Identification And Preliminary Validation Of Hepatic Carcinoma Related Proteins In Primary Hepatic Carcinoma Tissiue Interstitial Fluid

Primary hepatic carcinoma（PHC） refers to take place in the liver cells orintrahepatic bile duct cell’s malignancy.PHC is one of the most prevalentmalignancies in the world. In recent decades, AFP was considered as one ofserum markers on liver cancer diagnosis. But specificity and sensitivity of AFPis not ideal.It can’t satisfy the clinical need of diagnosis, Therefore, it is veryimportant to discover the new biomarkers for liver cancer diagnosis.At present, the research of liver cancer related biomarkers is mostly basedon liver cancer cell line or blood.However liver cancer cell line is cultured invitro and out of the body’s internal environment. So the changes of proteinsmay not reflect the pathological changes of body. The composition of serumproteins is very complicated. The candidate biomarkers secreted from livercancer cells may be cleared or greatly diluted.It is a challenge to search for newserum markers of liver cancer. As the medium of blood circulation and cancercells, tissue interstitial fluid (TIF) of tumor should contain all the candidateserum markers. Furthermore, TIF should have higher concentration than that of the serum. Therefore, TIF is believed to be a good source to find tumor markers.This study used matri-assisted laser desorption/ionization time of flightmass spectrometry(MALDI-TOF-MS) to analyze the different protein express inTIF between liver cancer and adjacent normal tissue from surgical removal ofthe liver cancer patients. Then AAT and PSMA1were screened as candidatebiomarkers. Afterwards, protein AAT and PSMA1were validated by proteomictechnology.Part one Two-dimensional gel electrophoresis and MS analysis ofprotein in TIF of PHC.Objective: To analyze the liver cancer and adjacent tissues TIFdifferentially expressed proteins by proteomic technology and screen thecandidate biomarker proteins.Methods: TIF of liver cancer and adjacent tissues were collected from8liver cancer patients.Two-dimensional(2D) gel electrophoresis andMALDI-TOF-MS technique were used to screen the protein biomarkers. Thencandidate biomarkers proteins were screened based on the bioinformaticsanalysis of protein peptide mass fingerprint and Mascot NCBI database.Result:32differentially expressed proteins were found in TIF betweenliver cancer and adjacent normal tissue,18proteins were up-regulated in livercancer tissue.14proteins were down-regulated in liver cancer tissue. ProteinAAT was obviously up-regulated and protein PSMA1protein was obviouslydown-regulated in liver cancer tissue.Conclusion: The study successfully identified differentially expressedproteins AAT and PSMA1between theTIF of liver cancer and adjacent normaltissue. The AAT and PSMA1proteins may be the biomarkers of liver cancer. Part two Expressed validation of candidates biomarker proteins in the celllines and tissue of liver cancer patients.Objective: To validate protein and gene expression of AAT and PSMA1proteins.Methods: In-cell western and RT-PCR were used to detect the protein andmRNA levals of AAT and PSMA1in normal liver cell line HL-7702,hepatomacarcinoma cell line SMMC-7721and HCCLM6. Immunohistochemistrytechnique was used to detect AAT protein level between liver cancer andadjacent normal tissue.Result:1. The results of in-cell western showed that AAT protein wasup-regulated in HCCLM6cell line than in HL-7702and SMMC-7721cell lines(P<0.01).It had no statistical significance between HL-7702and SMMC-7721cell lines (P>0.05).PSMA1protein were down-regulated in SMMC-7721andHCCLM6cell lines than in HL-7702cell line(P<0.01).PSMA1protein wasdown-regulated in HCCLM6cell line than in SMMC-7721cell line (P<0.01).2. The results of RT-PCR showed that AAT mRNA level in SMMC-7721cell line was up-regulated than in HL-7702cell line.The relative transcript levelwas1.49.AAT mRNA level in HCCLM6cell line was down-regulated than inHL-7702cell line.The relative transcript level was0.51. PSMA1mRNA levelwere down-regulated in SMMC-7721and HCCLM6cell lines than in HL-7702cell line.The relative transcript level respectively were0.52and0.57.3. The results of immunohistochemistry showed that AAT protein in livercancer tissue was significantly higher than that of the adjacent carcinomatissue(P<0.01). The AAT protein of different clinical features in liver cancertissue had no differences(P>0.05). Conclusion:1.Protein AAT in hepatitis B virus infection model of livercancer was significantly higher than that of the normal. Protein AAT may beused as a biomarker of the hepatitis B virus infection model of liver cancer.2.Protein PSMA1was down-regulated in liver cancer patients than that ofthe norma.It can be used as a biomarker of liver cancer.