The Study Of Gene Mutation And Alternative Splicing In Primary Hepatocellular Carcinoma Using DNA Sequencing And Oligonucleotide Chip

OBJECTIVES: To study the mutation of beta-catenin , p53 gene and the alternative splicing of DNMT3b, hTERT gene in primary hepatocellular carcinoma, and their effects on the development of hepatocellular carcinoma.METHODS: 30 hepatocellular carcinoma (HCC) tissues and their correspounding adjacent para-cancerous tissues, 10 normal liver tissues were examined ?RT-PCR, DNA sequencing and oligonucleotide chip were used to detect the mutation of p53 , beta-catenin gene and the alternative splicing of DNMT3b, hTERT gene. The relationship between the results and clinicopathological characteristics of HCC was also analyzed.RESULTS: RT-PCR showed that beta-catenin gene expression was detected in 96.7%(29/30)of HCC , 96. 7%(29/30)of para-cancerous tissues and al 1 normal liver tissues . There was a higher expression in HCC than in para-cancerous tissues and normal liver tissues. p53 gene expression was detected in 93. 3%(28/30)of HCC , 93. 3%(28/30)of para-cancerous tissues and 90. 0%(9/10) of normal liver tissues .There was no significant difference of the expression among HCC and other two groups. DNMT3b gene expression was detected in the forms of alternative spliced variant (DNMT3bl, DNMT3b3, DNMT3b4, DNMT3b5),their total expression were 93. 3%(28/30) in HCC, 93. 3%(28/30) in para-cancerous tissues and 100.0%(10/10) in all normal liver tissues. DNMT3b4 expression was higher in HCC than in para-cancerous tissues .hTERT gene expression was detected in the forms of alternative spliced variant(full-length transcripts, a -splicing variant P -splicing variant , and a P -splicing variant), their total expression were 77. 3%(22/30) in HCC , 43. 4%(13/30) in para-cancerous tissues and 30. 0%(3/10) in normal liver tissues. The expression of full-length transcripts of hTERTgene was higher in HCC than in para-cancerous tissues and all normal liver tissues. P-splicing variant expressed in the most cases of para-cancerous tissues and all normal liver tissues.DNA sequencing showed the mutation rate of beta-catenin gene was 43. 8% (7/16) in HCC , and the mutation sites focused in code 33, 37, 40, 45, 50. Beta-catenin gene mutation correlated with the high expression in HCC,but not correlated with the pathological differentiation and clinical CLIP valuation. The mutation rate of p53 gene was 15. 4%(2/13) in HCC, and the mutation sites focused in code235, 248, 278.Using Oligonucleotide chip hybridization , the signal corresponding to the mutation of TCT桾TT was detected in code 45 of beta-catenin gene . No mutation was detected in other sites of beta-catenin and all designed sites of p53 gene . The signals corresponding to 4 of alternative variants of DNMT3b gene and hTERT gene were all detected.CONCLUSIONS: Beta-catenin gene mutation and overexpression in HCC may have a critical role in malignant progression of HCC. DNMT3b4, one of the alternative spliced forms of DNMT3b, may play an important role in HCC. The overexpression of hTERT gene and reexpression of full-length transcripts may contribute to hepatic carcinogenesis.