Discovery Of Biomarkers Form Serum In Hepatocellular Carcinoma Using SELDI-TOF-MS Protein Chip Technique

0bjective: 1.To evaluate the expression of AFP and GPC3 gene and the value of diagnosis in hepatocellular carcinoma using Reverse transcriprase polymerase chain reaction(RT-PCR) method.2. To screen serum biomarkers in patients with hepatocellular carcinoma by using surface-enhanced laser desorption and ionization time-of-flight mass spectrometry(SELDI-TOF-MS)technique and to evaluate its clinical implication in the patients whose alpha-fetoprotein are negative in the sera.Method:1.Isolationing Total RNA in 20 pairs of the tumors and the surrounding nontumor liver tissues(1cm,5cm) ,then analyzing the expression of AFPmRNA and GPC3mRNA by using RT-PCR.2.Proteomic spectra were generated by mass spectroscopy in 118 cases,including 59 cases of hepatocellular carcinoma,and 59cases of healthy control.Among the 59 patients ,18 were AFP-negative hepatoma. The consequence was analyzed and the characteristic proteomic peaks was selected by using Biomarker Wizard . The modle of diagnosis was built by using Biomarker Patterns Systemspectra software. Relationship between the proteomic peaks and clinicopathological features was investigated.Results: 1.The expression of AFPmRNA and GPC3mRNA in the tumors was significantly higher than in the surrounding nontumor liver tissues.AFPmRNA was detectable in 14 cases of 20 HCCs(70%),and GPC3mRNA was 15 (75%).The expressions o f at least one of the two genes was elevated in 80.0% of all the HCC tissues.There were 2 cases of AFPmRNA and GPC3mRNA found in the surrounding nontumor liver tissues. AFPmRNA was related with the serum AFP ,while GPC3mRNA was irrelated to the serum AFP.The expression of GPC3mRNA is higher than AFPmRNA in HCCs that measured <5cm in diameter. 2.There were four distinguished proteomic peaks were detected in HCCs,which were all downregulated.The results yielded a sensitivity of 76.92%,66.67%,76.92%,89.74%, specificity of 92.31%,92.31%,89.74%,79.49% by the four proteomic peaks.The 18 cases of AFP-negative hepatoma were correctly diagnosed by 61.11%,72.22%,72.22%,88.89%.The peaks was not correlated with age, sex, HbsAg, AFP value ,tumor body size, pathology grading and cirrchosis in hepatocellular carcinoma.Conclusion:1.The expression of AFPmRNA and GPC3mRNA in the tumors was significantly higher than in the surrounding nontumor liver tissues. GPC3mRNA was irrelated to the serum AFP and might be a new genetic marker of HCC.2. There are significant differences between the serum of hepatocellular carcinoma and normal groups.SELDI-TOF-MS offers a unique platform for the proteomic detection of hepatocelllular carcinoma.It also offers an auxiliary diagnosis method to the patients whose alpha-fetoprotein are negative in the sera.