Clinical Implication Of Abnormal Methylation Of CpG Islands In SLIT2 Promoter Region And Its Mechanism In MDS/sAML Transition

Objective: Myelodysplastic syndrome(MDS)is a heterogeneous myeloid clonal disease that originates from hematopoietic stem/progenitor cells,often accompanied by pathological/ineffective hematopoiesis,and presents a high risk of progression to acute myeloid leukemia(s AML).So far,the specific mechanism of the onset and progression of MDS has not been clarified.Some MDS patients may have gene mutations such as DNMT3 A and TET2 or chromosomal abnormalities like 5q-and-7.However,the above-mentioned abnormal changes don’t occurr in all MDS patients,so gene mutations or chromosomal abnormalities cannot be used to explain the onset and progression of all MDS patients.In clinical work,decitabine,the demethylation drug,can reverse promoter DNA methylation of MDS-related tumor suppressor gene and delay the transformation to AML,suggesting that epigenetic modification plays an important role in MDS progression.This study aims to explore the clinical significance of the differentially methylated gene SLIT2 in MDS transformation,identify the target molecules regulated by CpG island hypermethylation in the promoter region of SLIT2,and analyze its specific anti-leukemia effects and molecular mechanisms.Methods: Reduced representation bisulfite sequencing was carried out in 5 cases of paired MDS progression samples and differentially methylated genes related to MDS disease progression were screened out.Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were used to enlarge the specimens to verify the changes of SLIT2 methylation before and after MDS progression,and its clinical significance was further analyzed.Real-time quantitative PCR was applied to detect the expression of possible target genes that may be regulated by SLIT2 methylation.The correlations between these target genes and SLIT2 methylation were further analyzed.MDS/AML cells were treated with demethylation drugs to further identify the target genes regulated by SLIT2 methylation.Cell lines with stable overexpression of the target genes were constructed and the changes in biological behaviors such as cell proliferation,cell cycle,apoptosis,differentiation,and colony formation were observed.Mouse leukemia models were established.The effects on leukemia and tumor growth were observed through live imaging,tissue HE staining/immunohistochemistry,bone marrow biopsy,and cellular immune typing.RNA-FISH was used to determine the intracellular localization of the target lnc RNA.The downstream genes of the methylation-dependent lnc RNA/miRNA molecules were screened via transcriptome sequencing and prediction websites.Dual luciferase experiments and AGO2-RIP experiments were applied to prove the direct binding between genes.By knocking down or restoring the m RNA expression of downstream target genes,the mechanisms involved in MDS transformation were further clarified.Results: 1.Screening of MDS progression-related differentially methylated gene SLIT2 and analyzing its clinical significances.After sequencing and analyzing 5matched MDS transformation specimens,the differentially methylated gene SLIT2 was screened out.By detecting larger specimens,SLIT2 promoter methylation level and density were significantly increased in s AML stage of 11 matched MDS/s AML patients,and SLIT2 methylation levels in AML patients were significantly higher than in controls and MDS patients,suggesting that SLIT2 methylation might be involved in MDS progression.By clinical parameters analysis,the ratios of poor karyotype and intermediate-risk-2/high-risk group in the SLIT2 hypermethylated group were significantly higher than that in the SLIT2 hypomethylated group in MDS,and the methylation of SLIT2 in complete remission(CR)phase was significantly lower than that at the initial onset in AML.Survival analysis suggested that SLIT2 methylation tended to be an independent factor affecting poor prognosis of MDS/non-APL/CN-AML patients.In non-MDS cytopenic diseases such as aplastic anemia,hemolytic anemia and megaloblastic anemia,SLIT2 methylation in bone marrow was significantly lower than in MDS patients.2.Identification of target genes regulated by CpG island hypermethylation in SLIT2 promoter region.The expression of SLIT2 in clinical specimens was significantly up-regulated compared with healthy controls,and our data and TCGA data showed that there were no correlation between SLIT2 methylation and its expression.When treated with SLIT2 recombinant protein,it was found that the recombinant SLIT2 protein can promote the proliferation of HEL and SKM-1,inhibit cell apoptosis,and might play a role in promoting leukemia.The above results suggested that CpG island hypermethylation in SLIT2 promoter region did not function by regulating SLIT2 expression.There were two non-coding RNAs lying in SLIT2 gene sequence in UCSC website: long non-coding RNA SLIT2-IT1 and miR-218-1.In clinical