Effects of RRM2 on malignant biological behavior and aerobic glycolysis of gastric cancer cells by regulating CDK1

doi:10.3760/cma.j.cn371439-20240607-00003

Abstract

Abstract:

Objective To investigate the effect of ribonucleotide reductase regulatory subunit M2 （RRM2） on the malignant biological behavior and aerobic glycolysis of gastric cancer cells by regulating cyclin-dependent kinase （CDK） 1. Methods Human gastric cancer MKN-45 cells were divided into si-NC group （transfected with blank fragment）， CoCl₂+si-NC group （hypoxia control transfected with blank fragment）， CoCl₂+si-RRM2 group （hypoxia with RRM2 silencing）， CoCl₂+si-RRM2+pcDNA3.1 NC group （hypoxia with RRM2 silencing and blank vector） and CoCl₂+si-RRM2+pcDNA3.1 CDK1 group （hypoxia with RRM2 silencing and CDK1 overexpression）. The mRNA relative expression levels of RRM2 and CDK1 were analyzed by real time fluorescent quantitative reverse transcription PCR. Co-immunoprecipitation （CoIP） was used to analyze the interaction between RRM2 and CDK1 protein. MTT assay was used to analyze the proliferation activity of cells. The cell migration distance was detected by cell scratch assay. Cell apoptosis was detected by flow cytometry. Adenosine triphosphate （ATP） and glucose kit were used to detect ATP production and glucose consumption. The protein expressions of ENO1， RRM2， HK2， PKM2， GLUT1 and p-CDK1/CDK1 were detected by Western blotting. Results Real time fluorescent quantitative reverse transcription PCR results showed that the relative expression levels of CDK1 mRNA in si-NC group， CoCl₂+si-NC group and CoCl₂+si-RRM2 group were 1.01±0.15， 1.30±0.06 and 0.51±0.18， and the relative expression levels of RRM2 mRNA were 1.03±0.32， 1.59±0.28 and 0.44±0.17， respectively， and there were statistically significant differences （F=25.52， P=0.001； F=14.47， P=0.005）. The mRNA expressions of RRM2 and CDK1 in CoCl₂+si-NC group were higher than those in si-NC group. Compared with the si-NC group and the CoCl₂+si-NC group， the mRNA expressions of RRM2 and CDK1 were lower in the CoCl₂+si-RRM2 group （all P<0.05）. CoIP results showed that there was interaction between RRM2 and CDK1. MTT assay， cell scratch assay and flow cytometry showed that the cell proliferation activity of si-NC group， CoCl₂+si-NC group， CoCl₂+si-RRM2 group， CoCl₂+si-RRM2+pcDNA3.1 NC group and CoCl₂+si-RRM2+pcDNA3.1 CDK1 group were 1.04±0.01， 1.18±0.04， 0.84±0.03， 0.81±0.03 and 0.93±0.05， respectively. The cell migration distances were （301.83±2.75），（369.67±0.76），（176.50±6.38），（175.83±3.69），（254.17±1.61） μm， respectively. The apoptosis rates were 8.05%±0.21%， 5.75%± 0.20%， 28.28%±0.04%， 30.18%±1.51% and 17.79%±0.22%， respectively， all with statistically significant differences （F=73.82， P<0.001； F=1 600.01， P<0.001； F=787.15， P<0.001）. Compared with the si-NC group and CoCl₂+si-NC group， the proliferation and migration ability of cells in the CoCl₂+si-RRM2 group， CoCl₂+si-RRM2+pcDNA3.1 NC group and CoCl₂+si-RRM2+pcDNA3.1 CDK1 group were weaker， and the apoptosis rates were higher （all P<0.05）. Compared with the CoCl₂+si-RRM2+pcDNA3.1 NC group， the proliferation and migration ability of cells in the CoCl₂+si-RRM2+pcDNA3.1 CDK1 group were stronger， and the apoptosis rate was lower （all P<0.05）. The results of ATP and glucose detection showed that there were statistically significant differences in the amount of ATP production and glucose consumption among the above five groups （F=12.53， P<0.001； F=19.21， P<0.001）. Compared with the si-NC group， the glucose consumption of cells was lower in the CoCl₂+si-RRM2+pcDNA3.1 CDK1 group （P<0.05）. Compared with the CoCl₂+si-NC group， the ATP production and glucose consumption of cells in the CoCl₂+si-RRM2+pcDNA3.1 CDK1 group were lower （both P<0.05）. Compared with the CoCl₂+si-RRM2+pcDNA3.1 NC group， the ATP production and glucose consumption of the CoCl₂+si-RRM2+pcDNA3.1 CDK1 group were higher （both P<0.05）. Western blotting showed that there were statistically significant differences in the protein expressions of ENO1， RRM2， HK2， PKM2， GLUT1， and p-CDK1/CDK1 among the above five groups （all P<0.001）. Compared with the si-NC group and the CoCl₂+si-NC group， the protein expressions of ENO1， RRM2， HK2， PKM2， GLUT1 and p-CDK1/CDK1 in the CoCl₂+si-RRM2+pcDNA3.1 CDK1 group were lower （all P<0.05）. Compared with the CoCl₂+si-RRM2+pcDNA3.1 NC group， the protein expressions of ENO1， RRM2， PKM2， GLUT1 and p-CDK1/CDK1 in the CoCl₂+si-RRM2+pcDNA3.1 CDK1 group were higher （all P<0.05）. Conclusions Silencing RRM2 can inhibit the malignant biological behavior of gastric cancer cells and the occurrence of aerobic glycolysis by regulating CDK1.

