| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 96 === Lung cancer is the most common cause of cancer deaths in the world, and the incidence is increasing each year. Since 1982, cancer has been the major cause leading to mortality for cancer patients that domestic ten major causes of the death ranked. In the previous studies, we have identified and characterized a novel tumor/invasion suppressor gene HLJ1 (human liver DnaJ-like protein, also called DNAJB4) in a lung cancer cell line model with different invasion capability (CL cell lines). HLJ1 is a member of Hsp40 family, and heat shock proteins are commonly involved in the essential defense mechanism for protein folding and assists cell against stress. According to the previous studies, over-expression of HLJ1 protein in lung cancer cell could reduce the abilities of proliferation, colony formation, invasion and migration. However, the suppressive mechanism of HLJ1 is unclear. Therefore, the objective of this study is to investigate and characterize the proteins interacting with HLJ1. To obtain the novel HLJ1-binding protein, the different density of tumor cell culture is employed to mimic metastatic characteristics in vitro. We have identified two proteins, p54nrb and PSF, that can interact with HLJ1 in high cell culture density by immunoprecipitation assay and MALDI-TOF analysis. The complex of HLJ1 and p54nrb was confirmed in vitro and in vivo by co-immunoprecipitation, GST pull-down and mammalian two-hybrid assays. In addition, the results of GST pull-down assay indicated that p54nrb can interact with HLJ1 through HTH motif. We also found that the expressions of both HLJ1 and p54nrb are increased in A549, CL1-0, CL1-5 and CL1-5F4 cancer cells while are culturing in high cell density. The results of immunofluorescent and cytosolic/nuclear protein fraction revealed that p54nrb is mainly located in the nucleus. Furthermore, we find that over-expression of p54nrb protein in highly invasion lung cancer cell line, CL1-5, can reduce cancer cell proliferation, colony formation, invasion and migration. On the other hand, p54nrb is decreased when most cells are in DNA synthesis phase. Once over-expression of p54nrb in CL1-5 cell line, the cell cycle would arrest in G1 phase to reduce cancer cell proliferation. Investigating the novel molecular mechanism about HLJ1-p54nrb interaction is important, it may not only provide a new insight to understand the pathway of metastasis and growth regulation but also be the new target molecule for anti-cancer therapy in the future.
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