Research On Candidate Protein Marker NAP-2(73) Of Primary Hepatic Carcinoma In Hepatocellular Carcinoma Cell Line And Hepatocellular Carcinoma Tissues

Primary Hepatic Carcinoma (PHC) is one of the most common human malignancies in our country. According to the statistics of WHO in 2005, there were 620000 new cases reported and 590000 cases died annually. There were 200000-300000 cases died of PHC in our country every year, which is a serious threat against our people's life and health. Therefore it has always been the research emphasis on the studies of the tumors we laid. It's hard to diagnosis PHC in the early stage. It would aggravate and worsen after they had clinical symptoms. In the recent year, the mortality of PHC is still the same even higher because of the very poor prognosis. One of the most important reasons is losing the optimal opportunities for operation. Therefore to find out the ideal protein marker is the emphasis on which we have been laid.It's possible to find out the new tumor bio-marker as the proteomics was available. We had successfully screened the PHC bio-markers from the blood serum of PHC patients by the SELDI-TOF-MS in our preliminary work. And the protein of molecular weight 7984Da was identified as neutrophil-activating peptide 2(NAP-2(73)) by protein isolation, protein purification, mass spectrometry identification, which would be a potential PHC serum bio-marker.Objective:To verify the reliability of SELDI screening test, NAP-2(73) polyclonal antibody was raised in rabbits. In this research, the level of expression of neutrophil-activating peptide 2(NAP-2(73)) was determined in hepatoma cell line SMMC-7721 and Hepatocellular carcinoma tissues.Methods: The epitope of NAP-2(73) was designed by DNAstart and raised in rabbits. To detect the mRNA and protein expression of NAP-2 in hepatoma cell line SMMC-7721 and human liver cell line HL-7702 by Real-time PCR, ICC and Western Blotting. 30 Hepatocellular carcinoma tissues and their adjacent tissues were obtained successfully from patients in the First Hospital of Guangxi Medical University. Immunohistochemical method was used to detect the expression of NAP-2(73) protein in 30 hepatocellular carcinoma tissues and their adjacent tissues.Results: The titre of antiserum was tested to be 1:729000 by the indirect ELISA. In SMCC-7721, the NAP-2 protein positive rates was 100% ,there was no NAP-2 protein expression in HL-7702 . In 30 hepatocellular carcinoma tissues, NAP-2 protein strong positive rates were 63.33%, but no strong expression in PHC adjacent tissues. Significant difference was found between the hepatocellular carcinoma tissues and their adjacent tissues (P < 0.01). NAP-2 mRNA expression in SMMC-7721 was significantly increased than the expression in HL-7702 by 7 times.Conclusions: In SMCC-7721, the NAP-2 protein positive rates was 100% ,there was no NAP-2 protein expression in HL-7702 . Its expression had significant difference between Hepatocellular carcinoma tissues and their adjacent tissues. This proved the reliability of the serum prescreening, verified the prediction and analysis of bio-information. It indicated that NAP-2(73) would be a new bio-marker of PHC.