Antimicrobial Peptide Merecidin Targeting MAPK1 To Affect The Biological Function Of Lung Cancer A549 Cells

Objective To study the effects of overexpression and knockout of human MAPK1 gene and the combined Antimicrobial peptide merecidinon biological functions such as proliferation,migration,invasion of lung cancer A549 cells,and the effect on ERK signaling pathways.Methods 1.The lung cancer A549 cells were divided into an experimental group and a Control group.The experimental lung cancer A549 cells were treated with 9 μmol / l Antimicrobial peptide merecidin.After 6 hours,the cells were collected and the whole protein was extracted for quantification by BCA method.The quality was determined by SDS-PAGE.After evaluation,trypsin was added to the whole protein solution for enzymatic hydrolysis to obtain all protein peptides.The peptides were sequentially labeled with TMT and HPLC fractionated.IMAC-Fe was used to enrich the phosphorylated peptides.Finally,high-resolution mass spectrometry was used to enrich the peptides.Detection of peptides.The library was identified and quantified using Max Quant software for the phosphorylated peptides obtained by mass spectrometry,and the functions and pathways of differentially phosphorylated proteins were analyzed in combination with bioinformatics.2.RT-PCR(reverse transcription PCR)was used to synthesize the CDS region of the MAPK1 target gene,which was digested with the overexpression vector p CDH-CMV-MCS-EF1α-Green Puro by restriction enzymes Xba I and Not I.The linearized vector was ligated,transformed into competent cells,and the over-expression recombinant vector PCDH-MAPK1 was synthesized and verified by sequencing.The liposome transfection method was used to transfect the over-expressed recombinant MAPK1 group(OE-MAPK1)and the empty vector Control group(OE-MAPK1-NC)to 293 T cells for virus packaging and further transfection to lung cancer cell A549,and use Puromycin was screened for stably overexpressing A549 cells.Real-time PCR and Western-blot experiments were used to detect MAPK1 m RNA and protein expression levels after overexpression.3.Design and synthesize the sg RNA target sequence,connect it to the knockout vector,then synthesize the recombinant vector MAPK1-U6-sg RNA-EF1a-Cas9-P2A-puro and transform it into competent cells.After sequencing and screening,use liposomes The transfection method was used to transfect the knock-out recombinant MAPK1 group(KO-MAPK1)and the empty vector Control group(KO-MAPK1-NC)to 293 T cells for virus packaging and further transfection to lung cancer cell A549,which was screened for stability with Puromycin A549 cells were knocked out of MAPK1.Real-time PCR and Western-blot experiments were used to detect MAPK1 m RNA and protein expression levels after stable knockout.4.CCK-8 method was used to detect the blank group(Control),over-expressed MAPK1group(OE-MAPK1),over-expressed MAPK1 empty vector group(OE-MAPK1-NC),MAPK1 knock-out group(KO-MAPK1),and MAPK1 knock In addition to the empty vector Control group(KO-MAPK1-NC),the change of the proliferation of lung cancer A549 cells in five groups,and the change of the drug inhibition rate of the Antimicrobial peptide merecidinon Control,OE-MAPK1,and KO-MAPK1.5.In the scratch test,compared with the blank group(Control)and the over-expressed MAPK1-empty vector group(OE-MAPk1-NC),the scratch healing rate of cells in the over-expressed MAPK1 group(OE-Map K1-NC)was significantly decreased,while that in the knockout group(KO-MAPK1-NC)was significantly decreased(P<0.05).Compared with the group without antimicrobial peptide Merecidin,the rate of cell scratch healing in the three groups was significantly lower(P<0.05),indicating that the antimicrobial peptide merecidininhibited cell migration in all three groups.6.Transwell assay observed that compared with the blank group(Control)and the overexpressed EMPTY vector group(OE-MAPK1-NC),the number of cell invasion in the overexpressed MAPK1 group(OE-Map K1-MAPK1-NC)was significantly increased,while the number of cell invasion in the knockout group(KO-MAPK1-NC)was significantly decreased(P<0.05).Compared with