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Title: Inhibition mechanism of a newly cloned candidate tumor suppressor gene JST during hepatocarcinogenesis and its abnormal expression in human hepatocellular carcinoma from Qidong liver cancer risk area, China.
Author: Dong-Dong L, Xi-Ran Z.
Journal: Hepatogastroenterology; 2004; 51(56):515-25. PubMed ID: 15086194.
Abstract:
BACKGROUND/AIMS: To study inhibition effect of a newly cloned candidate tumor suppressor gene (JST) during hepatocarcinogenesis and its normal expression in human hepatocellular carcinoma from Qidong liver cancer risk area, China. METHODOLOGY: By preparing rabbit anti-human JST polyclonal antibody, constructing of JST frameshift mutant and exploring RT-PCR, in situ hybridization, immunohistochemistry, Western blot, Northern blot, cDNA expression microarray, co-immunoprecipitation and the tumorigenicity assay in vivo and in vitro, gene treatment, etc, JST gene expression and inhibition tumor growth effects were analyzed, including 150 pairs of HCC specimens and their adjacent para-cancerous tissues, 8 cases of normal liver tissues and QGY7701, HepG2, Hep3B cell line. Its relationship with the invasiveness of HCC from Qidong was also investigated. RESULTS: Our results showed that there is expression difference for JST between liver cancer and para-cancerous tissue and the results of RT-PCR, in situ hybridization, immunohistochemistry, Western blot, Northern blot suggested that it is a down-regulation gene. The labeling index (LI) of cancer tissue and para-cancerous tissue was (70.2+/-8.7) and (9.4+/-2.8) respectively (p<0.01), lower LI was closely related with invasiveness of HCC, decreased expression of JST was also shown by Western blotting. Results of RT-PCR showed the JST gene expression index (EI) of HCCs was lower than that of para-cancerous tissue and normal liver tissue and there are some sequence differences between cancer and para-cancerous tissues. Northern blot showed JST having down-regulation expression among 92.20% (138/150) of patients. Using in situ hybridization, the signal corresponding to JST mRNA was particularly weak in cytoplasm of HCC when compared with those of para-cancerous and normal liver tissues. Less expression of JST was also found to be correlated with high metastasis potentiality of HCC. JST overexpression inhibits DNA synthesis and apoptosis in QGY7701 cells. QGY7701 cell transfected with JST is more inhibited in soft agar than that of vector transfected control cells (p<0.01) or QGY7701 cells stably transfected with the JST frameshift mutant. The tumor formation is more inhibited in the QGY7701-pcDNA3.1-JST group than that in the QGY7701-pcDNA3.1, QGY7701-pcDNA3.1-JST frameshift mutant group. cDNA expression microarray showed expression differences of 10% (20/200,18 up-regulation; 2 up-regulation) tumor genes were considered significant between QGY7701-pcDNA3.1-JST and QGY7701-pcDNA3.1. Using a co-immunoprecipitation approach, intracellular binding of JST and p53 was found. Higher levels of p53 were detected following infection with pcDNA3.1-JST when compared with pcDNA3.1. Induction of FasL could be demonstrated in Hep3B and in HepG2 cells following transfection pcDNA3.1-JST, but not following transfection pcDNA3.1. CONCLUSIONS: JST is a putative tumor suppressor gene. Overexpression of JST gene may contribute to inhibiting the occurrence, advancement and invasiveness of HCC from Qidong, a high risk area in China.

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