Effect of Bmal1 expression on radiotherapy sensitivity of nasopharyngeal carcinoma xenograft / 中国肿瘤临床

Objective: To explore the effects of up- and down-regulation of circadian clock gene Bmal1 on the growth and radiation sensitivity of nasopharyngeal carcinoma after CNE1 xenograft in nude mice. Methods: We produced four groups of cells using lentiviral transfection: cells overexpressing CNE1 (CNE1OE), negative control in which there was no overexpression of CNE1 (OENC), cells with short hairpin RNAi (CNE1sh3), and RNAi negative cells (CNE1shNC). We investigated the expression of Bmal1 protein in the aforementioned groups with Western blot. After subcutaneously injecting the four groups of cells in nude mice, the size of the xenograft was measured. Subsequently, the xenografts were irradiated with 15 Gy at 6 MeV, and variation in the xenograft volume was recorded. mRNA and protein expression levels of Bmal1, p53, and p21 in the xenograft were measured with RT-PCR and Western blot, respectively. Results: The CNE1OE group highly expressed Bmal1 protein whereas the CNE1sh3 group was silenced by RNAi as shown with the Western blot, indicating successful transfection. The xenograft in the nude mice developed well. The CNE1OE xenograft volume was lower than that of the CNE1OENC xenograft, whereas the CNE1sh3 xenograft was larger than the CNE1shNC xenograft (P<0.05). CNE1OE, CNE1OENC, and CNE1shNC xenograft volumes shrank after being irradiated (t=4.32, 5.38, 5.16, respectively; P<0.05) and the effect was the highest in the CNE1OE group. However, there was almost no variation in xenograft volume in the CNE1sh3 group. The relative amounts of mRNA and protein of Bmal1, P53, and P21 were higher in the CNE1OE group than in the CNE1OENC group,while they were lower in the CNE1sh3 group compared to the CNE1shNC group (P<0.05). Conclusions: Overexpression of Bmal1 inhibited the growth of the CNE1 xenograft in nude mice and enhanced its radiation sensitivity whereas silencing the Bmal1 gene by RNAi promoted the growth of the xenograft and led to radiation resistance. We believe that Bmal1 overexpression leads to P53 and P21 overexpression, thereby inhibiting the growth of the xenograft.

Increased glycogen synthase kinase 3β activity involves in the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 in HT22 cells / 中华神经科杂志

Objective@#To investigate the effect of glycogen synthase kinase 3β (GSK3β) on the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 (Aβ31-35) in HT22 cells.@*Methods@#HT22 mouse hippocampal cells were divided into control group, Aβ31-35 group and LiCl+Aβ31-35 group by random number table method in the present study. Cells were synchronized to G0/G1 phase by 1% serum starvation for 1 hour (circadian time 0 (CT0)). Cell viability was detected by the cell counting kit-8 assay. The mRNA expression of clock gene Bmal1 was examined by real-time PCR at different CT times. The expression of GSK3β and BMAL1 protein was detected by Western blotting.@*Results@#Compared with the control group, Aβ31-35 induced the decreased expression of Bmal1 mRNA; The expression of both Bmal1 mRNA and BMAL1 protein was decreased significantly at CT20 (Bmal1 mRNA: 0.38±0.06 vs 0.83±0.08, t=4.549, P=0.001; BMAL1 protein: 0.67±0.04 vs 1.00±0.04, t=5.943, P<0.001). In the Aβ31-35 group, GSK3β activity was increased and the ratio of phosphorylated GSK3βS9 to GSK3β was decreased compared to the control group (0.66±0.08 vs 1.02±0.14, t=2.217, P=0.025). Aβ31-35 decreased the viability of HT22 cells (71.85%±6.20% in the Aβ31-35 group vs 98.14%±2.68% in the control group, t=3.891, P=0.006), and the GSK3β inhibitor LiCl pretreatment effectively reversed the decline of the viability induced by Aβ31-35 (90.74%±5.74% in the LiCl+Aβ31-35 group vs 71.85%±6.20% in the Aβ31-35 group, t=3.412, P=0.010). LiCl (in the LiCl+Aβ31-35 group) increased the expression of Bmal1 mRNA and BMAL1 protein significantly at CT20 compared with the Aβ31-35 group (Bmal1 mRNA: 0.72±0.05 vs 0.38±0.06, t=4.378, P=0.001; BMAL1 protein: 0.90±0.04 vs 0.67±0.04, t=4.052, P=0.002).@*Conclusion@#Increased GSK3β activity involved in the decreased expression of Bmal1 induced by Aβ31-35 in HT22 cells.