specimens,SLIT2-IT1 was down-regulated in de novo AML,recovered slightly at CR phase,and the overall survival time of SLIT2-IT1 low-expressed patients was significantly shortened.Spearman correlation analysis indicated that SLIT2-IT1 expression was negatively correlated with SLIT2 methylation.For miR-218-1,the mature miR-218-1-3p expression was not associated with SLIT2 methylation,while the other mature miR-218-5p(abbreviated as miR-218)was down-regulated and negatively correlated with SLIT2 methylation in AML.After treated with demethylation drugs,SLIT2 promoter region was demethylated and the expression of SLIT2-IT1/miR-218 was up-regulated.The above results indicated that CpG island hypermethylation in SLIT2 promoter region might function by regulating the expression of SLIT2-IT1/miR-218.3.The anti-leukemia effect of lnc RNA SLIT2-IT1 and its mechanism.Cell lines stably overexpressing SLIT2-IT1 were constructed.Functional experiments found that SLIT2-IT1 overexpression didn’t affect the proliferation and apoptosis of erythroleukemia cell lines K562 and HEL,suggesting that SLIT2-IT1 was not involved in the occurrence and development of erythroleukemia.In HL60 and MDS-transformed leukocyte line SKM-1,SLIT2-IT1 overexpression decreased cell proliferation and colony forming ability,while increased apoptosis rates.SLIT2-IT1 could also block SKM-1 and HL60 in G0/G1 phase.In vivo experiments indicated that SLIT2-IT1 overexpression could delay the formation of AML in NCG mice.Mouse survival experiments also found that the survival period of mice in the SKM-1/SLIT2-IT1 group was significantly prolonged.The HE staining and immunohistochemistry of mice tissues indicated that the injected cells had metastasized to the liver,spleen,ovary and subcutaneous tissues of two groups to different degrees.Bone marrow biopsy,chromosome karyotype and cellular immunotyping revealed that the bone marrow of mice had infiltration of primitive cells,and the mice in control group developed myeloid leukemia.In order to explore the mechanism of the anti-leukemia effect of SLIT2-IT1,FISH was utilized to confirm the location of SLIT2-IT1 in cytoplasm,and the downstream mechanism was explored in the ce RNA mode.Illumina Hiseq sequencing and lnc RNA/miRNA target gene prediction websites were used to predict and screen the potential downstream miRNA and m RNA of SLIT2-IT1—miR-3156-3p and BMF,respectively.AGO2-RIP and dual luciferase experiments confirmed that miR-3156-3p could target SLIT2-IT1 and BMF.After knocking down BMF expression,cell proliferation speed and colony forming ability increased,apoptosis ratio decreased,G0/G1 phase ratio also decreased,and G2/M phase ratio increased,which reversed the effects of SLIT2-IT1 overexpression on cell proliferation,apoptosis and cell cycle.4.The anti-leukemia effect of miR-218 and its mechanism.Cell lines stably overexpressing miR-218 were constructed.Functional studies found that miR-218 overexpression could inhibit cell proliferation and colony formation,and promote cell apoptosis.Cell cycle experiments revealed that miR-218 might block HEL,SKM-1 and HL60 in G0/G1 phase.In addition,miR-218 overexpression increased the expression of CD235 a in HL60 cells,suggesting that miR-218 can promote the differentiation of HL60 cells into erythroid.In vivo experiments displayed that miR-218 overexpression also delayed the formation of AML in NCG mice.The HE staining and immunohistochemistry of mice tissues showed positive metastases in ovaries of the control mice.Bone marrow biopsy,chromosome karyotype and cellular immunotyping revealed that the bone marrow of mice had infiltrated primitive cells,and both groups developed myeloid leukemia.In order to explore the molecular mechanism of the anti-leukemia effect of miR-218,the downstream target gene HOXA1 was screened out through Illumina Hiseq sequencing and website prediction,and it was further confirmed by dual luciferase expriments.Rescue experiments presented that restoring HOXA1 expression increased cell proliferation and colony forming ability,reduced the apoptotic ratio,decreased the ratio of cells in G0/G1 phase,and increase the ratio of G2/M phase,which reversed the effects of miR-218 overexpression on cell proliferation,apoptosis and cell cycle.Conclusions: 1.CpG islands in SLIT2 promoter region of MDS and AML patients were hypermethylated,and abnormal hypermethylation might be involved in MDS progression.2.Abnormal methylation of the CpG islands in SLIT2 promoter region correlated with poor prognosis in MDS and AML patients,and could be used to monitor AML treatment.3.CpG islands hypermethylation in SLIT2 promoter region might regulate the expression of contained genes SLIT2-IT1/miR-218 instead of SLIT2.4.Lnc RNA SLIT2-IT1 could regulate the downstream BMF expression by competitively adsorbing miR-3156-3p and play an anti-leukemia effect.5.Mi R-218 could regulate HOXA1 expression by targeting the downstream HOXA1 3’UTR,and also play an anti-leukemia effect.