Molecular Mechanisms Of O-GlcNAc Glycosylation In Occurrence And Development Of Children’s Precursor B-cell Lymphoblastic Leukemia

Background:Precursor B-cell lymphoblastic leukemia(pre-B-ALL)is a common malignant disease in children.Although the current long-term disease-free survival rate increased significantly,but there are still 20%-30%of children eventually died,the reason lies in the process of chemotherapy without remission and recurrence.In recent years,there have been a lot of reports on the regulation of nucleic acid and protein levels in pre-B-ALL and their development.However,few reports on glycosylation abnormalities have been reported,especially about O-GlcNAc glycosylation.O-GlcNAc glycosylation is an important protein post-translational modification,first discovered by Gerald W.Hart.In recent years,it has been found that there are hundreds of post-translational modifications of proteins,of which glycosylation,especially O-GlcNAc glycosylation is particularly noticeable.O-GlcNAc glycosylation is a dynamically reversible process in whichβ-N-acetylglucosaminyltransferase(OGT)is responsible for glycosylation andβ-Nacetylglucosaminidase(OGA)is responsible for glycosylation removal.In vivo,O-GlcNAc glycosylation controls basic life processes such as protein synthesis,chromatin structure,demethylation of DNA and biological circadian patterns.Some signaling proteins or transcription factors such as Akt,c-Myc,p53 and NF-κB are glycosylated by O-GlcNAc.In recent years,a large number of literatures have proved that O-GlcNAc glycosylated protein plays an important role in various cancers,such as breast cancer,prostate cancer and bladder cancer,but not in pre-B-ALL and its drug resistance.In this study,bone marrow specimens of pre-B-ALL children patients and pre-B-ALL cell line Nalm-6 cells were selected as the main research objects.From the perspective of glycogen biology,the roles of O-GlcNAc glycosylation and related enzymes OGT and OGA in the development and drug resistance of pre-B-ALL were analyzed,and its mechanism from the perspective of cellular glycolytic metabolism was explored.Objective:1.To investigate the expression of O-GlcNAc glycosylation and related enzymes in pre-B-ALL patients leukemia cells and the effect of abnormal O-GlcNAc glycosylation on the biology of Nalm-6 cells;2.To investigate the regulatory role of abnormal O-GIcNAc glycosylation in the glycolysis of Nalm-6 cells via the PI3K/Akt/c-Myc pathway.3.To investigate the effect of combined use of glycolysis inhibitor 2-DG and targeted inhibition of OGT on the killing effect of Nalm-6.Methods:1.To investigate the expression of O-GlcNAc glycosylation and related enzymes in pre-B-ALL patients leukemia cells and the effect of abnormal O-GlcNAc glycosylation on the biology of Nalm-6 cells;1.1 Bone marrow specimens were collected from newly diagnosed pre-B-ALL patients at from September 2015 to September 2016.Specimens were collected using heparin anticoagulation tubes.Immediately after the specimen was collected,lymphocytes were separated and bone marrow mononuclear cells(BM-MNCs)were separated using lymphocyte separation fluid by density gradient centrifugation.CD19+B lymphocytes were sorted by immunomagnetic beads.The purity of the transfected cells was detected by flow cytometry.1.2 The overall O-GlcNAc glycosylation and OGT,OGA protein expression levels in pre-B-ALL children and healthy children were detected by Western-Blotting.1.3 The acute pre-B lymphocyte leukemia cell line Nalm-6 cells were used as the research object,and the biological behavior of Nalm-6 cells was detected by the RNA interference technology and Alloxan,an OGT specific inhibitor.The RNA interference method:siRNAs for OGT genes(OGT-siRNA)and NC-siRNAs for negative controls were synthesized.OGT-siRNA and NC-siRNA were transfected into Nalm-6 cells by Lipofectamine2000.After 48h,the expression of OGT and the level of O-GlcNAc glycosylation were detected by Western-Bloting to detect the interference effect.Inhibitor method:Nalm-6 cells treated with Alloxan for 24h,Western-Bloting was used to detecte O-GlcNAc glycosylation levels,OGT expression levels to verify the inhibition;After the inhibitory effect was verified,the effect on the biological behavior was subsequently examined after exposure to Nalm-6 cells with Alloxan at a concentration of 10mmol/L.1.4 Cell proliferation:The absorbance at 450 nm was detected