RRM2 Regulating Apoptosis,Proliferation and Resistance of Lung Adenocarcinoma Cells Through Mdm2/P53 Pathway

JIAN Shibo; XIAO Xuyang

doi:10.3969/j.issn.2096-305X.2025.01.003

Abstract：Objective This study aims to investigate the expression and biological functions of ribonucleotide reductase M2(RRM2)in lung adenocarcinoma,particularly its role in regulating cell proliferation,apoptosis,and drug resistance through the MDM2/P53 signaling pathway.Methods Human lung adenocarcinoma cell lines A-427,A549,and PC-9,as well as normal bronchial epithelial cells BEAS-2B,were selected.RT-PCR was used to screen for high RRM2 expression in lung adenocarcinoma cell lines.PC-9 cells were divided into four groups:si-NC(low-expression control),oe-NC(high-expression control),si-RRM2(low-expression group),and oe-RRM2(high-expression group).The proliferation and apoptosis rates of cells in each group were assessed.The STRING online analysis platform was utilized to construct a protein-protein interaction(PPI)network,with Cytoscape software analyzing the core nodes involving RRM2,MDM2,and P53.Dual-luciferase reporter assays validated the targeting relationships between RRM2 and MDM2,as well as between MDM2 and P53.The KEGG database was employed for enrichment analysis of the MDM2/P53 signaling pathway.Finally,the IC50 levels and expression of related proteins,including RRM2,MDM2,P53,Cyclin D1,Bax,Bcl-2,Caspase-3,MRP,MDR1,and GST-π,were measured.Results RT-PCR analysis revealed that the relative mRNA expression levels of RRM2 in lung adenocarcinoma cell lines A-427,A549,and PC-9 were significantly higher than those in BEAS-2B(P<0.05),with the highest expression observed in PC-9 cells(P<0.05),making them the choice for subsequent experiments.Compared to the oe-NC group,the oe-RRM2 group exhibited a significantly increased proliferation rate and a significantly decreased apoptosis rate.Conversely,the si-RRM2 group showed a significantly reduced proliferation rate and an increased apoptosis rate compared to the si-NC group.PPI network analysis indicated that MDM2 had the highest score among the core genes of RRM2,while P53 had the highest score among the core genes of MDM2.Dual-luciferase reporter assays demonstrated that the wild-type(WT)MDM2 luciferase activity in the oe-RRM2 group was significantly higher than in the oe-NC group(P<0.05).Meanwhile,the WT P53 luciferase activity in the oe-MDM2 group was significantly lower than in the oe-NC group(P<0.05).It is known through the enrichment analysis of the MDM2 and P53 signaling pathways that MDM2 can inhibit the expression of P53,and P53 can regulate the expression of downstream P21 and Cyclin D1,thereby affecting the expression levels of Bax and Bcl-2.Based on these results,we selected Bax,Bcl-2,and Cyclin D1 as key proteins for subsequent proliferation and apoptosis assays.The findings indicated that the IC50 levels in the oe-RRM2 group were elevated compared to the oe-NC group,with increased expression of RRM2,MDM2,Bcl-2,Cyclin D1,MRP,MDR1,and GST-π,and decreased expression of Bax,Caspase-3,and P53.In contrast,the si-RRM2 group exhibited lower IC50 levels compared to the si-NC group,with decreased expression of RRM2,MDM2,P53,Cyclin D1,Bax,Bcl-2,Caspase-3,MRP,MDR1,and GST-π,and increased expression of Bax,Caspase-3,and P53.Conclusion RRM2 regulates apoptosis,proliferation,and drug resistance in lung adenocarcinoma cells through the MDM2/P53 pathway.