| Background and PurposeLeukemia is one of the common malignant tumors in the blood system,and it is a malignant clonal disease with abnormal hematopoietic stem cells.According to statistics,the incidence of leukemia accounts for about 3% of the total incidence of tumors,which seriously endangers human life and health.The main cause of leukemia is the growth disorder of white blood cells,which causes the white blood cells to stagnate at different stages of cell development,and then a large number of immature cells accumulate in the bone marrow and other hematopoietic tissues,cell differentiation is blocked,and the normal apoptosis process is disordered,leading to normal human body Hematopoietic function is affected.Although the roles and mechanisms of many genes and pathways in leukemia have been preliminarily clarified,the abnormal genes discovered so far cannot explain all the mechanisms of leukemia.Therefore,finding new key factors and related molecular pathways that regulate the proliferation,apoptosis and differentiation of leukemia cells and conducting in-depth research on them are of great significance for the treatment of leukemia.Protein 4.1R is a member of the protein 4.1 family.Protein 4.1R was originally a cell membrane skeletal protein molecule found in mature red blood cells and it is a bridge molecule that connects the red blood cell transmembrane protein and the spectrin-actin skeleton.Protein 4.1R plays an important role in maintaining the shape,fragility,adhesion and normal function of red blood cells.The protein 4.1R encoding gene EPB41 is not only expressed in red blood cells,but also expressed in various immune cells that also originate from bone marrow hematopoietic stem cells and plays an important regulatory role,such as lymphocytes,dendritic cells,and macrophages.Loss or mutation of human protein 4.1R expression can cause hereditary oval polycythemia,leading to the occurrence of hemolytic anemia.Studies have shown that the expression of protein 4.1R is absent in clinical bone marrow malignancies.Whether protein 4.1R affects the proliferation,differentiation and apoptosis of leukemia cells and other related functions,thereby affecting the occurrence and development of leukemia,there is no relevant report at home and abroad.In this study,the Lentiviral interference technology was used to establish a K562 cell model of leukemia with 4.1R gene knockdown.Taking 4.1R gene knockdown type and wild-type K562 cells as the research objects,the effect of protein 4.1R on the malignant biological behavior of leukemia K562 cells was preliminarily explored.ThroughRNA whole transcription sequencing to understand the regulatory molecular mechanism of protein 4.1R in the occurrence and development of leukemia,and discover the function of protein 4.1R in leukemia cells.Methods1.The qRT-PCR and Western Blot method was used to detect the expression of protein 4.1R in different leukemia cell lines.2.RT-PCR and Western Blot technology were used to verify the expression of protein 4.1R in K562 cells.3.Design and screen the shRNA of protein 4.1R,and sequence it.Construct a lentiviral interference plasmid,transfect 293 T cells,and observe the transfection efficiency under a fluorescence microscope.4.Lentiviral interference technology was used to construct a K562 cell line with stable knockdown protein 4.1R,and qRT-PCR and Western Blot methods were used to verify the interference efficiency.5.CCK-8 method was to detect the effect of protein 4.1R knockdown on the proliferation of leukemia K562 cells.Flow cytometry to detect the effect of protein4.1R knockdown on K562 cell cycle.The effects of protein 4.1R knockdown on the expression of cell cycle-related proteins Cyclin-D1 and Cyclin-D2 were detected by qRT-PCR and Western Blot methods.Tunel method was used to detect the effect of protein 4.1R on the apoptosis of leukemia K562 cells.The effects of protein 4.1R knockdown on the expression of apoptosis-related proteins BAX 、 BCL-2 and Caspase-9 were detected by qRT-PCR and Western Blot methods,respectively.6.Hemin was used to induce K562 cells to differentiate into erythroid,and the effect of protein 4.1R on erythroid differentiation of K562 cells was detected;qRT-PCR method was used to detect the expression of erythroid transcription factors GATA-1 and GFI-1B.7.A subcutaneous xenograft tumor model was established in nude mice,and the effect of protein 4.1R knockdown on the tumor formation ability of K562 cells was tested in vivo.8.Transcriptome sequencing was performed on wild-type K562 cells and 4.1R knockdown K562 cells.Differential genes were screened by P value and log FC,and signal pathways related to protein 4.1R were analyzed and screened by KEGG method.Western Blot method was used to detect the expression of related signaling pathway molecular proteins that may be affected by knockdown of protein 4.1R.9.Glucose kit and lactic acid production kit were used to detect the effect of protein 4.1R knockdown on the glycolysis function of K562 cells;qRT-PCR method was used to detect the expression of glycolysis-related genes.10.The qRT-PCR method verifies the influence on the expression of downstream differential genes.11.Western blot method was used to detect the effect of protein 4.1R knockdown on downstream signaling pathways.Results1.At the mRNA and protein levels,it was found that the expression level of protein 4.1R in K562 cells was higher than other leukemia cells.2.mRNA and protein levels found that protein 4.1R was expressed in leukemia K562 cells.3.The protein 4.1R shRNA and shRNA-NC plasmids were successfully constructed,and the sequencing results were consistent with the expected results.4.The knockdown efficiency of the lentiviral vector transfected into K562 cells was higher than 80%,which laid the foundation for subsequent experiments.5.Knockdown of protein 4.1R promoted the proliferation ability of leukemia K562 cells,and inhibited the apoptotic ability and erythroid differentiation ability of leukemia K562 cells.6.Successfully constructed a nude mouse subcutaneous xenograft tumor model,knockdown of protein 4.1R enhances the subcutaneous tumor formation ability of K562 cells.7.A total of 615 differentially expressed genes were identified in the protein4.1R knockdown group and the control group through transcriptomics testing.Compared with control cells,455 regulated genes were up-regulated and 160 genes were down-regulated in knockdown cells.Differentially expressed genes involve energy metabolism pathways such as non-codingRNA,glucose and amino acid metabolism.8.KEGG analysis showed that protein 4.1R is related to metabolic pathways,and protein 4.1R gene knockdown enhanced the glycolysis level of K562 cells.9.The long non-codingRNA CCDC26(lncRNA CCDC26)and C-kit of the differentially expressed genes were screened through the screening conditions of P ≤0.001 and log FC ≥ 2.10.The expression of lncRNA CCDC26 increased when protein 4.1R gene knocked down.In sh-K562 cells,the expression of lncRNA CCDC26 was silenced,and the expression of protein 4.1R increased.11.Knockdown of protein 4.1R increases the expression of C-kit and activates the expression of RAS,p-JNK,and p-ERK proteins in the MAPK signaling pathway.Conclusions1.Knockdown of protein 4.1R promotes the proliferation of K562 cells,and inhibits the apoptosis and erythroid differentiation of K562 cells.2.Knockdown of protein 4.1R promotes tumor growth in vivo.3.Knockdown of protein 4.1R enhances the glycolysis level of K562 cells.4.Knockdown of protein 4.1R increases the expression of lncRNA CCDC26 and C-kit,also increases the expression of RAS,p-JNK,and p-ERK proteins in the MAPK signaling pathway.Protein 4.1R may affect the expression of C-kit through the related effects of lncRNA CCDC26,thereby affecting the downstream MAPK signaling pathway and then affecting the occurrence and development of leukemia. |
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