Different Analysis Protein Profile Of Multicentric Occurrence And Intrahepatic Metastasis Of Multinodular HCC

Hepatocellular carcinoma (HCC) is one of the malignant tumor to threaten human's health. Multiple cancer nodules of HCC are frequently detected in the hepatocarcinogenensis. The simultaneous occurrence of multiple HCCs may reflect either the dissemination of malignant cells from a single primary tumour to form satellite tumour nodules (intrahepatic metastasis, IM), or the synchronous development of several independent tumours(multicentric occurrence, MO). The two possible mechanisms of development of multiple HCCs reflect important differences in pathogenesis that appear to have an impact on treatment and prognosis. To distinguish between these two categories of HCC for any individual case that contains multiple tumours requires that the clonality of the individual tumours be determined. The most accurate method to assess the nature of multiple HCCs is to determine whether the various individual tumours are monoclonal or polyclonal. Monoclonality among all of the multiple tumours in a liver indicates that all were derived from a common precursor lesion, whereas disparate clonality of each of the tumours would suggest that each developed as a unique and separate tumour. Distinction between these two categories, that is, MO and IM, in multinodular HCC has conventionally been determined by pathological criteria, but they are relatively subjective. The most precise and specific methods for assessing tumour clonality depend on the detection of common patterns of aberrations in DNA among the various tumours. The classic method to determin the origin of tumour cell is to compare the HBV integration pattern among tumours. Use of the HBV integration pattern can be applied only to livers infected with HBV. Relatively abundant DNA and fresh frozen tissue are required for analysis; although there are other molecular methods to evaluate the HBV integration pattern that require less DNA, their use is not widely adopted. Gene p53 is important tumour suppressor gene. The mutation or deletion of p53 can be to promote hepatocarcinogenensis and development of tumour. Codon 249 (exon 7) of the putative tumour suppressor gene p53 is a mutational hot-spot for hepatocellular carcinoma (HCC) but not other tumors. And the mutation patterns are different from each tumour nodular. In addition, mtDNA contains a non-coding region that includes a unique displacement loop (D-loop), which controls replication and transcription of mtDNA. The D-loop region is responsible for the control of replication and transcription of mtDNA, mutations in this region might cause a decrease in the copy number and/or a repression of gene expression of the mitochondrial genome. It is reported that the mutation of D-Loop region is frequent in HCC. Mutation of D-Loop region seems to be useful for differentiating the origin of multinodular HCC.Presently, there has not been unified gold standard to identified multinodular HCC. Proteomics is the most classical method to analyze protein profile entirety. Here we have used two-dimensional electrophoresis (2-DE) for liver tissue proteomics, followed by matrix assisted laser desorption/ ionization- time of flight mass spectrometry (MALDI-TOF-MS/MS), to screen different proteins between IM and MO. To analysis the protein expression profile of MO and IM of multinodular HCC, so as to improve foundation of molecular typing of MO and IM. 2-DE and mass spectrogram technique could be as to methods of molecular typing of IM and MO. It could be of help in determining clinical therapeutic regimen and judge prognosis to HCC patients.Part 1 THE RESEARCH OF COMPARATIVE PROTEOME IN IM AND MO OF MULTINODULAR HCCObjective: To analysis the protein expression profile of multicentric occurrence (MO) and intrahepatic metastasis (IM) of multinodular HCC, so as to improve foundation of molecular typing of MO and IM.Method: According to the size of nodular, the multinodular of 5 IM and 6 MO were divided into groups of IM1, IM2, MO1 and MO2. Two dimensional gel electrophoresis (2-DE) and mass spectrogram technique were used to analysis protein expression of multinodular HCC.Result: By comparing the 2-DE profiles of IM1, IM2, MO1, MO2, the average number of each group spots was 1053±13 (n=3) (mean±SD), 1127±2 (n=3), 1066±141 (n=3), 1062±73 (n=3), respectively. And the average number of match spots was 771±44,match rate up to 72%. Comparing IM1and IM2, IM1 and MO1, MO1 and MO2, IM2 and MO2, Image master 6.0 software showed a more than 2-fold difference in intensity between the two mixtures with statistical significance and identified by MALDI-TOF-MS to be 25 proteins after de-redundance (protein scored≥64). We found these changeable patterns as follows: the difference of S100 calcium-binding protein A8 was only in MO, over-expressed in MO1 and down-expressed in MO2; Chain A, Crystal Structure Of Lipid-Free Human Apolipoprotein A-I is special in MO2; Both Acy1 andβ-actin were down-expressed in IM and over-expressed in MO; HSP27 and selenium binding protein 1 were different from IM1 and MO1; Transferrin and Chain B, Cathepsin D were different from IM2 and MO2; and so on. The gene ontology classification displays that the proteins were associated to cell movement, signal transduction, oxidoreduction, lipid metabolism, amino acid metabolism and so on.Conclusions: The protein profiles were different to IM and MO, 2-DE and mass spectrogram technique could be as to methods of molecular typing of IM and MO. It could be of help in determining clinical therapeutic regimen and judge prognosis to HCC patients, though further validation is needed.Part 2 VERIFICATION ON DIFFERENTIAL EXPRESSION OF IM AND MO OF MULTINODULAR HCCObjective: To verificate the mass results of identificating IM and MO. Method: 8 HCC IM nodular from 4 IM HCC patients, and 8 HCC MO nodular from 4 MO HCC patients. Every patient's 2 nodular were different on size. We used western blot to confirm the 2-DE results that showed the expressions of HSP27 and L-FABP. Reference protein was GAPDH.Result: The results from western blot for HSP27 and L-FABP, confirm to the 2-DE results that showed the expressions of HSP27 and L-FABP in four groups of HCC tissue. As to compare with MO2, L-FABP was low-expression in IM2 and MO1. HSP27 was up-expression in MO1 and low-expression in IM1.Conclusions: We validated the change on L-FABP and HSP27 protein expression levels in IM1, IM2, MO1, MO2. The results of western blot were with one accord the mass results.Conclusion1. We established and optimized a 2-DE and mass spectrogram technique for 4 HCC groups of IM1, IM2, MO1, MO2, and identified twenty-five proteins by MALDI-TOF-MS.2. By mass spectrum, some proteins were found, such as HSP27, PrxⅡ, L-FABP, Acy1 and so on. They may be special biomarkers to identificate IM and MO.3. It is feasible to appreciate IM and MO from complicate HCC tissue by comparison proteomics technique. And it is ideal to find the change of HCC proteome influenced by many factors. This technique can widen the idea to identify IM and MO. Novelty of this project1. The molecule typing method that 2-DE and MALDI-TOF-MS technique identify IM and MO was firstly reported in four groups. These proteins play some roles in four groups were firstly validated by our works.2. By applicating proteomics technique, the proteins expression disparation among different nodular of multinodular HCC was firstly researched in China by our works. The 25 proteins that were appraised by 2-DE were used to distinguish IM and MO.The potential application of this work1. 2-DE and MALDI-TOF-MS technique could be a method to identify IM and MO, and to make up the defect of the molecule typing reported.2. We found some proteins biomarks that could distinguish IM and MO of multinodular HCC by 2-DE and MALDI-TOF-MS technique. This could help doctors to make evaluation before treatment, to determate treat scheme and to pretest survival rate of post-treatment. It could play a role to raise the treat level of multinodular HCC.