Increased glycogen synthase kinase 3β activity involves in the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 in HT22 cells / 中华神经科杂志

Objective@#To investigate the effect of glycogen synthase kinase 3β (GSK3β) on the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 (Aβ31-35) in HT22 cells.@*Methods@#HT22 mouse hippocampal cells were divided into control group, Aβ31-35 group and LiCl+Aβ 31-35 group by random number table method in the present study. Cells were synchronized to G0/G1 phase by 1% serum starvation for 1 hour (circadian time 0 (CT0)). Cell viability was detected by the cell counting kit-8 assay. The mRNA expression of clock gene Bmal1 was examined by real-time PCR at different CT times. The expression of GSK3β and BMAL1 protein was detected by Western blotting.@*Results@#Compared with the control group, Aβ31-35 induced the decreased expression of Bmal1 mRNA; The expression of both Bmal1 mRNA and BMAL1 protein was decreased significantly at CT20 (Bmal1 mRNA: 0.38±0.06 vs 0.83±0.08, t=4.549, P=0.001; BMAL1 protein: 0.67±0.04 vs 1.00±0.04, t=5.943, P<0.001). In the Aβ31-35 group, GSK3β activity was increased and the ratio of phosphorylated GSK3βS9 to GSK3β was decreased compared to the control group (0.66±0.08 vs 1.02±0.14, t=2.217, P=0.025). Aβ31-35 decreased the viability of HT22 cells (71.85%±6.20% in the Aβ31-35 group vs 98.14%±2.68% in the control group, t=3.891, P=0.006), and the GSK3β inhibitor LiCl pretreatment effectively reversed the decline of the viability induced by Aβ31-35 (90.74%±5.74% in the LiCl+Aβ31-35 group vs 71.85%±6.20% in the Aβ31-35 group, t=3.412, P=0.010). LiCl (in the LiCl+Aβ31-35 group) increased the expression of Bmal1 mRNA and BMAL1 protein significantly at CT20 compared with the Aβ31-35 group (Bmal1 mRNA: 0.72±0.05 vs 0.38±0.06, t=4.378, P=0.001; BMAL1 protein: 0.90±0.04 vs 0.67±0.04, t=4.052, P=0.002).@*Conclusion@#Increased GSK3β activity involved in the decreased expression of Bmal1 induced by Aβ31-35 in HT22 cells.

Increased glycogen synthase kinase 3β activity involves in the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 in HT22 cells / 中华神经科杂志

Objective To investigate the effect of glycogen synthase kinase 3β (GSK3β) on the decreased expression of Bmal1 induced by amyloid-beta protein 31-35 (Aβ31-35) in HT22 cells.Methods HT22 mouse hippocampal cells were divided into control group,Aβ31-35 group and LiCl+Aβ 31-35 group by random number table method in the present study.Cells were synchronized to G0/G1 phase by 1％ serum starvation for 1 hour (circadian time 0 (CT0)).Cell viability was detected by the cell counting kit-8 assay.The mRNA expression of clock gene Bmal1 was examined by real-time PCR at different CT times.The expression of GSK3β and BMAL1 protein was detected by Western blotting.Results Compared with the control group,Aβ31-35 induced the decreased expression of Bmal1 mRNA;The expression of both Bmal1 mRNA and BMAL1 protein was decreased significantly at CT20 (Bmal1 mRNA:0.38±0.06 vs 0.83±0.08,t=4.549,P=0.001;BMAL1 protein:0.67±0.04 vs 1.00±0.04,t=5.943,P＜0.001).In the Aβ31-35group,GSK3β activity was increased and the ratio of phosphorylated GSK3βS9 to GSK3β was decreased compared to the control group (0.66±0.08 vs 1.02±0.14,t=2.217,P=0.025).Aβ31-35 decreased the viability of HT22 cells (71.85％±6.20％ in the Aβ31-35 group vs 98.14％±2.68％ in the control group,t=3.891,P=0.006),and the GSK3β inhibitor LiC1 pretreatment effectively reversed the decline of the viability induced by Aβ31-35 (90.74％±5.74％ in the LiCl+Aβ31-35 group vs 71.85％±6.20％ in the Aβ31-35 group,t=3.412,P=0.010).LiCl (in the LiCl+Aβ31-35 group) increased the expression of Bmal1 mRNA and BMAL1 protein significantly at CT20 compared with the Aβ31-35 group (Bmal1 mRNA:0.72±0.05 vs 0.38±0.06,t=4.378,P=0.001;BMAL1 protein:0.90±0.04 vs 0.67±0.04,t=4.052,P=0.002).Conclusion Increased GSK3β activity involved in the decreased expression of Bmal 1 induced by Aβ31-35 in HT22 cells.

Research progress and new ideas in circadian gene Bmal1 and oncotherapy / 中国肿瘤临床

Circadian rhythm plays a role in regulating life activities and contributes to periodicity, orderliness, and synergy in organ-isms . The Bmal1 gene is one of the core components of the circadian rhythm system; the level of Bmal1 expression differs between tu-mor and normal tissues. Studies have shown that Bmal1 plays different roles in different tumors. Therefore, understanding the role of Bmal1 in different tumorigenesis could provide a new theoretical basis for early detection or effective treatment of tumors. In recent years, with the in-depth studies on time-based approaches, chrono-chemotherapy, which combines pharmacokinetics and biotic rhythm, has become one of the important methods of tumor treatment. Radiotherapy at different times can lead to differences in ra-diosensitivity, which makes chrono-radiotherapy increasingly prominent in tumor treatment. In-depth studies on circadian rhythm in cancer biology could provide new perspectives for tumor treatment.