Screening Of Differentially Expressed Genes In Rat Hepatocarcinogenesis Induced By Aflatoxin B1 And Study On The Candidate Gene Cullin7

Hepatocellular carcinoma (HCC) is one of the most common malignancies with difficulty in early diagnosis and extremely poor prognosis. As the formation of HCC is a multi-factor, multi-step and multi-gene process, the study on HCC with the methods that only focus on single factor or single gene can not accurately reflect the complexity of its pathological mechanism.In recent years, with the rapid development of the functional genomics and the proteomics,the research of HCC in pathogenesis , diagnosis and treatment have been greatly promoted.This study applied gene array technology to compare the differentially expressed genes among the normal tissue, preneoplastic tissue and HCC tissue during rat hepatocarcinogenesis induced by Aflatoxin B1 (AFB1). The preneoplastic tissue was distinguished byγ-glutamyl transpeptidase (γ-GT) staining which marks the foci of liver cell proliferation positive. Among the candidates, the changes of Cullin7 expression were confirmed by Western blot and RT-PCR. The biological functions and mechanism of Cullin7 were further studied with RNA interference (RNAi) technique. Its expression in a larger scale of human HCC samples and the clinical significance were studied by RT-PCR and immunohistochemical techniques.The results indicated that Cullin7 probably affects cell proliferation and apoptosis by regulating the cell cycle, and thereby plays a role in the occurrence and development of HCC.Cullin7 codes the component of the E3 sumo-protein ligase,which involves in sumo depended protein degradation.The up-expression of Cullin7 in HCC indicates that Cullin7 could be a molecular marker for the early diagnosis and the prognosis analysis of HCC.The entire study includes three parts.Part One Study on differentially expressed genes in the rat hepatocarcinogenesis induced by AFB1Objective: Part one applied gene array technology to compare the differentially expressed genes among the normal tissue, preneoplastic tissue and HCC tissue during rat hepatocarcinogenesis induced by Aflatoxin B1 (AFB1).Methods: Male Wistar rats, 4 weeks old, were divided into AFB1 group and control group. Rats in AFB1 group were injected AFB1 in order to induce HCC, and the rats in control group were raised normally. The animal experiment lasted 64 weeks, during which all the animals from each group were biopsied periodically for collecting liver tissues samples(14W,28W,42W,55W,64W). The samples were stained withγ-GT and HE to distinguish the normal, preneoplastic and HCC tissue.3 samples were picked randomly from each kind of above tissues for extracting the total RNA.Gene array technology was applied to screen the differenetially expressed genes.The molecular function,biological process,cell component,and the biological pathways were analyzed by the Gene Ontology,KEGG and NCBI database.Results: The animal experiment was end in 64 weeks. A total of 19 rats in AFB1 group developed HCC until the end of animal experiment. The earliest HCC occurred at the 51st week in the animals in AFB1 group, while none in control group. With the extension of time by AFB1-induced HCC, the number and size ofγ-GT foci in the liver of animals in AFB1 group increased, and the totalγ-GT positive area reached 29%～43% of the whole liver tissue area. HCC tissues wereγ-GT positive entirely. There was noγ-GT focus in control group.The results of gene array (limit differenece times≥2.0 or≤0.5)showed that 3753 genes expressed upwards while 3042 genes downwards in the HCC and normal group, 4885 genes expressed upwards while 3447 genes downwards in the HCC and preneoplastic group, 1027 genes expressed upwards while 1117 genes downwards in the normal and preneoplastic group. Though the bioinformatics, the conclusion suggested that such genes were relate to the cell proliferation and apoptosis or the signal pathway.Conclusion: The results showed the methods thatγ-GT staining combined with HE were able to distinguish the normal, preneoplastic and HCC tissue.The significant difference of gene expression existed in the developing stage of HCC. The happening and development of HCC may be related to the change of gene expression.Part two Validation on differential expression of Cullin7 in rat and human HCCObjective: To verify the candidate Cullin7 expression in rat and human HCC.Methods: The changes of Cullin7 expression were confirmed by Western blot and RT-PCR.Results:①mRNA and protein expression of Cullin7 increased in the rat normal, preneoplastic and HCC tissue.②Cullin7 protein expression also turned up in the human normal, preneoplastic and HCC tissue. Conclusion: Above results from mRNA and protein levels were consistent with the gene microarray, which indicates Cullin7 may be involved in the development and progression of human HCC.Part three Biological function and mechanism study of Cullin7 in hepatocarcinogenesisObjective: To explore the role of Cullin7 in hepatocarcinogenesis, further study on its biological function and mechanism was carried on by RNAi technique on the HCC cell line of SMMC-7721.Methods: Chemically synthesized small interfering RNA (siRNA) targeted on Cullin7 were transfected into SMMC-7721 cells. mRNA and protein levels of the Cullin7 in HCC cell lines SMMC-7721 were examinated by RT- PCR and Western blot.Establishing the RNAi,negtive and control group,the function and mechanism study were designed to analyze the change of apoptosis, proliferation, adhesion, and invasion of the above three cell groups.Results: Flow cytometry analysis showed that the proportion of apoptotic cells in the RNAi , Mock and Con group were 67.45±5.83%, 13.74±4.25%, 16.69±3.18% respectively. MTT results showed that the value of OD490 RNAi group in 24h, 48h, and 72h were 0.941±0.04, 0.924±0.13, 0.783±0.12 respectively. Movement test in vitro shows that the porous cells in the RNAi, negtive and control group were 26.35±1.55, 68.33±3.55 and 65.77±3.18. Cell invasion assay showed the average invading cell numbers perfield in the group RNAi, mock and control were 14.38±2.37,45.38±4.16,and 48.67±2.51 respectively. There was siginificant difference between RNAi and the other two groups.Conclusion: By promoting cell proliferation, inhibiting cell apoptosis, Cullin7 may be lead to the transformation from normal cells to the malignant.Part four The expression and clinical significance of Cullin7 in a larger scale of human HCC samplesObjective: To analyze the clinical relationship between Cullin7 expression and human hepatocellular carcinoma .Methods: To explore the clinical significance of Cullin7 expression in HCC and evaluate the potential value of Cullin7 as a new molecular marker for diagnosis early HCC, the Cullin7 protein expression level was examinated in 62 cases of human HCC and the adjacent liver tissues, as well as in 12 cases of normal human liver tissue, by Real-Time PCR and immunohistochemical techniques.Results: The results showed the expression level of Cullin7 was up-regulated in all kinds of tissues. There was siginificant difference between human HCC and the adjacent liver tissues (p<0.05), and no siginificant difference between the adjacent liver tissues and normal liver tissues. The Cullin7 level had significant correlation with Edmondson stage, recurrence and metastasisof the HCC cases.(p<0.05).Conclusions: Cullin7 expression turned up in the HCC tissue,which indicates that Cullin7 could be a molecular marker for the diagnosis and the prognosis analysis of HCC.