Function Study Of Timp-1 In Liver Cancer

Tissue Inhibitor of Metalloproteinases (TIMP) are natural inhibitors of Metalloproteinases in tissues. Maintenance of the balance between TIMPs and MMPs is most important for the homeostasis of ECM. TIMP-1 is the first member discovered in TIMPs family, and with the researches further on, it was found not only to be an inhibitor for MMPs, but also a multi-function molecule.Hepatoma carcinoma is one of the most serious diseases in both China and the world; the diagnosis and therapy are always the focus for researchers. In this thesis, the function of TIMP-1 was studied in a hepatoma carcinoma cell line BEL-7402.1. Using immuno-staining technology, the amount of TIMP-1 was analyzed in HCC, liver cirrhosis and normal liver tissues. The results showed the expression of TIMP-1 in HCC is higher than it in normal livers and the semi-quantitative RT-PCR also support the high expression of TIMP-1 in liver cancer cells.2. The nuclear distribution of TIMP-1 in liver cancer cells was observed. Immuno-staining results showed the nuclear signals both in BEL-7402 and HCC tissues, while GFP subcell study also support the interesting distribution pattern. PCNA was found to be negatively correlated with TIMP-1 nuclear distribution, suggesting the TIMP-1-Nuclear-Positive cells might not be in the active proliferation state. The number of nuclear-located TIMP-1 cells was increased by HiO? treatment. We hypothesized some probability for it; however, the definitive biological significance of this phenomenon remains unresolved.3. The function of TIMP-1 in cell cycle regulation was analyzed. The MTT assay and growth curve results suggest the growth inhibition of TIMP-1 on BEL-7402 and QSG-7701, a cell line derived from peripheral tissue of liver carcinoma, while no similar effects was observed in L-02. Further study on its mechanisms showed that TIMP-1 could upregulate the expression of p21WA ' reporter in luciferase assay, suggesting that p21WAFI is involved in the growthinhibition. The results are also supported by the semi-quantitative RT-PCR research.4. In both Wound Assay and single cell migration detection assay, TIMP-1 was found to decrease the migration ability of BEL-7402 cell. To explorer the mechanism, the variation of distribution pattern and structure of F-actin by TIMP-1 was monitored, but no obvious change could be detected in cytoplasm and nuclear regions between TIMP-1 transfected cells and untransfected cells.5. TIMP-1 was found might to be a signal molecule. By upregulating p21WAF1, TIMP-1 is at least partly involved in the cell proliferation inhibition by IL-6. MTT assay and growth curve results both suggested that IL-6 could inhibit BEL-7402 proliferation. Luciferase Assay revealed that in BEL-7402 cell, IL-6 could upregulate TIMP-1 by JAK-STAT3 signal pathway, and TIMP-1 could inhibit cell growth by p21NVAFI as we mentioned previously, so TIMP-1 is at least partly involved in the proliferation inhibition by IL-6.6. The function of TIMP-1 was further studied in vivo. The preliminary study on nude mice also support a of suppress-carcinoma function of TIMP-1.