The Effects Of 3-Methyladenine And SIK2 On Autophagy Induced By Ionizing Radiation

Objective: To investigate the cell proliferation, apoptosis, cell autophagy effects produced by ionizing radiation in the He La cells, and the modifications on these radiobiological effects mediated by PI3 K inhibitor 3-MA, or knocking-down SIK2 expression and irradiation with fractionated dose.Methods:1. Hela- sh SIK2 with depressed SIK2 by sh RNA and the control cells were used in this study. 3-methyadenine was used as PI3 K inhibitor.2. CCK8 test was used to investigate the cell proliferation and the toxicity effects after treated with 4 Gy 60 Co γ, or /and combined treatment with 3-MA at early time(immediately after irradiation for 12 h) and at late time(from 12 h to 24 h post-irradiation) at 0 h, 12 h, 24 h, 48 h and 72 h post irradiation.3. Using Annexin V- FITC/PI marker flow cytometry to investigate Hela-sh SIK2 and control cells apoptosis after treat with 4 Gy 60 Co γ, 3-MA treatment at early time and at late time.4. To detect the radiation sensitivity of Hela- sh SIK2 and control cells after treated with 2 Gy, fractionated dose 2+2 Gy and single dose 4 Gy of X-ray irradiation and combined treatment of 3-MA.5. Western blot was used to detect the changes of LC3 protein expression level after treated with 2 Gy, fractionated dose 2 + 2 Gy and a single dose 4 Gy of X-ray irradiation.6. Using immunofluorescence to observe the formation of autophagic body of Hela- sh SIK2 and its control cells after treated with 2 Gy, fractionated dose 2 + 2 Gy and single dose 4 Gy of X-ray irradiation.Results:1. The results of CCK8 analyses showed that compared to the control group, an increased-cytotoxicity at 12 h time-point was found in all treatment groups, except the group of 3-MA treatment at late, P < 0.05. Compared to the 4 Gy irradiation group, the group of 4 Gy irradiation combined with 3-MA treatment at early time had greater toxicity in all of the observed time-points from 12 h to 72 h, the differences were statistically significant, P < 0.01. Compared to the 4 Gy irradiation group, the group of 4 Gy irradiation combined with 3-MA at late time had greater toxicity at the time points of 24 h, 48 h and 72 h, the differences were statistically significant, P < 0.01. Compared to the group of 4 Gy irradiation combined with 3-MA at late time, the group combined with 3-MA at the early time had more toxic effect, the difference was statistically significant, P < 0.05.2. The incidence of apoptosis detected by flow cytometry analysis of Annexin V positive cells in all treatment groups was significant higher as compared with that of the control group, P < 0.01. Compared to the 4 Gy irradiation alone group, the group combined with 3-MA at early time had more toxic effect, the difference was statistically significant, P < 0.05. Compared to the group of 4 Gy irradiation combined with 3-MA at early time, the group combined with 3-MA at late time had relative less effect, the difference was statistically significant, P < 0.05..3. Clone formation ability experiment showed that the cell survival rate of the group of cells treated with 3-MA combined fractionated dose of 2+2 Gy irradiation was higher than that of the cells group treated with 3-MA combined with 4 Gy single dose irradiation, the difference was statistically significant, P < 0.05. All group which added 3-MA treatment had more toxic effect, the difference was statistically significant, P < 0.01. With the treatment of 3-MA combined with radiation, Hela-sh SIK2 cells exhibited a higher death rate as compared to the control Hela-sh NC cells, P < 0.05. 4. Western Blot results showed that X-ray irradiation with three doses(2 Gy, 2+2 Gy fractionated dose, 4 Gy) can cause the autophagy biomarker LC3 II increase in He La cells, and which reached a peak at 24 h. The changed levels of LC3 II expression are different for different irradiation doses, LC3 II expression level after irradiation with fractionated dose 2+2 Gy was higher than that of 4 Gy single dose exposure. therefore, irradiation with fractionated dose seems to induce more cell autophagy. In SIK2 Knockdown cells, the LC3 II expression level was lower than that of control cells, which could explain the decreased autophagy incidence in SIK2 knockdown cells.5. Immunofluorescence staining of autophagy marker protein LC3 demonstrated that the fluorescence intensity of typical LC3 staining in SIK2 knockdown cell was less than that of the control cells. It’s confirmed that SIK2 is negative regulator of cell autophagy associated with ionizing radiation. And after irradiation with fractionated dose 2+2 Gy, the fluorescence intensity of LC3 staining was higher than that of the other exposure groups at the same time.Conclusions: The combined treatments of 3-MA at early and late time have different effect on cell autophagy and apoptosis induced by ionizing radiation. SIK2 and fractionated dose irradiation can promote cell autophagy. Knockdown of SIK2 sensitizes cells to ionizing radiation with the decreased autophagy.