| [Objective] To investigate the effect of HMGB1 (high mobility group protein box 1) gene knockdown with siRNA on the radiosensitivity of esophageal squamous cell carcinoma (ESCC) TE-1 cell line, and to preliminarily explore its molecular mechanisms.[Methods](1) By siRNA interference technique, the NC siRNA or siHMGBl was transfected into TE-1 cell line through Lipofectamine 2000. Three groups of TE-1 cells were designed according to the experimental requirements:TE-1 group (control group), TE-1 NC siRNA group (negative control group, NC group), and TE-1 siHMGB1 group (HMGB1 silencing group). The expression of HMGB1 mRNA and protein were detected by quantitative real-time polymerase chain reaction (PCR) and Western blot assay respectively.(2) The three groups of TE-1 cells were irradiated with different doses (0,2,4,6,8 Gy) of x-rays after transfection for 24 h, and cultured for the certain period of time (24,48,72,96 h), and then cell proliferation of TE-1 cells was detected by MTT.(3) The groups of cells were irradiated with different doses (0,2,4,6,8 Gy) of x-rays after transfection for 24 h, then cultured for 14 days to allow the formation of colonies, and the survival fraction of TE-1 cells was detected by clony formation assay.(4) The apoptotic rate, ROS (reactive oxygen species) content and γH2AX level of the cells were detected by flow cytometry after transfection for 24 h prior to 4 Gy x-rays irradiation; The changes of expression of NOX1 and mRNA NOX5 were detected by PCR after transfection for 24 h prior to 4 Gy x-rays irradiation; The changes of Caspase-3, Cleaved PARP, p-p38, p38, JNK and p-JNK protein expression were detected by Western blot assay after transfection for 24 h prior to 4 Gy x-rays irradiation.[Results](1) Compared with the control group or negative control group, the levels of HMGB1 mRNA and protein in the HMGB1 silencing group were significantly reduced (P<0.001).001).(2) MTT assay showed that the rate of cell proliferation of HMGB1 silencing group was lower than control group and negative control group after transfection for 24,48,72,96 h (P <0.05). The proliferation rate of the three groups of cells was inhibited after transfection for 24 h and 4 Gy x-rays irradiation for 24,48,72,96 h, but the proliferation rate of the HMGB1 silencing group was lower than that of the two control group (P<0.05). With the increase of the irradiation dose, the proliferation rate of cells was all inhibited after transfection for 24 h and different doses (2,4,6,8 Gy) of X-rays irradiation for 48 h, but the proliferation rate of the HMGB1 silencing group was lower than that of the two control group (P<0.05).(3) Sigmaplot software analysis showed that D0, Dq, N values of the HMGB1 silence group were lower than the control group and negative control group, and HMGB1 silence group prior to irradiation caused radiosensitization with an SER of 1.26 compared with the control group.(4) After transfection for 24 h, the results of flow cytometry showed that the apoptotic rate, ROS content and γH2AX level in the HMGB1 silencing group were higher than the two control groups’. And the results of PCR showed that the expression levels of NOX1 and NOX5 mRNA in the HMGB1 silencing group were higher than in the two control group, the expression levels of Caspase-3, Cleaved PARP, p-p38 and p-JNK protein in the HMGB1 silence group were higher than in the two control group (all P<0.05). After transfection for 24 h prior to 4 Gy x-rays irradiation, the results of flow cytometry showed that the apoptotic rate, ROS content and γH2AX level in the HMGB1 silencing coprocessing 4 Gy group were higher than in the simple radiotherapy group. The results of PCR showed that the expression levels of NOX1 and NOX5 mRNA in the synergistic treatment of HMGB1 silencing and 4 Gy group were higher than in the simple radiotherapy group, and the expression levels of Caspase-3, Cleaved PARP, p-p38 and p-JNK protein were higher than in the other radiotherapy groups (all P<0.05)[Conclusions]Downregulation of HMGB1 expression can inhibit the proliferation and reduce the rate of clone formation in the TE-1 cell line after radiotherapy in vitro. Namely, HMGB1 knockdown can enhance radiosensitivity of TE-1 cells. The molecular mechanisms are as follows:downregulation of HMGB1 expression may up regulate the levels of pro-apoptotic Caspase-3, Cleaved PARP protein, ROS production and DNA damage; through the activation of p38 and JNK pathways and jointing the up-regulation of p-p38 and p-JNK proteins expression. Together regulating radiation-induced proliferation and apoptosis of TE-1 cells, and then enhance the sensitivity of TE-1 cells to radiotherapy. |
PDF Full Text Request