| Objective: To screen out the growth factor interacted with GPC5 in prostate cancer, and preliminarily explore the potential mechanism of their interaction on the regulation of tumor progression.Methods: 1.Using CO-immunoprecipitation and mass spectrometry, to selecte out the growth factor interacted with GPC5 in prostate cancer cells. 2.Taking advantage of immunofluorescence confocal technique, to detect the distribution of GPC5 and the target protein in the cell substructure of prostate cancer cells. 3.Using CO-immunoprecipitation to extract GPC5-target protein complexes, after Western blotting, to verify their interaction between GPC5 and its target protein. 4.Utilizing plasmid DNA transfection technique in the help of liposome, to establish prostate cancer cell lines of the abnormal expression of GPC5. 5.Taking advantage of Western blot technology, to detecte the content change of intracellular target protein after the abnormal expression of GPC5 in prostate cancer cells.Results: 1.CO-immunoprecipitation complexes of GPC5 find three kinds of growth factors: HDGF, IGF2BP1, GRB2, among which HDGF is detected in three samples, and it has high protein score. 2.Immunofluorescence Confocal results show that there is widely overlapping distribution between GPC5 and HDGF in the cytoplasm. 3.HDGF was detected by Western blot in co-immunoprecipitation complexes induced by GPC5, and GPC5 was also detected by Western blot in co-immunoprecipitation complexes induced by HDGF. 4.Establish a stably transfected prostate cancer PC3 cell line of GPC5 overexpression.5.Because of GPC5 overexpression, the content of HDGF decreased and the content of its downstream protein such as PI3-K and SERPINE1 increased in prostate cancer cells.Conclusion: 1.HDGF is a target of GPC5 in prostate cancer. 2.GPC5 can decrease the intracellular HDGF level in prostate cancer, which indicates that GPC5 may inhibit the transmembrane signal transduction of HDGF. |
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