P75NTR Inhibits Growth Of Hepatocellular Carcinoma

The nerve growth factor receptor p75 (p75NTR), which contains 427 amino acids, is a low-affinity receptor of neurotrophin. P75NTR plays an important role not only in the development of nerve system, but also in the carcinogenesis and development of cancers.Previous studies have shown that p75NTR has been identified as a potential tumor suppressor associated with growth inhibition, which could negatively regulate cell growth and proliferation in prostate cancer, however, it was also reported that p75NTR acted as a survival receptor in brain-metastatic melanoma cells. This results indicated that p75NTR play different roles in different kinds of tumors and cell context. Our previous showed that the expression of p75NTR was significantly decreased in gastric cancer tissues than the adjacent noncancerous counterparts, and p75NTR could inhibit the invasion and metastasis of gastric cancer.Until now, it is unclear whether p75NTR was expressed in hepatocellular carcinoma and the effects of p75NTR on cell proliferation and apoptosis in hepatocellular carcinoma is still unknown. Our purposes in this study are as following:【Objectives】(1) To investigate the expression of p75NTR in hepatocellular carcinoma tissues and the adjacent noncancerous counterparts. (2) To study the relationship between p75NTR and growth of hepatocellular carcinoma by expressional and functional studies; (3) To examine the possible mechanisms of cell cycle arrest in hepatocellular carcinoma induced by p75NTR.【Methods】(1) The expression of p75NTR protein in hepatocellular carcinoma tissues and their adjacent noncancerous counterparts was investigated by immunohistochemistry assay. (2) The expression of p75NTR protein in hepatocellular carcinoma tissues and their adjacent noncancerous counterparts was measured by Western blot. And the expression of p75NTR protein in hepatocellular carcinoma cell lines and normal liver cell lines was also measured by Western blot. (3) Construct the siRNA vector of p75NTR, transfect it into Chang and select stable clones by G418 screening. Construct the sense expression vector of p75NTR, transfect it into HepG2 and select stable clones by G418 screening. (4) Perform Western blot on different stable clones to identify the effect of transfection. (5) Depict the growth curves of the transfected cells and the control cells. (6) Flow CytoMeter (FCM) is used to identify the cell cycle distribution of these cells. (7) To study the effect of p75NTR on cell apotosis, Hoechst/PI staining and Annexin V flow cytometric experiments were performed. (8) Cloning formation assay on soft agar is applied to test the in vitro tumorigenesis of the transfected HCC cells. (9) Tumor xenograft in nude mice is performed to test the in vivo tumorigenesis of the transfected HCC cells. (10) Identify the expression of the cell cycle associated molecules (cyclin D1, Rb, p-Rb, and PCNA) by Western blot.【Results】(1) The expression of p75NTR was decreased significantly in hepatocellular carcinoma tissues as compared with their adjacent noncancerous counterparts, and its expression levels were correlated with the degree of tumor differentiation. (2) The expression of p75NTR was also significantly decreased in various human hepatocellular carcinoma cell lines. (3) The siRNA vector of p75NTR was successfully constructed and transfected into Chang cells. The expression vector of p75NTR was also successfully constructed and transfected into HepG2 cells. The stable clones were selected and identified. (4) The growth of the p75NTR-siRNA transfected Chang cells was accelerated, but the growth of the p75NTR-expressing vector transfected HepG2 cells was slowed down. (5) The FCM indicated that p75NTR inhibited hepatocellular carcinoma cells entering S phase from G1 phase and the proliferative index was decreased. (6) The results of FCM and Hoechst33258/PI staining showed that p75NTR induced apoptosis in hepatocellular carcinoma cells. (7) The cloning formation assay showed that the clone formation rate of p75NTR-expressing vector transfected HepG2 cells was lower than the control cells. (8) The tumorigenicity of p75NTR-expressing vector transfected HepG2 cells was reduced compared with the control cells. (9) Western blot revealed that up-regulating p75NTR could down-regulate the expression of cyclin D1, p-Rb and PCNA, but up-regulate the expression of Rb. Conversely, the results were inverse when p75NTR was down-regulated by specific siRNA.【Conclusion】 (1) The expression of p75NTR was decreased significantly in hepatocellular carcinoma tissues as compared with their adjacent noncancerous counterparts, and its expression levels were correlated with the degree of tumor differentiation. (2) P75NTR can inhibit the proliferation and induce apoptosis of hepatocellular carcinoma cells. (3) P75NTR can induce G1/S phase arrest throgh down-regulating the expression of cyclin D1, p-Rb and PCNA and up-regulating the expression of Rb.