The Clinical Study On Expression And The Methylation Pattern Of DOK6 And LINC00324 In Acute Myeloid Leukemia

Objective:The purpose of this study was to investigate the expression and methylation status of Downstream of tyrosine kinase 6(DOK6)and Long Intergenic Non-Protein Coding RNA 00324(LINC00324)in patients with acute myeloid leukemia(AML),and to further analyze their clinical significance and the effect on the biological phenotype of leukemia cell lines.Methods:In this study,bone marrow samples and corresponding clinical data were collected from 32 normal people and 108 newly diagnosed adult patients with AML.The relative expression levels of DOK6 and LINC00324 in bone marrow samples of normal and AML patients were detected by qPCR technology.The RQ-MSP technology was used to detect the promoter methylation levels of DOK6 and LINC00324 in bone marrow samples from healthy donors and AML patients.BSP technology was used to verify the accuracy of MSP test results.Demethylation drug 5-aza-dC was used to reduce the level of genomic DNA methylation in leukemia cell lines,and possible epigenetic regulation mechanism was analyzed by observing the changes in DOK6 and LINC00324 expression and promoter methylation levels between treatment groups.Cell proliferation was used to detect the proliferation of leukemia cell lines after overexpression of LINC00324.FCM was used to detect the change of apoptosis in leukemia cell lines after overexpression of LINC00324.Results:1.DOK6 is lowly expressed and hypermethylated in AML and is significantly correlated with the prognosis of AML patients.The mRNA expression of DOK6 in bone marrow samples of newly diagnosed AML patients was significantly lower than that in the control group,and the difference was statistically significant(P < 0.001).According to the cutoff value determined by the ROC curve,AML was divided into two groups with high and low expression.It was found that the higher expression group of DOK6 low expression group had a smaller age of onset(median: 54 years vs.59 years,P = 0.031),fewer platelet(PLT)counts(median: 29.5 * 10^9/L vs.48.0* 10^9/L,P = 0.046)and higher complete response rates(55.8% vs.25.0%,P = 0.014).Kaplan-Meier analysis showed that the DOK6 low expression group had longer overall survival(OS)in both overall AML and non-acute promyelocytic leukemia(non-APL)than the high expression group(P = 0.010 and P = 0.045).Cox univariate and multivariate analysis also found that low DOK6 expression was an independent influencing factor for overall survival in patients with AML(P = 0.016).Compared with the control group,the DOK6 promoter region of AML patients showed significantly higher levels of methylation(P = 0.037).There was a statistically significant difference in FAB distribution between patients with hypermethylated AML and patients with hypomethylated AML in the DOK6 promoter region(P = 0.037).In overall AML cases,patients with hypermethylated DOK6 promoter region had significantly longer overall survival(OS)than patients with hypomethylated DOK6 promoter region(P = 0.042).In addition,in non-APL patients and AML patients younger than 60 years of age,the OS of patients with hypermethylation in the DOK6 promoter region was also significantly longer than that in patients with hypomethylation in the DOK6 promoter region(P = 0.036,P = 0.031).Multivariate analysis showed that methylation of the DOK6 promoter region was an independent factor for overall survival in patients with non-APL and AML patients younger than 60 years of age(P = 0.052,P = 0.043).After 5-aza-dC treatment of leukemia cell line THP-1,the methylation level of the DOK6 promoter region decreased and the transcription level increased,and the changes were concentration-dependent within a certain range(0-10mmol/l).2.LINC00324 is hypoexpressed and hypomethylated in AML and is associated with the proliferation of leukemia cell lines.LINC00324 had significantly lower transcription levels in patients with overall AML,non-APL,and cytogenetically normal AML(CN-AML)than the control group,and the differences were statistically significant(P < 0.001,P < 0.001 and P < 0.001).In the overall AML case,the higher expression group of the LINC00324 low expression group had higher hemoglobin(Hb)content,and the difference was close to statistical significance(median: 80.0g/l and 75.0g/l,P = 0.058).Kaplan-Meier analysis found that the LINC00324 low-expression group had a longer OS compared to higher expression group in overall AML cases,and the difference was statistically significant(P = 0.029).LINC00324 was significantly hypomethylated in patients with overall AML and non-APL(P = 0.038 and P = 0.045).There was a difference in FAB typing between the two groups of LINC00324 high and low methylation(P = 0.032).Overexpression of LINC00324 in leukemia cell lines HL60 and THP1 can promote proliferation and inhibit apoptosis of leukemia cell lines.Conclusions:1.The low expression of DOK6 and the hypermethylation of the promoter region are common molecular events in newly diagnosed AML patients and are significantly related to the overall survival time of AML patients.2.In the leukemia cell line THP-1,the transcription level of DOK6 is partially affected by its promoter region.3.Low expression of LINC00324 and hypomethylation in the promoter region are common molecular events in newly diagnosed AML patients,and the expression level of LINC00324 does not depend on the regulation of the methylation status of its promoter region.4.LINC00324 promotes proliferation and inhibit the occurrence of apoptosis in leukemia cell lines.