羟基脲联合辐射对沉默ATRX后细胞周期及凋亡的影响

doi:10.13481/j.1671-587X.20230306

Abstract

Abstract:

Objective To investigate the effects of hydroxyurea （HU） combined with irradiation on the cycle cycle and apoptosis of the A549 cells after silencing α-thalassemia/mental retardation syndrome X stain-associated protein （ATRX）， and to clarify the molecular mechanism. Methods The A549 cell model （shATRX-A549） stable silencing ATRX was constructed， the infection situation of the cells was observed under fluorescence microscope，the expression of ATRX protein in the ATRX cells was detected by Western blotting method to verify the cell model， and the shNC-A549 cells were chosen as negative control. The experiment were divided into control group， HU group，radiation group （given 8 Gy X-ray radiation）， and hydroxyurea+radiation group （given HU+8 Gy X-ray radiation）.The percentages of cells at different cell cycle and apoptotic rates of the cells were detected by flow cytometry， and the expressions of mRNA in the cells after silencing ATRX were detected by RNA-sequencing （RNA-seq），and the expression amonts of the cell division cyclin 25B（CDC25B）， Cyclin B1， and cyclin dependent kinase 1（CDK1） proteins in the cells in various groups were detected by Western blotting method. Results The shNC-A549 cells and shATRX-A549 cells expressed green fluorescence protein（GFP） under fluorescence microscope；the Western blotting results showed that compared with shNC-A549 cells，the expression amount of ATRX protein in the shATRX-A549 cells was decreased. The flow cytometry results showed that compared with control group， the percentage of the shNC-A549 cells at G₀/G₁ phase in HU group was increased （P<0.05）， the percentages of the shNC-A549 cells at S phase and G₂/M phase were significantly decreased （P<0.05 or P<0.01）， and the percentages of the shNC-A549 cells at G₀/G₁ phase and S phase in radiation group were significantly decreased （P<0.01）， while the percentage of the shNC-A549 cells at G₂/M phage was significantly increased （P<0.01）， the percentage of the shNC-A549 cells at G₀/G₁phase in HU+radiation group was significantly decreased（P<0.01）， and the percentages of the cells at S phase and G₂/M phase were significantly increased （P<0.01）.Compared with control group，the percentage of the shATRX-A549 cells at G₀/G₁ phase in HU group was increased（P<0.05），and the percentage of the shATRX-A549 cells at G₂/M phase was decreased（P<0.01）；the percentage of the shATRX-A549 cells at G₂/M phase in radiation group was significantly increased（P<0.01）， but the percentages of the shNC-A549 cells at G₀/G₁ phase and S phase were significantly decreased（P<0.01）， while the percentage of the shATRX-A549 cells at G₀/G₁ phase in HU+radiation group was decreased（P<0.01），and the percentages of the shNC-A549 cells at S phase and G₂/M phase were significantly increased （P<0.05）. Compared with the shNC-A549 cells，the percentage of the shATRX-A549 cells at G₀/G₁ phase in radiation group was increased（P<0.05），the percentage of the shATRX-A549 cells at S phase in HU+radiation group was increased（P<0.05）.Compared with control group， the apoptotic rates of the shNC-A549 cells and shATRX-A549 cells in HU group， radiation group， and HU+radiation group were significantly increased（P<0.05 or P<0.01）. Compared with shNC-A549 cells， the apoptotic rates of the shATRX-A549 cells in HU group and HU+radiation group were increased （P<0.05）. The differential expressions of mRNAs after silencing ATRX involved c-Myc， Esp1， Cdc20， Plk1， CycA/B， Cip1， and PCNA.The Western blotting results showed that compared with control group， the expression amounts of CDC25B， Cyclin B1， and CDK1 proteins in the shNC-A549 cells and shATRX-A549 cells in HU group， radation group and HU+radiation group were decreased；compared with the shNC-A549 cells， the expressions amounts of Cyclin B1 protein in the shATRX-A549 cells in control and HU groups were decreased， while the expression amounts of CDC25B，cyclin B1， and CDK1 proteins in the shATRX-A549 cells in radiation group and HU+radiation group were increased. Conclusion HU and radiation can cause the cell cycle arrest and the apoptosis of the A549 cells after silencing ATRX， and its mechanism is related to the CDC25B/Cyclin B/CDK1 pathway.

Key words: Ionizing radiation, hydroxyurea, Cell cycle arrest, apoptosis, α-Thalassemia/mental retardation syndrome X chromosome-associated protein

CLC Number:

R749.94

Hongyuan TIAN,Caiyun YIN,Li WANG,Peiyun HU,Chenyang ZHANG,Qiuyue LI,Qingzhao ZHENG,Yali QI,Fang FANG,Zhicheng WANG. Effects of hydroxyurea combined with radiation on cell cycle and apoptosis of cells after silencing ATRX[J].Journal of Jilin University(Medicine Edition), 2023, 49(3): 590-598.

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References 25

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Group	Percentage of cells
Group	G₀/G₁	S	G₂/M
Control	69.66±5.60	23.53±6.97	6.81±1.40
HU	80.66±1.04^*	17.72±3.70^*	1.62±0.35^**
Radiation	45.93±0.88^**	4.54±0.34^**	49.53±0.56^**
HU+radiation	19.63±0.84^**	32.96±1.39^**	47.41±0.94^**

Group	Percentage of cells
Group	G₀/G₁	S	G₂/M
Control	73.71±0.35	19.08±0.67	7.22±0.38
HU	78.04±0.58^*	19.71±1.23	2.25±0.23^**
Radiation	50.06±1.44^**	5.26±0.78^**	44.68±0.76^**
HU+radiation	18.01±0.12^**	37.64±0.18^**	44.35±0.23^**

Group	Apoptotic rate
Control	1.91±0.38
HU	6.21±0.68^**
Radiation	5.53±0.72^*
HU+radiation	24.37±0.83^**

Group	Apoptotic rate
Control	2.88±0.53
HU	9.69±1.28^**
Radiation	6.66±1.03^*
HU+radiation	27.86±0.77^**

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Effects of hydroxyurea combined with radiation on cell cycle and apoptosis of cells after silencing ATRX

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