Protein Modification Of New Biomarker IPO-38/Histone And Its Diagnostic Methods For Gastric Cancer

ObjectivesIPO-38/histone is a novel serum marker for gastric cancer discovered by our group previously.The transformation of laboratory research results into clinically available diagnostic methods requires breakthroughs in several key technical aspects,including highpurity antigens used as standards,antibody recognizing two antigenic epitopes,clinically practical and highly sensitive diagnostic method,and the like.This study will focus on the above key issues,including protein modification identification of histone antigens,possible regulatory mechanisms of protein modification,selection of clinically practical diagnostic methods,and methodological establishment,in order to promote the early clinical transformation of laboratory research results.Methods1.Identification of protein modification of IPO-38/histone biomarker.Clarify the specific histone modification type labeled by IPO-38 monoclonal antibody by modified histone peptide array;detect the expression of IPO-38 and the candidate histone modification using immunoblotting.2.Regulatory mechanism of novel histone modification.Investigate the expression of candidate histone modification enzymes in gastric cancer tissues and cell lines by immunoblotting,realtime PCR and immunofluorescence.The candidate histone modification regulatory genes were overexpressed and knocked down by lentiviral-mediated gene overexpression and CRISPR/Cas9-based gene editing technology to detect the influence on the candidate and its adjacent histone modification.The changes in histone modifications were again tested using PADIs inhibitors and EZH2 inhibitors.Finally,the association between candidate histone modification and its adjacent modifications was investigated by immunoprecipitation.3.The establishment of clinical practicability and high-sensitivity diagnostic methods.We try to explore and establish detection methods targeting the candidate histone modification,including standard product preparation,paried antibodies screening,coating material screening,and signal detection method selection.The standard antigen would be chemically synthesized.The antibody would be first screened from the available commercial antibodies.The coating material would be selected according to different detection methods,and the signal detection would be respectively performed by the traditional double antibody sandwich ELISA method and the new high sensitivity electrochemiluminescence.Results1.Modified histone peptide array indicated that the highest specificity of histone modification recognized by IPO-38 monoclonal antibody was H3R26 Cit,which is much higher than other modifications,and the top five positions of the signal intensity from high to low were H3R26 Cit with dimethylation modification of adjacent 27 lysine(H3R26CitK27me2),monomethylation modification(H3R26Cit-K27me1),and trimethylation modification(H3R26Cit-K27me3),H3R26 Cit,acetylation of lysine at position 27 of histone H3(H3K27ac).Then immunoblotting revealed that the expression of H3R26 Cit was highly correlated with the protein labeled by IPO-38 antibody in various tumor cell lines including gastric cancer.2.The protein level detection showed that there was a significant positive correlation between PADI4 and H3R26 Cit expressiion,while PADI2 did not.And the immunofluorescence showed that PADI4 was both localized to the cell nucleus in gastric cacner tissues and cell lines,while PADI2 was located to cytoplasmic in gastric cancer tissues,both cytoplasmic and nucleus in gastric cancer cell lines.Then the eukaryotic expression vector of PADI2 and PADI4 and PADI4 gene editing vector were successfully constructed,and they were transfected into gastric cancer cell lines using lentivirus.The results showed that PADI4 overexpression and knockdown could significantly up-regulate and down-regulate H3R26 Cit expression,while PADI2 overexpression did not.PADI4 overexpression significantly up-regulated the expression of H3K27me3 adjacent to H3R26 Cit,while the PADIs inhibitor could up-regulate H3K27me3 expression and downregulated H3K27 ac expression.It was also found that PADI4 overexpression significantly reduced the expression of H3K27me3 methyltransferase EZH2 and increased the expression of demethylases KDM6 A and KDM6 B.EZH2 inhibitor could down-regulate H3K27me3 expression and up-regulated H3R26 Cit expression.At the same time,the results of immunoprecipitation showed that H3K27 ac and H3R26 Cit could be co-expressed,while H3K27me3 was not co-expressed with H3R26 Cit.3.Establishment of clinical applicable and highly sensitive diagnostic methods: First,we successfully synthesized the full-length H3R26Cit-K27me2 polypeptide antigen used as a standard,labeled the detection antibody with HRP and SULFO-TAG,and tested the effectiveness of the detection antibody.A histone detection method based on the traditional double antibody sandwich ELISA method and a novel highly sensitive electrochemiluminescence method was initially established.ConclusionsFor the first time,we confirmed that the protein labeled by IPO-38 monoclonal antibody was the citrullinated 26 th arginine of histone 3(H3R26Cit),and the arginine methyltransferase family member 4(PADI4)was identified as the key regulatory enzyme that catalyzed the conversion of arginine to citrulline.We also found that H3R26 Cit and the adjacent trimethylated 27 th lysine histone 3(H3K27me3)had a mutual inhibition relationship.An ELISA assay methodology and a highly sensitive electrochemical assay methodology targeting IPO-38 were initially established.