The Effects of Coronin-1 with Different Expression Levels on iNOS and TLRs Expression and Apoptosis in RAW264.7 Cells

Abstract: Coronin-1 is widely expressed in eukaryotic cells， which have been reported to be involved in a wide array of cellular functions including calcium homeostasis， cytoskeletal dynamics， immune and inflammatory responses. In order to investigate the effect and molecular mechanisms of different levels of Coronin-1 on the protein levels of iNOS and TLRs and apoptosis in RAW264.7 cell line， the experiment was divided into three groups (RAW264.7-Cor.Plus， RAW264.7 and RAW264.7-Cor.Minus) according to the differences of Coronin-1 expression level. Before and after treated with the CsA (a Calcineurin inhibitor) for 24 hours or cytD (an actin polymerization inhibitor) for 48 hours respectively， the level of iNOS mRNA was tested by Real-time PCR. The protein levels of TLR2， TLR4， TLR9 and calcineurin were detected by Western blot， and the apoptosis percent was analyzed by flow cytometry. Three groups of cells were stained with TRITC-phalloidin， then the percentage of F-actin rearrangement was calculated. The results were showed that the mRNA level of iNOS， the protein level of TLRs and apoptosis percent were significantly decreased in cells with over-expression of Coronin-1 (RAW264.7-Cor.Plus) (P<0.05). After treated with CsA， the mRNA levels of iNOS， the protein levels of TLR2， TLR4 and TLR9 and apoptosis rate in RAW264.7-Cor.Plus cells were significantly increased compared with the untreated control cells (P<0.05)， but the CaN level of RAW264.7 and RAW264.7-Cor.Plus cells were significantly lower than that of untreated control cells (P<0.05). After treated with cytD， between the treated cells and their untreated control cells， there were no significant difference in the mRNA levels of iNOS and apoptosis rate (P>0.05)， but the mRNA level of TLRs was significantly decreased (P<0.05). By phalloidin staining， the F-actin rearrangement rate of RAW264.7-Cor.Plus cells was significantly higher than that of RAW264.7 cells (P<0.05). These results indicated that the over-expression of Coronin-1 not only promotes the F-actin rearrangement， but also down-regulates the expression of TLRs and iNOS and inhibits cell apoptosis， of which the molecular mechanism is associated with that Coronin-1 promotes Ca2+ signaling pathway regulated by Calcineurin.

CSTR: 32200.14.cjcb.2017.02.0003