The Role And Mechanisms Of NR3C1 Gene Mutations In Drug Resistance Of Acute Lymphoblastic Leukemia

Background:Acute lymphoblastic leukemia?ALL?,one of the common malignant tumors in children,is a kind of hematological malignant disease with highly heterogeneous genetic background.With the constant improvement of ALL treatment,survival rate of ALL have got remarkable improvement.However,there are a considerable portion of ALL patients develop resistance and relapse to treatment.Glucocorticoid is the primary medicine to treat ALL,through out different chemotherapy regimens of ALL.Response to glucocorticoid is an independent prognostic factor in patients with ALL[I]Glucocorticoid resistance is one of the important factors of the treatment failure in patients with ALL,while the mechanism of drug resistance is not completely clear.lt has been reported that the deletion of NR3C1 gene was specifically found in the relapse of ALL patients after chemotherapy[2].In our previous study,we performed longitudinal whole-exome sequencing analysis in matched samples from adult patients with Philadelphia chromosome-negative?Ph—?B-cell acute lymphoblastic leukemia?Ph-B-ALL?from diagnosis to relapse after allogeneic hematopoietic stem cell transplantation?allo-HSCT?.We found that the NR3C1 gene,encoding a transcription factor/glucocorticoid receptor,was selectively mutated in relapsed samples and is therefore a putative relapse-specific mutation in adult ALL.Furthermore,mutations within the NR3C1 genes occurred exclusively in sites that are highly conserved across species and were distributed in glucocorticoid receptor functional domains,which would be predicted to result in the loss of protein function?exon2:c.431₄31delinsAG?p.D144Efs×11??.[3]Based on our previous study findings,in the present study,we attempt to elucidate the role and mechanisms of NR3C1 gene mutations in drug-resistance ALL by ex vivo cell studies.Objective:Based on our previous study,our object is to study the role NR3C1 gene mutations in the protein function of glucocorticoid receptor,to provide valuable information to research of the mechanisms of leukemia drug-resistance and improve the survival of ALL patients.Methods:?1?We treated ALL cell Iines?Reh,Jurkat,CCRF-CEM,6T-CEM,Nalm6?with different concentration?0.1umol/1,0.5umol/l,1.Oumol/1,2.5 umol/1,5 umol/1?of Dexamethasone?DEX?,and performed CCK8 assay to detect its cell proliferation rate after 24h or 48h.We used western blot technique/fluorescence quantitative PCR assay to detect the expression of NR3C1 gene of ALL cell lines.Based on the results,we analyzed the relationship between the expression of endogenous NR3C1 in ALL cell lines and the sensitivity to DEX.?2?We adopted lentiviral transfection method to make ALL resistant strains overexpress NR3C1 protein,and treated the cells with 1.Oumol/1 DEX for 48h.After these steps,we used flow cytometry and CCK8 assay to detect their apoptosis rate and cell proliferation rate,and determined whether NR3C1 can reverse drug resistance of ALL cell lines.?3?We adopted CRISRP/CAS9 system to knock out NR3C1 gene of ALL sensitive strains,and treated the cells with 1.0umol/1 DEX for 48h.After these steps,we used flow cytometry and CCK8 assay to detect their apoptosis rate and cell proliferation rate,and further determined the relation between the expression of NR3C1 and ALL's sensitivity to dexamethasone.?4?We employed lentivirus with wild type NR3C1 gene to infect DEX resistant cell line Reh.Then we extracted the total RNA,and prepared library for mRNA sequence.After examination of library,we performed the sequencing of the whole genome at the transcriptome level,and filtered the raw reads,analyzed the clean reads.?5?We used 1.0umol/l DEX