The Impact Of EZH2 Down-regulation On The Radiosensitivity Of T Cell Lymphoma

Objective:To investigate the anti-tumor function of EZH2 down-regulation combined with irradiation on T cell non-Hodgkin’s lymphoma(T-NHL) in vitro, and elucidate the effects on cell proliferation as well as apoptosis in T-NHL cell lines Hut78 and Jurkat.Demonstrating if inhibition of EZH2 could enhance the radiosensitivity of T-NHL,we try to provide new ideas and theoretical basis for the gene therapy in combination with conventional radiotherapy.Methods:1. The T-NHL cell lines Hut78 and Jurkat were infected by lentivirus-delivered EZH2-sh RNA and negative control(empty vector) respectively.2. The EZH2 expression in m RNA and protein levels of Hut78 and Jurkat were detected by RT-q PCR and Western-blot.3. MTS/PMS cell proliferation assay was conducted to evaluate the effect of EZH2-sh RNA on cell proliferation and radiosensitivity to X ray in Hut78 and Jurkat cells.4. EZH2 inhibitor(UNC1999) was used to treat Hut78 and Jurkat cells, and caused pharmacological inhibition of EZH2 confirmed by RT-q PCR and Western-blot.5. MTS/PMS cell proliferation assay was conducted to evaluate the effect of EZH2 inhibitor(UNC1999) on cell proliferation and radiosensitivity to X ray in Hut78 and Jurkat cells.6. Flow cytometry was used to to evaluate apoptosis of EZH2 inhibitor(UNC1999) in combination with irradiation in Hut78 and Jurkat cells7. Rhodamine 123 staining was used to observe mitochondria membrane potential of EZH2 inhibitor(UNC1999) in combination with irradiation in Hut78 and Jurkat cells8. Protein level of Bcl-2 and cleaved PARP were tested by Western-blot in Hut78 and Jurkat cells treated with EZH2 inhibitor(UNC1999) and irradiation.Results:1.Hut78 and Jurkat cells were successfully infected by lentivirus-delivered EZH2-sh RNA and empty virus, and we got two transfected cell lines of Hut78 and Jurkat respectively. RT-q PCR and Western-blot were performed to detect the expression level of EZH2. EZH2 m RNA levels of Hut78 and Jurkat cells infected with EZH2-sh RNA were only 31.6% and 46.7%, lower than other two groups(P<0.05), while no significant difference was observed between untreated cells and negative control. Western-blot showed that expression of EZH2 protein in Hut78EZH2-sh RNA and Jurkat EZH2-sh RNA cells were significantly lower than other two groups.2.MTS/PMS assay was conducted to detect cell proliferation of Hut78 and Jurkat cells infected by lentivirus-delivered EZH2-sh RNA alone, or together with X ray irradiation. Compared with other two groups, the survival fraction of Hut78 and Jurkat cells transfected with EZH2-sh RNA was lower(P<0.05), indicating an enhanced radiosensitivity in cells with EZH2 inhibition.3.EZH2 inhibitor(UNC1999) was used to treat Hut78 and Jurkat cells, and caused pharmacological inhibition of EZH2 confirmed by RT-q PCR and Western-blot.EZH2 down-regulation could exacerbate the inhibition of cell growth caused by irradiation.4.Flow cytometry test on apoptosis: Pharmacological inhibition of EZH2 with small molecule inhibitor(UNC1999) could promote apoptosis induced by irradiation in Hut78 and Jurkat cells.5.Rhodamine 123 staining: Irradiation could decrease mitochondria membrane potential of Hut78 and Jurkat cells, and treatment with EZH2 inhibitor(UNC1999)resulted in further mitochondria membrane potential loss induced by irradiation.6.Western blot experiments: Pharmacological inhibition of EZH2 with small molecule inhibitor(UNC1999) could promote the expression of cleaved PARP and inhibit anti-apoptotic protein Bcl-2 expression induced by irradiation in Hut78 and Jurkat cells.Conclusion:The lentivirus-delivered EZH2-sh RNA and EZH2 inhibitor were used todown-regulate the expression of EZH2 in Hut78 and Jurkat cells. Compared with the conventional irradiation alone, EZH2 inhibition could exacerbate the suppression of cell growth and promote apoptosis induced by irradiation.