by CCK-8 at 24h,48h,72h,and 96h after OGT inhibition(OGT-siRNA or Alloxan)and the corresponding proliferation curve was drawn.1.5 Cell apoptosis:Annexin V-PI double staining method was used to detect apoptosis ratio after OGT inhibition(OGT-siRNA 48h or Alloxan 24h)by flow cytometry.2.To explore the regulatory role of abnormal O-GlcNAc glycosylation in the glycolysis of Nalm-6 cells via the PI3K/Akt/c-Myc pathway.2.1 To verify that c-Myc is regulated by the PI3K/Akt pathway:Nalm-6 cells were treated with LY294002(PI3K/Akt pathway inhibitor)at different concentrations(0,1,5,and 10 μmol/L),the protein expression of Akt,phosphorylated Akt(Ser473),c-Myc,and phosphorylated c-Myc(Thr58)were detected by Western-Blotting.2.2 To compare activation of PI3K/Akt/c-Myc in CD 19 + cells and serum lactate dehydrogenase(H-LDH)children with ALL with low serum lactate dehydrogenase(L-LDH):Compared with 400u/L as the cut-off point to distinguish L-LDH group and H-LDH group,the detection method is the same as 2.1.2.3 Activity test of glycolysis:Nalm-6 cells were treated with different concentrations of LY294002(0,1,5,10p μmol/L)for 48h.The extracellular acidification rate(ECAR)was measured by seahorse XFe24 cell energy analyzer.The changes of baseline,glycolytic capacity,and glycolytic reserve were statistically analyzed.2.4 Analyse O-GlcNAc glycosylation levels and OGT,OGA expression in leukemia children with L-LDH and H-LDH level.2.5 Activity test of glycolysis:Nalm-6 cells were divided into three groups:the experimental group,transfected with OGT-siRNA group,called the OGT-siRNA group;the negative control group,ransfected with negative control siRNA,called the NC-siRNA group;the blank control group,transfected with only Lipofectamine2000.ECAR detection of the above three groups,the specific method with 4.3.2.6 Relative molecular detection of glycolysis catalysis:The mRNA expression levels of HK2,Glutl,HIF-1α,LDHA and PFK1 in the above three groups were detected by real-time fluorescence quantitative PCR,and β-actin was taken as the internal control for statistical analysis.2.7 Detection of PI3K/Akt/c-Myc pathway activation:detect the phosphorylation of key molecules in the pathway in the above three groups.The detection is the same as 2.1.2.8 The effect of IGF-1 on OGT-siRNA-induced glycolysis inhibition:OGT-siRNA+ IGF-1 group:Nalm-6 cells were treated by OGT-siRNA and IGF-1 simultaneously for 48h.OGT-siRNA group:Nalm-6 cells were treated with only OGT-siRNA for 48h.In the control group,only Lipofectamine2000 and an equal amount of PBS were added.The glycolytic test assay was performed as described in 4.3.3.To investigate the effect of combined use of glycolysis inhibitor 2-DG and targeted inhibition of OGT on the killing effect of Nalm-6.3.1 The effect of 2-DG on O-GlcNAc glycosylation and OGT in Nalm-6 cells:The effects of 2-DG at different concentrations(0,2.5,5mmol/L)on the O-GlcNAc glycosylation and OGT protein expression were examined by Western-Blotting.3.2 The effect of 2-DG on OGT expression and the proliferation of Nalm-6 cells:The OD value of 450 nm of OGT-siRNA transfected Nalm-6 cells at 48,72,and 96 h was detected by CCK-8 method.3.3 To examine the effect of 2-DG combined with targeted inhibition of OGT on the proliferation of Nalm-6 cells:Nalm-6 cells were divided into three groups:The 2-DG group was treated with 2-DG(2.5 mmol/L);The 2-DG + NC group was treated with 2-DG(2.5 mmol/L)and NC-siRNA;The 2-DG + OGT-siRNA group was treated with 2-DG(2.5 mmol/L)and OGT-siRNA.The proliferation detection method is the same as 1.4.Results:1.Abnormal expression of O-GlcNAc glycosylation and related catalytic enzymes in myeloid cells of pre-B-ALL and influence the biological behavior of Nalm-6 cells1.1 In our study,24 bone marrow samples from 24 newly diagnosed pre-B-ALL children and 10 healthy children were collected.There was no significant difference in age and sex between the two groups.The CD19+ BM-MNCs was up to 90.47%sorted by immunomagnetic beads.1.2 Compared with healthy children,the overall O-GlcNAc glycosylation levels(P<0.01)and OGT(P<0.001)and OGA protein expression levels(P<0.001)in children with B-ALL were significantly lower.Differences were statistically significant.1.3 OGT-siRNA and NC-siRNA were successfully synthesized.Western Bloting results showed that the expression of OGT and the level of O-GlcNAc glycosylation were significantly down-regulated