Experimental study on correlation of Coronin-1 with non-Mtb induced autophagy in macrophages / 中国免疫学杂志

ABSTRACT

Objective:To explore the correlation between Coronin-1 and non-Mtb induced autophagy in macrophages and its possible signal pathway.Methods: The pEGFP-C1-Coronin-1 plasmids encoding the over-expression of Coronin-1 and pGenesil-1-Coronin-1 plasmids encoding the siRNA of Coronin-1 gene were constructed and transfected into RAW264.7 respectively in order to obtain their stable transfected cell line ( RAW264.7-Cor.Plus and RAW264.7-Cor.Minus ).After the RAW264.7, RAW264.7-Cor.Plus and RAW264.7-Cor.Minus group cells were treated with complete medium, starvation, Rapamycin and Cyclosporine A respectively,the transcription level of Beclin-1 gene was detected by RT-PCR,and the protein expression level of Coronin-1 and Beclin-1 and the ratio of LC3BⅡ/LC3BⅠwas analyzed by Western blot.Results:The over-expression plasmids,the siRNA plasmids and their stable transfected cell lines which can over-express the Coronin-1 or inhibit its expression were successfully constructed.When the three groups cell treated with blank control,the ratio of LC3BⅡ/LC3BⅠwas found that:RAW264.7-Cor.Minus>RAW264.7>RAW264.7-Cor.Plus significantly,and the transcription level of Beclin-1 gene and its protein detected by RT-PCR and Western blot respectively showed the same trend with the ratio of LC3BⅡ/LC3BⅠ;When the three groups cell treated with starvation,the ratio of LC3BⅡ/LC3BⅠjust become the opposite of the first result significantly;When the RAW264.7 cells treated with Rapamycin, the expression of Coronin-1 was inhibited significantly.The ratio of LC3BⅡ/LC3BⅠin the RAW264.7-Cor.Plus group treated with Rapamycin and CsA respectively was higher than its corresponding control treatment group.Conclusion:There is a correlation between Coronin-1 and non-Mtb induced cell autophagy in macrophage.In the nutrition sufficient condition, Coronin-1 can inhibit autophagy;in the hungry condition,Coronin-1 can promote autophagy.The regulation of autophagy by Coronin-1 may be associated with mTOR and calcium signaling pathway.