evidence for miRNA involvement in lung epithelial-mesenchymal interactions. Finally, this work provides new insights into the pathogenesis of lung fibrosis, by suggesting a role for miRNAs in the lung fibrotic process. Materials and Methods Ethics statement All animal care and experimental protocols were conducted in accordance with the regulations of the institutional care and use committee at the University of Lille 2. Personnel from the laboratory carried out all experimental protocols under strict guidelines to insure careful and consistent handling of the mice. Cell Culture and Treatments The human fibroblast cells HFL1 derived from normal lung tissue, adenocarcinoma A549 cells, NIH 3T3 and HEK 293 were purchased from the American Type Culture Collection and maintained in monolayer culture in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. IL-1b, TNF-a and TGF-b were used at final concentration of 10 ng/ml. RNA isolation from cell samples Total RNA were extracted from the samples with TRIzol solution, and the integrity of RNA was assessed by using an Agilent BioAnalyser 2100 . The miRvana miRNA isolation kit was used for isolation and enrichment of small RNA fractions, according to the manufacturer’s protocol. Animal treatment 912 weeks old male C57BL/6 and Balb/C mice were purchased from Charles River, France. To induce fibrotic miR-155 Function in Fibroblast changes, mice were intratracheally instillated with bleomycin or PBS as previously described. Briefly, mice were anesthetized with sevoflurane inhalation and placed in dorsal recumbency. Transtracheal insertion of a 24-G animal feeding needle was used to instillate bleomycin or vehicle, in a volume of 80 ml. Mice were sacrificed 7 and 14 days after instillation and lungs were removed for further analysis. Lung fibrosis was assessed by histopathology after fixation with 4% paraformaldehyde/PBS overnight at 4uC. The major criteria examined included interstitial thickening of alveolar or bronchiolar walls, collagen deposition, and inflammatory cell infiltration. Total RNA was extracted from lungs removed from PBS- and bleomycin-treated mice as described above and used for assessment of miR-155 expression by quantitative RT-PCR. 21505263 Microarray procedure miRNA microarray procedure. The oligonucleotide sequences corresponding to 2054 mature miRNAs found in the miRNA registry are available on http://www.microarray.fr:8080/merge/index ”. Each oligonucleotide was spotted four times on each microarray, in order to reduce positional bias of the fluorescence readout. Experimental data and associated microarray designs have been deposited in the NBCI Gene Expression Omnibus under series GSE12033/GSE12035 and platform record GPL4717. GSK1363089 site target preparation and array hybridization were performed as previously described. Briefly, small R