Study of differentially expressed genes in human hepatocellular carcinoma and the adjacent non-tumorous liver.

摘要

Hepatocellular carcinoma is one of the most common cancers in the world. Altered expression of a wide spectrum of genes has been associated with the development of hepatocellular carcinoma. However, the molecular mechanism(s) of hepatocarcinogenesis is not yet clear due to insufficient knowledge of the related genomic abnormalities. Using mRNA differential display technique, a 386 bp transcript was shown to be expressed at higher level in hepatocellular carcinoma as compared to the corresponding non-tumorous liver samples. Nucleotide sequence analysis revealed that this transcript had 99% similarity with the mRNA encoding human homogentisate 1,2-dioxygenase, the enzyme that converts homogentisate to maleylacetoacetate in the phenylalanine/tyrosine catabolic pathway. Northern analysis using HepG2 cell RNA further confirmed that this transcript and homogentisate 1,2-dioxygenase mRNA are identical. Elevated expression of homogentisate 1,2-dioxygenase mRNA was found in most of the tumors by RNA dot blot analysis. The activity of homogentisate 1,2-dioxygenase was also significantly elevated in hepatocellular carcinoma as compared to the non-tumorous liver while glyceraldehyde-3-phosphate dehydrogenase activity remained similar. To further study the involvement of homogentisate metabolism in hepatocarcinogenesis, the activity of fumarylacetoacetate hydrolase, the enzyme downstream of homogentisate 1,2-dioxygenase, converting fumarylacetoacetate into acetoacetate and fumarate, was investigated. Decreased activity of this enzyme was found in most of the tumors. Based on this small series of study, there was also a trend for increasing of the ratio of the activities of homogentisate 1,2-dioxygenase to fumarylacetoacetate hydrolase in tumor versus the non-tumorous liver in more advanced tumors. In the presence of homogentisate, the establishment of homogentisate metabolism in NIH 3T3 cells by the overexpression of homogentisate 1,2-dioxygenase led to several transformed phenotypes, i.e., decrease in population doubling time, increase in colony-formation efficiency and anchorage-independent growth in soft agar. These studies suggest that an abnormal homogentisate metabolism exists in the course of hepatocarcinogenesis and may play an important role during tumor formation. The results presented in this thesis will lead to a better knowledge of the genetic and epigenetic abnormalities related to hepatocellular carcinoma.