to treat ALL cell lines with NR3C1 overexpression or NR3C1 gene knocked out for 48h,then extracted the total protein.We adopted western blot technique to detect their expression of protein related to PI3K?AKT?GSK3? pathway or BCL-2 family.Statistical methods:We applied SPSS 20.0 statistical software to analyze data,and all experiments were repeated at least three times.The values between two groups were analyzed by independent sample T test.The values among more than two groups were analyzed by multivariate analysis.Measurement data were expressed in?X+S?.The inspection level was equal to 0.05 and two-tailed test.Results:?1?Detected by CCK8 assay,ALL cell lines Reh and Jurkat were resistant to DEX,but CCRF-CEM,6T-CEM and Nalm6 cell were sensitive to DEX with time and dose dependence.In addition,through western blot technique and fluorescence quantitative PCR assay,we found that Reh and Jurkat low expressed NR3C1 while CCRF-CEM,6T-CEM and Nalm6 high expressed NR3C1.These results indicated that the expression of endogenous NR3C1 in ALL cell lines was positively associated with the sensitivity to DEX.?2?Overexpression of NR3C1 protein of ALL resistant strains could reverse their resistance to DEX.Under the treatment of lumol/L DEX for 48h,compared to the negative control group,Reh group with NR3C1 overexpressing had lower cell proliferation rate?0.6467±0.03215,1.06±0.19123;P<0.05?and much higher cell apotosis rate?33.6%±0.34641%,6.5%±0.17321%;p<0.001?.Under the same condition,compared to the negative control group,Jurkat group with NR3C1 overexpressing had lower cell proliferation rate?0.45±0.0721,0.9733±0.00577;p<0.001?and much higher cell apotosis rate?57.4%±0.8888%,4.2333%±0.3214%;p<0.001?.?3?Knock out of NR3C1 gene of ALL sensitive strains could help ALL cells gain resistance to dexamethasone.Under the treatment of lumol/L DEX for 48h,compared to the negative control group,CCRF-CEM group with NR3C1 knocked out had higher cell proliferation rate?0.93±0.12 vs 0.15±0.04;p<0.001?and much lower cell apotosis rate?5.1 ± 1.7%vs 79.4±15.4%;p<0.001?.Under the same condition,compared to the negative control group,6T-CEM group with NR3C1 knocked out had higher cell proliferation rate?0.95±0.055 vs 0.16±0.006;p<0.001?and much lower cell apotosis rate?2.05±1.2%vs 67.4±4.2%;p<0.001?.Indentically,compared to the negative control group,Nalm6 group with NR3C1 knocked out had higher cell proliferation rate?0.91±0.11 vs 0.21 ±0.007;p<0.001?and much lower cell apotosis rate?3.2±1.5%vs 67.8±0.5%;p<0.001?.?4?Under the treatment of lumol/L DEX wthin 48h,compared to the negative control group,Reh group with NR3C1 overexpressing,at the transcriptome level,had lower expression of PIK3CD,BCL2 and BAG2,much higher expression of AKT3,GSK3?,PIK3R6,PIK3R5,PIK3R2,BCL2L11,BMF and BOK.?5?After the treatment of lumol/L DEX within 48h,related proteins of ALL cell lines with NR3C1 overexpressing or NR3C1 knocked out were detected by western blot technique.The results showed that overexpression of NR3C1 could promote PI3K?AKT?GSK3? signal pathway,promote the expression of pro-apoptotic proteins?Bad?Bim?and inhibit the expression of anti-apoptotic proteins?BCL-2,BCL-XL?.Interestingly,knock outNR3C1 gene could reverse this phenomenon.Conclusion:?1?The expression of endogenous NR3C1 in ALL cell lines was positively associated with the sensitivity to DEX.?2?Overexpression of NR3C1 protein of ALL resistant strains could reverse their resistance to DEX.?3?Knock out of NR3C1 gene of ALL sensitive strains could help ALL cells gain resistance to dexamethasone.?4?NR3C1 promoted PI3K?AKT?GSK3? signal pathway,promoted the expression of pro-apoptotic proteins?Bad,Bim?and inhibited the expression of anti-apoptotic proteins?BCL-2,BCL-XL?.