The Effect And Molecular Mechanism Of Pyrroline-5-carboxylate Reductase 1 On The Biological Phenotype Of Chronic Myeloid Leukemia

Background: Chronic myeloid leukemia(CML)is a clonal myeloproliferative neoplasm characterized by a t(9:22)(q34:q11)translocation between chromosomes 9and 22 and the formation of a BCR/ABL fusion gene.The BCR/ABL fusion protein encoded by the BCR/ABL fusion gene has constitutive tyrosine kinase activity and activates a series of downstream signaling pathways to promote CML cell proliferation.Tyrosine kinase inhibitors(TKIs),including imatinib(IM),have significantly improved the prognosis and survival of CML patients.However,with the widespread use of TKI in the clinic,some patients are experiencing poor treatment outcomes and drug resistance,so it is important to explore the specific mechanisms of CML progression and new therapeutic targets.Proline metabolism plays a crucial part in the metabolic reprogramming of tumors and is implicated in the synthesis of ATP,protein and nucleotide synthesis and redox homeostasis in tumor cells.Pyrroline-5-carboxylate reductase(PYCR1),as an important key enzyme for proline biosynthesis,is closely associated with the progression of several solid tumors and has been considered as a potential target for anticancer drug development,but studies of PYCR1 in CML have not been reported.Objective: The present study was designed to determine the effect of PYCR1 on the biological effects of CML cells and the mechanism of its action,and to provide a theoretical and experimental basis for exploring new targets and new ideas for CML treatment.Methods: 1.The expression levels of PYCR1 in CML cells from GEO,TCGA and other databases were analyzed by bioinformatics,and then clinical samples were collected to isolate single nucleated cells,and the practical expression levels of PYCR1 m RNA were detected by RT-q PCR.2.A stable transfection strain of CML cells silencing and overexpressing PYCR1 was constructed by lentiviral transfection technique,followed by RT-q PCR and Western blot to detect the effect of transfection;the effects of silencing and overexpression of PYCR1 on CML cell proliferation,apoptosis,cycle,migration,and imatinib sensitivity were examined by CCK-8,flow cytometry,transwell,and cytotoxicity assays.3.The effect of PYCR1 silencing on the BCR-ABL pathway was first detected by Western blot;screening for differentially expressed genes and associated pathways affected after knockdown of PYCR1 by RNA-seq;further validation of gene expression levels of relevant pathways identified in RNA-seq by RT-q PCR;finally,the effect of PYCR1 silencing on mitochondrial ROS levels in CML cells was detected by flow cytometry.Results: 1.Analysis of the GEO database showed that PYCR1 expression was sequentially upregulated in healthy individuals,in the chronic phase of CML and in the blastic crisis of CML,and was significantly higher in bone marrow stem and progenitor cells of CML patients than in normal individuals.Pan-cancer analysis in the TCGA database also indicated that PYCR1 was highly expressed in most tumors.Further clinical samples were collected to verify the expression of PYCR1 m RNA in CML patients,and the results demonstrated that PYCR1 showed high expression levels.2.A stable transgenic strain of CML cells silencing and overexpressing PYCR1 was successfully constructed.Knockdown of PYCR1 inhibited CML cell proliferation,migration,promoted apoptosis,cell cycle arrest in the G1 phase and enhanced the sensitivity of CML cells to imatinib.Overexpression of PYCR1 promoted CML cell proliferation,migration and cell cycle transition from G1 to S phase,and inhibited CML cell apoptosis and sensitivity to imatinib.3.Knockdown of PYCR1 inhibited BCR-ABL and its downstream Stat5 and Crkl signaling pathways.Transcriptome sequencing analysis revealed that knockdown of PYCR1 inhibited mitochondrial energy metabolic pathways such as tricarboxylic acid cycle and oxidative phosphorylation in CML cells,and induced an increase in mitochondrial ROS levels.Conclusion: PYCR1 was highly expressed in CML and promoted the proliferation and migration of CML cells and inhibited apoptosis and sensitivity to imatinib.Inhibition of PYCR1 expression induced apoptosis in CML cells by downregulating the BCR-ABL downstream signaling pathway,disrupting mitochondrial energy metabolism,and inducing elevated mitochondrial ROS levels,thereby suppressing the malignant phenotype of CML.