the group without the corresponding antimicrobial peptide Merecidin,the number of cell invasion in the three groups was significantly reduced(P<0.05),indicating that the antimicrobial peptide merecidinhas an inhibitory effect on cell invasion in all three groups.7.In the MAPK/ERK signaling pathway,the upstream and downstream proteins of MAPK1,ERK1/2,RSK1,MEK2 and ETS-1,were increased in the OVER-expressed MAPK1group(OE-MAPK1)cells compared with the Control group(P<0.05).In the cells with MAPK1 knockout(KO-MAPK1),the expression levels of ERK1/2 and RSK1 decreased(P<0.05).Compared with the group without antimicrobial peptide Merecidin,the expression levels of ERK1/2,RSK1,ETS1 and C-Jun in the treated group were decreased,while the expression levels of MEK2 were increased(P<0.05).Results 1.Phosphorylation proteomics finally detected 753 differentially phosphorylated proteins after treatment with the antimicrobial peptide merecidin.Among them,RB1,MAPK1,ARAF,PTK2,FOXO,MARCKS and other proteins have changed their phosphorylation sites.2.Successful construction of MAPK1 over-expressing stable transfection strain in human lung cancer A549 cells using lentiviral packaging system3.The CRISPR / CAS9 system was successfully used to construct a permanent knockout MAPK1 knockout cell line in human lung adenocarcinoma cell A549.4.CCK 8 method results show that increases over time,and the blank group(Control),MAPK1 empty expression vector group(OE-MAPK1-NC),express MAPK1 compared group(OE-MAPK1)cells absorbance(OD)value increased significantly,compared with blank group(Control),the knockout MAPK1 empty carrier group(KO-MAPK1-NC)comparison,knockout(KO-MAPK1)MAPK1 group cell absorbance(OD)value decreased significantly(P < 0.05).After the antimicrobial peptide Merecidin was applied to the five groups of cells,the inhibition rate of the five groups of cells increased with the increase of drug concentration and time.Among them,compared with the corresponding Control group and NC group,the increase trend of cell inhibition rate was more obvious after the addition of KO-MAPK1,while the increase was slower in the OE-MAPK1 group(P<0.05).5.In the scratch test,compared with the blank group(Control)and the over-expressed MAPK1-empty vector group(OE-MAPk1-NC),the scratch healing rate of cells in the over-expressed MAPK1 group(OE-Map K1-NC)was significantly increased,while that in the knockout group(KO-MAPK1-NC)was significantly decreased(P<0.05).Compared with the group without antimicrobial peptide Merecidin,the rate of cell scratch healing in the three groups was significantly lower(P<0.05).6.Transwell assay observed that compared with the blank group(Control)and the overexpressed EMPTY vector group(OE-MAPK1-NC),the number of cell invasion in the overexpressed MAPK1 group(OE-Map K1-MAPK1-NC)was significantly increased,while the number of cell invasion in the knockout group(KO-MAPK1-NC)was significantly decreased(P<0.05).Compared with the group without antimicrobial peptide Merecidin,the number of cell invasion in the three groups was reduced(P<0.05).7.The MAPK1 upstream and downstream proteins ERK1 / 2,RSK1,MEK2,and ETS1 in the MAPK / ERK signaling pathway increased in the overexpressed MAPK1 group(OE-MAPK1)cells compared to the Control group(Control)cells.However,the expression of ERK1 / 2 and RSK1 decreased in MAPK1 knockout cells(KO-MAPK1).Compared with the corresponding non-antimicrobial peptide merecidingroup,the expression levels of ERK1 /2,RSK1,ETS1,and c-jun in the drug-added group decreased,while the expression levels of MEK2 increased.Conclusion 1.After the Antimicrobial peptide merecidinacts on lung cancer A549 cells,the quantitative proteomics results indicate that MAPK1 is closely related to the antitumor effect of the Antimicrobial peptide merecidin.2.Overexpression of MAPK1 can promote proliferation,migration and invasion of lung cancer A549 cells;Knockout of MAPK1 inhibits proliferation,migration and invasion of lung cancer A549 cells.3.The antimicrobial peptide merecidinmay inhibit the proliferation,migration and invasion of lung cancer A549 by targeting the MAPK/ERK signaling pathway.