Moreover, improved cytotoxicity by IL-6 treatment was significantly attenuated in cells overexpressing miR-181c (Number 4E). Open in a separate window Figure 4 Inhibition of IL-6-induced beta cell apoptosis via miR-181c upregulation. understanding the part of miRNAs in IL-6-induced beta cell death will be beneficial in the pursuit of regulatory mechanisms involved in IL-6-induced apoptosis. To identify mRNAs and miRNAs associated with IL-6-induced beta cell apoptosis, we investigated changes in gene manifestation in IL-6-treated INS-1 cells. In this study, we found that TNF- manifestation was highly upregulated in IL-6-treated INS-1 cells, and that miR-181c contributed to IL-6-induced beta cell apoptosis through rules of TNF- manifestation. 2. Results 2.1. Induction of Apoptosis in IL-6 Treated Cells To investigate induction of apoptosis by chronic IL-6 treatment, cell viability and annexin V- stained INS-1 cells were measured after 48 h treatment. As demonstrated in Number 1, we confirmed that treatment with 20 ng/mL of IL-6 improved early apoptotic cell populations, and cell viability was significantly reduced (Number 1A,B). Open in a separate window Number 1 Effect of Interleukin (IL)-6 on apoptosis and cell viability in beta cells. INS-1 cells were treated with 20 ng/mL IL-6 for 48 h and (A) stained with FITC-annexin V/PI and analyzed by circulation cytometry to determine the human population of cells in early apoptosis. (B) Cells were treated as explained in (A), and cell viability was determined by MTT assay. The results represent the mean SEM from experiments performed in triplicate and normalized to control (CON) cells. * < 0.05 in comparison with CON. 2.2. Differential Gene Manifestation during Apoptosis in IL-6-Treated Cells To identify apoptotic mechanisms triggered in response to IL-6 treatment, variations in mRNA levels of apoptosis-related genes were examined using a custom RT2 profiler PCR array by comparing IL-6-treated cells with control, untreated cells. We observed significant upregulation or downregulation of many genes (Supplementary Table S1). A total of 26 genes were upregulated (>2-collapse difference in manifestation) in IL-6 treated cells compared to untreated cells. Among them, manifestation levels of tumor necrosis element (and and < 0.05 in comparison with 0 h, ? < 0.05 in comparison with DMSO treated CON, ?? < 0.05 in JAK1-IN-4 comparison with DMSO treated with IL6, * < 0.05 in comparison with CON Ab treated CON, # < 0.05 in comparison with CON Ab treated with IL6. 2.4. Downregulation of miR-181c during IL-6-Induced Beta Cell Apoptosis To determine the involvement of miRNAs in IL-6-induced apoptosis, we analyzed global miRNA manifestation in INS-1 cells using Rat miRNome RT2 miRNA PCR array and miRDB ( prediction algorithm. We found that miR-101a, -122, and -181c were significantly downregulated about two-fold in IL-6-treated cells compared with control cells. JAK1-IN-4 To evaluate these results, we quantified miRNA manifestation using qRT-PCR. Among the three miRNAs, only the level of miR-181c was significantly decreased in INS-1 cells exposed to 20 ng/mL of IL-6 compared with untreated cells (Number 3A). As our earlier JAK1-IN-4 study showed that STAT-3 signaling mediated IL-6-induced beta-cell apoptosis, a STAT-3 inhibitor, AG490 [10], was used to determine whether miR-181c manifestation was controlled by STAT-3 inactivation. A treatment of 10 M of AG490 efficiently reduced STAT-3 phosphorylation in INS-1 cells [10] and downregulation of miR-181c by IL-6 treatment was reversed by co-treatment with AG490 (Number 3B). Open in Rabbit polyclonal to ZNF33A a separate window Number 3 Downregulation of miR-181c during IL-6-induced beta cell apoptosis. (A) INS-1 cells were treated with 20 ng/mL IL-6 for 24 h and miRNA levels were analyzed by quantitative RT-PCR. (B) INS-1 cells were pretreated with or without AG490 (10 M) for 3 h and then incubated with 20 ng/mL IL-6 for 24 h. miR-181c levels were analyzed by quantitative RT-PCR and normalized to endogenous RNU6. The results demonstrated represent the mean SEM from experiments performed in triplicate. * < 0.05 in comparison with CON, # JAK1-IN-4 < 0.05 in comparison with IL6. 2.5. Inhibition of IL-6 Induced Beta Cell Cytotoxicity via miR-181c Upregulation As it was reported that miR-181c is definitely a new regulator of TNF- manifestation [23], we tested whether miR-181c has an effect on apoptosis induced by IL-6. First, to investigate whether TNF--mediated beta cell apoptosis, in JAK1-IN-4 response to IL-6 treatment, is definitely regulated by miR-181c manifestation, we examined the population of apoptotic cells in cells overexpressing miR-181c. We observed that cells transfected having a miR-181c mimic, ranging from ~1 to 20 pmol, showed elevated miR-181c levels. Cells.