The Acquisition Of FoxM1-DBD Targeting Peptide 9R-P49 And Its Effects And Preliminary Molecular Mechanism On HCCLM3 Hepatocellular Carcinoma Cells

Cancer is a major disease that seriously endangers human life and health.Malignant tumors,including liver cancer,have become the number one killer of human disease deaths.Targeted molecular markers or targeted drug research targeting closely related proteins of tumors is an important direction for cancer diagnosis and anticancer drug development.Among many target proteins,Fox M1 plays a very important role in the occurrence and development of tumors,as well as metastasis and drug resistance.Fox M1 is considered to be an important drug target and marker for anticancer drug treatment and intervention.In the early stage of our laboratory,Fox M1-DBD was used as the target,and a high-affinity polypeptide P201 was obtained in the phage dodecapeptide library by phage display technology.The study found that it has an inhibitory effect on liver cancer Hep G2 cells,in addition,followed by alanine-scanning mutagenesis,five key amino acid sites were identified in P201.In order to screen and obtain optimized peptides with more strong effects and specificities for tumors,in this study,we adopted the strategy that the five key amino acids were fixed while the remaining sites were randomly synthesized in P201.Hence,the new and biased phage peptide library were constructed and screened,the anti-tumor P49 peptide with more strong affinity and targeting was obtained,.its effect and molecular mechanism in liver cancer HCCLM3 cells were then explored.In this study,a phage secondary biased peptide library with a library capacity of 6.67×105 pfu was successfully constructed.Five rounds of affinity panning were performed on the constructed peptide library with Fox M1-DBD as the target,and the P/N value was as high as 16071 was reached.33 groups of positive phage clones were obtained by random sequencing.ELISA results showed that 11 monoclonal phages had higher affinity than the original sequence P201.The results of reverse screening showed that the binding ability of peptides P2,P18,P19,P21 and P49 to the target protein was higher than that of P201.The top four peptide sequences: P2,P8,P21 and P49 were determined after comprehensive analysis by molecular simulation docking and bioinformatics methods for peptide synthesis and further study.The synthesized four optimized polypeptides had different degrees of inhibitory effects on the four tumor cells including A549,HCCLM3,Hep G2 and MDA-MB-231 by CCK-8 assay.,Among them,9R-P49 had strong inhibitory effects for all the four tumor cells,especially in HCCLM3 cells.The IC50 value of 9R-P49 group for HCCLM3 cells after 24 h treatment was 10.24±0.45 μM,which was significantly better than that of P201 polypeptide.Subsequently,we focused on HCCLM3 cells,through AO/EB double staining,flow cytometry,plate cloning formation and Transwell experiments,it was found that 9R-P49 polypeptide can significantly promote their apoptosis,inhibit cell proliferation and migration.RT-PCR and Western blot showed that the polypeptide could regulate the physiological function of cells by down-regulating the transcription and translation levels of Fox M1.Further research found that 9R-P49 could down-regulate the transcription levels of cancer-promoting genes CCNB1,MMP2 and Survivin and the protein expression levels of C-myc.Finally,HCCLM3 cells were treated with the IC50 dose of 9R-P49 polypeptide and RNA transcriptome sequencing analysis was performed.Differential genes were screened according to the conditions of Q-value≤0.05 and Fold change≥2.The results showed that there were 1154 differentially expressed genes,of which 489 genes were was down-regulated while 665 genes were up-regulated.GO enrichment analysis found that the 9R-P49 treatment group was mainly enriched in the regulation of vascular permeability and neutrophil chemotaxis.KEGG analysis of differentially expressed genes found that the 9R-P49 treatment group was involved in immune regulation,cancer,signal transduction and other pathways.Among them,the most enriched genes were under the entry of human diseases,suggesting that the treatment of this polypeptide is closely related to the therapy of human diseases.In conclusion,this study successfully screened and obtained the peptides with better affinity and targeting than that of P201,and the peptide 9R-P49 can inhibit the proliferation,migration and promote apoptosis of HCCLM3 cells by down-regulating the expression of Fox M1.As a molecular targeting Fox M1.The thesis provides important working basis and idea for the in-depth development of anti-tumor peptidemolecularly targeting Fox M1.