Antimicrobial Peptide Merecidin Induce Autophagy Of Lung Adenocarcinoma A549 Cells Regulated Via MAPK1

Objective:Detection the expression difference of autophagy-related proteins and enrichment the autophagy regulatory pathways in lung adenocarcinoma A549 after acted on the antibacterial peptide merecidin,and exploration the mechanism of autophagy of A549 cells induced by merecidin regulated via MAPK1.Methods1.CCK-8 assay was used to detect the inhibitory effect of 0-16μmol·L^-1 merecidin on the proliferation of lung adenocarcinoma A549 cells,and its IC50 value was determined.2.The differential expression levels of proteins in lung adenocarcinoma A549 cells treated with 9μmol·L^-1merecidin were detected by mass spectrometry,and the significantly differentially expressed proteins were screened out and the signal pathways were enriched.3.Transmission electron microscopy was used to detect the effects of A549 cells,KO-MAPK1（knockout group）,NC-KO-MAPK1（knockout no-load group）,NC-OE-MAPK1（overexpressed no-load group）,OE-MAPK1（overexpressed group）,and were treated with 0and 9μmol·L^-1merecidin.4.MDC staining was used to detect the effects on A549 cells,KO-MAPK1,NC-KO-MAPK1,OE-MAPK1 and NC-OE-MAPK1 cell lines were treated with 0 and9μmol·L^-1 merecidin.5.CYTO-ID was used to detect the effect on A549 cells,KO-MAPK1,NC-KO-MAPK1,OE-MAPK1 and NC-OE-MAPK1 cell lines were treated with 0 and 9μmol·L^-1 merecidin.6.AO staining used to detect the effect on A549 cells,KO-MAPK1,NC-KO-MAPK1,OE-MAPK1 and NC-OE-MAPK1 cell lines were treated with 0 and 9μmol·L^-1 merecidin.7.Western blot assay was used detected the expressions of ATG5,ATG7,ATG13,ULK1,LC3,MAPK1,P62 andβ-Tublin with 0,9,18μmol·L^-1 in A549 cells group,OE-MAPK1 and KO-MAPK1 group.Results.1.The proliferation of A549 cells inhibited by the antimicrobial peptide merecidin,and the half inhibitory concentration IC50 was 9.05μmol·L^-1 at 24h.2.Proteomic analysis:After merecidin was treated on A549 cells,peptide section was detected by mass spectrometry,and Max Quant database was searched and analyzed.The results showed that protein expressions of ATG2B,ATG13 and ULK1 were up-regulated.MAPK1 and EGFR,etc proteins were down-regulated,and these differentially expressed proteins were all involved in autophagy.KEEG pathway enrichment showed that PI3K-Akt,mTOR and Wnt signaling pathways were involved in autophagy regulation.3.Transmission electron microscopy observation of autophagosomes:A549 cells and KO-MAPK1 cells treatement with merecidin were showed that autophagosomes containing organelles with two-mode structure in the cytoplasm.4.MDC staining for detection of autophagosomes:A549 cells and KO-MAPK1 cells treatement with merecidin were stained by MDC,and observed under fluorescence microscope,green fluorescent particles of different sizes and dense staining were found in the cytoplasm.5.AO staining for detection of autophagosolysosomes:A549 cells and KO-MAPK1 cells treatement with merecidin were stained with AO,and the nucleus showed green fluorescence and the cytoplasm showed red fluorescence under fluorescence microscope.6.CYTO-ID detection of autophagosomes and autophagosolysosomes:A549 cells and KO-MAPK1 cells treatement with merecidin after CYTO-ID treatment.Fluorescence microscopy showed that green fluorescent particles were present in the cytoplasm.7.Western blot detection of protein expression:Western blot results showed that the protein expressions of ATG5,ATG7,LC3,ULK1 and ATG13 were up-regulated,while the protein expressions of P62 and MAPK1 were down-regulated after merecidin treatment.Conclusion1.The antimicrobial peptide merecidin induce autophagy in lung adenocarcinoma A549.2.The antimicrobial peptide merecidin regulated the autophagy of A549 cells via MAPK1.