Key words: Stomach neoplasms, Ribonucleotide reductases, Cyclin-dependent kinases

Tan Rongjian, Ou Wenting, Zhai Jiawei, Quan Zhenhao, Sun Lijun, Zhou Caijin. Effects of RRM2 on malignant biological behavior and aerobic glycolysis of gastric cancer cells by regulating CDK1[J]. Journal of International Oncology, 2025, 52(1): 23-30.

Figures/Tables 10

References 17

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组别	CDK1	RRM2
si-NC组	1.01±0.15	1.03±0.32
CoCl₂+si-NC组	1.30±0.06^a	1.59±0.28^a
CoCl₂+si-RRM2组	0.51±0.18^ab	0.44±0.17^ab
F值	25.52	14.47
P值	0.001	0.005

组别	RRM2	CDK1
阳性对照组（Input）	1.00±0.11	1.00±0.04
阴性对照组（IgG）	0.00±0.00^a	0.00±0.00^a
RRM2共沉淀组	3.13±0.12^ab	0.54±0.02^ab
F值	827.06	985.89
P值	<0.001	<0.001

组别	CDK1	RRM2
阳性对照组（Input）	1.00±0.02	1.00±0.05
阴性对照组（IgG）	0.00±0.00^a	0.00±0.00^a
CDK1共沉淀组	1.77±0.59^ab	0.49±0.02^ab
F值	1 799.21	796.86
P值	<0.001	<0.001
注：RRM2为核糖核苷酸还原酶调节亚基M2；CDK1为周期蛋白依赖性激酶1；^a为与阳性对照组比较，P<0.05；^b为与阴性对照组比较，P<0.05

组别	细胞增殖活性	细胞迁移（μm）	细胞凋亡率（%）
si-NC组	1.04±0.01	301.83±2.75	8.05±0.21
CoCl₂+si-NC组	1.18±0.04^a	369.67±0.76^a	5.75±0.20^a
CoCl₂+si-RRM2组	0.84±0.03^ab	176.50±6.38^ab	28.28±0.04^ab
CoCl₂+si-RRM2+ pcDNA3.1 NC组	0.81±0.03^ab	175.83±3.69^ab	30.18±1.51^ab
CoCl₂+si-RRM2+ pcDNA3.1 CDK1组	0.93±0.05^abc	254.17±1.61^abc	17.79±0.22^abc
F值	73.82	1 600.01	787.15
P值	<0.001	<0.001	<0.001

组别	ATP（mmol/g prot）	葡萄糖（mmol/g蛋白）
si-NC组	715.44±60.13	5.41±0.40
CoCl₂+si-NC组	901.65±82.20^a	6.21±0.37^a
CoCl₂+si-RRM2组	458.27±92.50^ab	3.62±0.43^ab
CoCl₂+si-RRM2+ pcDNA3.1 NC组	454.52±109.91^ab	3.80±0.56^ab
CoCl₂+si-RRM2+ pcDNA3.1 CDK1组	667.85±107.76^bc	4.67±0.36^abc
F值	12.53	19.21
P值	<0.001	<0.001