| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 99 === Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, and a leading cause of death in many countries. The epidemiology of HCC has marked demographic and geographic variations, occurring mainly in Africa and Asia.
The long-term object of studies in our lab is to investigate the molecules and pathways involved in regulation of apoptosis evasion in human hepatoma cells. We have used optimized UV to induce cellular hermetic responses, and identified both the proteins and pathways regulating cellular death（Hsieh et al., J Proteome Res 2009）.
Of them, the Histidine triad nucleotide binding protein 1 (HINT 1) was of particularly interesting, since silencing of its expression endowed cells with resistance to
UV-induced cell apoptosis. Since apoptosis evasion is an essential process for carcinogenic tumor formation of cells, HINT1 is a candidate tumor suppressor gene for
human cancers inducing HCC. We thus hypothesized that HINT1 is a tumor suppressor gene playing significant roles in hepatocellular carcinogenesis.The aims of this study were
1) to examine whether there was any evidence showing loss of pro-apoptotic function of HINT1 in human HCC；2) to examine whether there was down-regulation of HINT1 in
HCC；3) to find any mutations that led to loss of HINT1’s function. First, we used XTT assay by ionic radiation compare cell sensitivity to apoptosis induction before and after silencing the expression of HINT 1. Our data showed that when HINT 1 had been silenced, HCC cells became more sensitive to apoptosis in Mahlavu and Hep 3B, but not
in SK-Hep 1、Hep G2、Tong and Huh7. Second, we used both quantitative polymerase chain reaction and immunoblotting assays to determine the expression level of patients.
Our data showed that HINT 1 was down-regulated in cases 50% and up-regulated HINT1 in 21% patients with hepatoma tissues compare to 5 normal liver tissues. We then
examined whether HINT1 loses its function by inactivation mutation at its mRNA. The entire coding sequences of HINT1 cDNA of six different HCC cell lines were cloned and
sequenced. We found 5 of them（Mahlavu、SK-Hep 1、Hep G2、Huh7 and Tong） had no mis-sense mutation. Only one no-sense mutation was found in Hep 3B cells.
We concluded that loss of pro-apoptotic function of HINT1 was found in some of the HCC cells. The functional loss of HINT1might be attributable to down-regulation of the
expression HINT1,though which was only found in approximate 21% of HCC. Our data did not suggest any mutation related to loss of proapoptotic function of HINT1 in HCC
cell lines.
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