after OGT-siRNA transfection for 48 h,indicating that interfering with the expression of OGT was effective.1.4 Western blotting showed that O-GIcNAc glycosylation level and OGT expression level decreased gradually after Alloxan treatment on Nalm-6 cells for 24h.Alloxan could be used as an inhibitor of OGT.1.5 The CCK-8 proliferation assay showed that the proliferation of Nalm-6 cells was significantly slowed down after OGT-siRNA or Alloxan treatment.The differences were statistically significant.1.6 The analyse of apoptosis showed Alloxan induced the apoptosis of Nalm-6 cells[Alloxan group vs control group(15.19±2.539)%vs(21.91±4.105)%,P<0.05].Similarly,Nalm-6 cells also showed apoptosis[OGT-siRNA group vs control group(21.72 ± 1.903%vs 11.07 ± 1.069%),p<0.05].2.Abnormal O-GlcNAc Glycosylation regulates the Nalm-6 cell glycolytic process via the PI3K/Akt/c-Myc pathway.2.1 LY294002 resulted in a significant downregulation of phosphorylated Akt(Akt Ser473/Akt)and phosphorylated c-Myc(c-Myc Thr58/c-Myc)in Nalm-6 cells,indicating that c-Myc is a downstream PI3K/Akt molecule,LY294002 can inhibit the activity of this pathway.2.2 The results of Western-Blotting showed that the expression of PI3K protein and the phosphorylation of Akt and c-Myc in CD19+ BM-MNC group were higher than those in L-LDH group,which suggested that there is over-activation of PI3K/Akt/c-Myc pathway in children with H-LDH.2.3 EACR test reults using Seahorse XF24 cell energy analyzer showed that NLY294002 could lead to significant changes in the extracellular acidification rate of Nalm-6 cells and the changes of various statistical indicators,suggesting that PI3K/Akt/c-Myc pathway can down regulate intracellular glycolysis level.2.4 Analysis of Western-Blotting results of clinical samples:Compared with children with L-LDH,O-GlcNAc glycosyl levels were higher in children with H-LDH children,OGT expression was increased,but OGA expression was not significantly different.2.5 The results of glycolytic activation showed that the level of ECAR and related indicators in OGT-siRNA group were significantly decreased compared with the control group,but there was no significant difference between the control group and the NC-siRNA group.2.6 The results of real-time PCR showed that the expression of HK-2,HIF-1α,LDHA and PFK1 were down-regulated by interfering OGT(p<0.05),but Glutl had no significant change.2.7 Activation of PI3K/Akt/c-Myc pathway:The protein expression of PI3K was down-regulated and the phosphorylation levels of Akt and c-Myc were down-regulated after OGT was interfered,suggesting that OGT interfered with the activation of PI3K/Akt/c-Myc pathway.2.8 The results of glycolytic activation showed that compared with OGT-siRNA group,the level of glycolysis in OGT-siRNA + IGF-1 group was increased,indicating that activation of PI3K/Akt pathway can antagonize the inhibition of OGT-induced glycolysis inhibition.3.Combination of glycolysis inhibitor 2-DG and targeted inhibition of OGT can enhance the killing effect on Nalm-63.1 Western-Blotting results showed that O-GlcNAc glycosylation and OGT in Nalm-6 cells gradually decreased after 2-DG treatment.3.2 The results of CCK-8 showed that the proliferation of Nalm-6 cells gradually decreased after interfering with OGT expression.3.3 The results of CCK-8 showed that the OD value of 2-DG + OGT-siRNA group was significantly lower than the other 3 groups,indicating that combined application of 2-DG and targeted inhibition of OGT can enhance the inhibition of proliferation of Nalm-6 cells.3.4 Cell apoptosis assay results showed that the proportion of apoptosis in 2-DG +OGT-siRNA group was significantly higher than the other 3 groups,the difference was statistically significant.Conclusion:1.Overexpression of O-GlcNAc and increased expression of the key catalytic enzyme OGT exist in CD19+ BM-MNC in pre-B-ALL children,especially in children with high serum LDH.2.Interfering with OGT to decrease the level of O-GIcNAc modification can reduce the phosphorylation of important signaling proteins in PI3K/Akt/c-Myc pathway and further affect the metabolic process of glycolytic.3.Targeting the key enzymes in glycolysis process,interfering with inhibiting the expression of OGT and their combination can both inhibit the proliferation of Nalm-6 cells and promote apoptosis,which is of extremely important theoretical significance for the treatment of childhood leukemia.