ABSTRACT
Objective To investigate the effects of silencing the gene of single-stranded DNA-binding protein 1 (SSB1) on proliferation and DNA repair of rat submandibular gland (SMG) cells after irradiation, and explore the relationship between SSB1 and DNA damage repair. Methods Primary rat SMG cells were obtained by mechanical-enzyme digestion and identified by immunohistochemistry. The cells were divided into three groups, including blank control, negative control and shRNA transfection group. The shRNA was transfected into cells by recombinant adenovirus vector. Real-time quantitative PCR ( qRT-PCR) was used to detect the expression of SSB1 after silencing. The cell viability was detected by CCK-8 assay. Immunofluorescence analysis was performed to observe the dynamic formation of γ-H2AX foci. Results The SMG cells were positively stained for both Pan CK and α-Amylase. The efficiency of shRNA transfection was about 90%at 72 h post-transfection. Compared with the blank control group, the expression of SSB1 was significantly decreased in the cells transfected with shRNA (t=16. 24, P<0. 05). The cell viability of shRNA transfection group without irradiation was decreased indistinctively and became lower than the blank control group significantly until 120 h(t=3. 29, P<0. 05). After radiation with 5 Gy of γ-rays, the cell viability of shRNA transfection group was lower than that of the control groups significantly (F=10. 19-30. 13, P<0. 05). Silencing the expression of SSB1 could increase the number ofγ-H2AX foci in SMG cells at different time of radiation. Conclusions After silencing of the expression of SSB1, the SMG cells could be more radiosensitive, which indicats that SSB1 may play an important role in DNA damage repair after radiation.
ABSTRACT
Objective To investigate the effects of silencing the gene of single-stranded DNA-binding protein 1 (SSB1) on proliferation and DNA repair of rat submandibular gland (SMG) cells after irradiation, and explore the relationship between SSB1 and DNA damage repair. Methods Primary rat SMG cells were obtained by mechanical-enzyme digestion and identified by immunohistochemistry. The cells were divided into three groups, including blank control, negative control and shRNA transfection group. The shRNA was transfected into cells by recombinant adenovirus vector. Real-time quantitative PCR ( qRT-PCR) was used to detect the expression of SSB1 after silencing. The cell viability was detected by CCK-8 assay. Immunofluorescence analysis was performed to observe the dynamic formation of γ-H2AX foci. Results The SMG cells were positively stained for both Pan CK and α-Amylase. The efficiency of shRNA transfection was about 90%at 72 h post-transfection. Compared with the blank control group, the expression of SSB1 was significantly decreased in the cells transfected with shRNA (t=16. 24, P<0. 05). The cell viability of shRNA transfection group without irradiation was decreased indistinctively and became lower than the blank control group significantly until 120 h(t=3. 29, P<0. 05). After radiation with 5 Gy of γ-rays, the cell viability of shRNA transfection group was lower than that of the control groups significantly (F=10. 19-30. 13, P<0. 05). Silencing the expression of SSB1 could increase the number ofγ-H2AX foci in SMG cells at different time of radiation. Conclusions After silencing of the expression of SSB1, the SMG cells could be more radiosensitive, which indicats that SSB1 may play an important role in DNA damage repair after radiation.