Application Of Cross-species Screening Strategy To Explore Crucial Molecule(s) For Hepatocarcinogenesis And Verification Of Candidate E-FABP At MRNA And Protein Level

Objective:Applying cross-species screening strategy to explore the crucial molecule(s) for hepatocarcinogenesis, verifying candidate's differential expression and related function, by which to locate the key molecule(s) that could be used as the targets for preventing or treating hepatocellular carcinoma (HCC) and provide basic clue for understanding the mechanism of HCC.Method:1. By collecting the data on differentially expressed genes and proteins in HCC of tree shrew and rat that were achieved by using gene chips, two dimensional electrophoresis (2-DE) and mass-spectrometric technique in this laboratory previously, and searching the literatures related to differentially expressed gene or protein of HCC in MEDLINE, CNKI and Chongqing VIP databases, to establish a data set that included all the up-to-date HCC-related genes and proteins differentially expressed in different species.2. Screened out firstly four candidates including E-FABP and then verified their expression in human HCC, Para-HCC and normal liver tissues by RT-PCR.3. Selected E-FABP that showed the most obvious differential expression. Apply RT-PCR to further verify its differential expression in a larger sample size including HCC, Para-HCC and normal liver samples of human, tree shrew and rat. Then Western blot and immunohistochemistry method were applied to verify the expression of E-FABP protein in HCC, Para-HCC and normal liver samples from human, tree shrew and rat.4. Analyzed the relationship between the expression of E-FABP and the clinicopathologic characteristics of HCC patients, as well as the serum AFP level of HCC patients.Result:1. A data set was constructed which contains 45 HCC-related molecules that were reported from researches on sample of human, tree shrew, rat and mouse.2. Four candidate molecules, whose differently expressed level were more obvious, were selected firstly which included epidermal-type fatty acid binding protein (E-FABP), liver-type fatty acid binding protein (L-FABP), transketolase (TKT) and cytokeratin 8 (CK8). RT-PCR result showed that among the four candidates, the different expression of E-FABP was the most obvious.3. On a larger sample size which included 60 cases of human HCC/Para-HCC and 15 cases of normal liver tissues,20 cases of tree shrew HCC/Para-HCC and 15 cases of normal liver tissues, and 10 cases of rat HCC/Para-HCC and 10 cases of normal liver tissue, the result from RT-PCR on E-FABP mRNA was consistent with the former. The result of Western blot and immunohistochemistry on E-FABP protein were consistent with the result of E-FABP mRNA in the samples from human, tree shrew and rat. Both E-FABP mRNA and E-FABP protein level were up-regulated in HCC tissues from all the three species.4. The results of analyzing the relationship between expression of E-FABP and clinicopathologic characteristics of HCC patients showed that E-FABP might be related with HCC metastasis, and combined detection of E-FABP and serum alpha-fetoprotein (AFP) might improve the early detection rate of HCC.Conclusion:1. The expression level of E-FABP is higher in HCC than the corresponding Para-HCC and normal liver tissues of human as well as tree shrew and rat, therefore it might play an important role in hepatocarcinogenesis. E-FABP may be applied as a biomarker for predicting HCC metastasis or HCC early diagnosis, and a target for preventing or treating HCC.2. Cross-species screening strategy is expected to accelerate the research on locating the key molecules for human tumors.