The Study On The Biological Function And Mechanism Research Of Anti-tumor M1-138 Peptide Based On FOXM1

Cancer as the worldundefineds second largest human death factor after cardiovascular disease.With the development of global science and technology,the death toll from infectious diseases decreases and life expectancy increases,cancer is likely to become a greater human challenge.Biological targeted therapy is an important method for the treatment of tumors.The transcription factor FOXM1 is involved in stimulating cell proliferation,enhancing DNA damage repair,and promoting cancer cell metastasis,and has been shown to prevent the occurrence and development of multiple cancers by inhibiting FOXM1.At present,FOXM1 has been recognized as an anti-tumor target.The N-terminus of FOXM1 binds to the C-terminus of FOXM1 as an inhibitory domain in the cell cycle,and also binds to LIN9 of the MuvB complex to regulate the activity of FoxM1.FOXM1 interacts with SMAD3 through the N-terminus,promotes nuclear translocation of SMAD3,and promotes tumor metastasis.In the present study,M1-138 acts as an anti-tumor peptide,blocking the direct and indirect promoter binding mechanism of FOXM1 by replacing the N-terminus of full-length FOXM1 with the C-terminus or LIN9;M1-138 competitively inhibits the interaction between FOXM1 and SMAD3 by replacing full-length FOXM1,thereby inhibiting the transcriptional activities of FOXM1 and SMAD3,and exhibits an effective anti-tumor ability.Polyarginine(Arg)9,a powerful vector for intracellular delivery of large molecules,could transport therapeutic agents across cell membrane.The purpose of this study is to construct M1-138 Anti-tumor peptide and investigate its effects on cancer cells,expecting to provide potential new approaches for anti-cancer therapy.The main results were listed as followed:(1)Detection of R9 trans-membrane peptide carrying GFP can efficiently enter cells and be widely distributed in cytoplasm and nucleus by fluorescence inverted microscope.WB technology analyzed the entry of M1-138 into cells,and that M1-138 can effectively enter cells and remain for at least 48 h in the cells.At the same time,CCK-8 was used to find that M1-138 inhibited the proliferation of each tumor cell(MCF-7,231,A549,HepG2 and Hela cells).The inhibition rate of M1-138 on 231 cells was significantly higher than that of MCF-7 cells.(2)The mRNA and protein levels of FOXM1 in 231 cells were significantly higher than those in MCF-7 cells using qPCR and WB,and the sensitivity of 231 cells to M1-138 was significantly higher than that of MCF-7 cells.A cell line M1 high expressing transcription factor FOXM1 was constructed,and CCK-8 detected the inhibitory effect of M1 and MCF-7 on M1-138,and it was found that M1-138 had a more significant inhibitory effect on M1 cells.It indicated that M1-138 inhibited the activity of FOXM1 high expression cells in tumor cells significantly higher than that of FOXM1 low expression.The above results show that the inhibitory effect of M1-138 on tumor cells is positively correlated with the expression level of the transcription factor FOXM1.(3)It was found that M1-138 inhibited the migration,proliferation and tumorigenic ability of tumor cells using transswell assay,monoclonal formation assay and nude mouse assay.(4)It was confirmed that M1-138 can effectively enter the cell and mainly enter the nucleus by immunohistochemistry,which is consistent with the transcription factor FOXM1 in the nucleus.We confirmed that M1-138 interacts with FOXM1 and directly binds to the C-terminus(688-748aa)of the activation domain of FOXM1 by pulldown assay.It was found that M1-138 significantly affected the direct and indirect promoter binding mechanism of the transcription factor FOXM1 and reduced its transcriptional activity by luciferase assay.These results indicate that M1-138 may inhibit the transcriptional activity and inhibit tumor cell proliferation,migration and tumorigenic ability by binding to the C-terminus of the activation domain of FOXM1.(5)The results showed that M1-138 interacted directly with SMAD3 using pulldown assay.The further confirmed that M1-138 could competitively inhibit the interaction between FOXM1 and SMAD3.At the same time,M1-138 was found to inhibit SMAD3 transcriptional activity by luciferase assay.Taken together,M1-138 inhibits both FOXM1 transcriptional activity and SMAD3 transcription.(6)The expression of the target genes of FOXM1 and SMAD3 was detected by qPCR and WB,we chose four genes as typical examples from the list of multiple targets regulated by these two transcription factors: proliferation-related CyclinB1,CDC25 B,and PLK1 as typical FOXM1 targets,and migration-related SLUG as a typical SMAD3 target.It was found that M1-138 inhibited the expression of the target genes of FOXM1 and SMAD3.(7)M1-138 inhibited the growth of tumors by nude mice xenograft experiments.M1-138 inhibited the expression of the target genes of FOXM1 and SMAD3 in tumors by qPCR and WB experiments.Immunohistochemistry confirmed the proliferation-related factor KI-67 and PCNA expression were down-regulated.(8)The effects of M1-138 on body weight,hemolysis of blood cells and animal toxicity were examined by mouse experiments,hemolysis test and toxicity test.The results showed that mice and blood cells were well tolerated by M1-138.