| Objective:During recent years,various studies have confirmed that long non-coding RNA(lnc RNA)is differentially expressed in tumors and plays an important role in tumorigenesis and development,including acute myeloid leukemia(AML).PRR34-AS1(PRR34 antisense RNA1)is a lnc RNA of 1651 nucleotides and located on chromosome 22 q13.31,which is the antisense lnc RNA of PRR34 gene.At present,studies have reported that PRR34-AS1 plays a role as an oncogene in solid tumors such as hepatocellular carcinoma,but the role of PRR34-AS1 in hematological malignancies has not yet been studied.This study was aimed to preliminarily explore the expression and methylation level of PRR34-AS1 gene in newly diagnosed adult AML patients,and their impact on clinical prognosis,in order to provide an effective biomarker for prognosis stratification,treatment effect prediction and disease monitoring of AML patients.Methods:In this study,bone marrow(BM)specimens from AML patients and healthy controls and their corresponding clinical data were collected,bone marrow mononuclear cells(BMMNCs)were extracted from BM samples for a series of related experiments.RNA was reverse transcribed into c DNA,while DNA was treated for bisulfite modification.Using real-time quantitative PCR for detecting the relative expression of PRR34-AS1 in BM from healthy controls and AML patients.The methylation level of PRR34-AS1 promoter region was detected by real-time quantitative methylation specific PCR,and the density of PRR34-AS1 methylation was further verified by bisulfite sequencing PCR.Si RNA was used to silence the expression of PRR34-AS1 in leukemic cell line,and the proliferation ability of leukemic cell line after PRR34-AS1 interference was detected by CCK-8.Flow cytometry was used to detect the changes in apoptosis of leukemia cell line after PRR34-AS1 interference.Results:1.PRR34-AS1 gene expression in AMLThe GEO data sets GSE24006 and GSE63270 were analyzed and screened,and it was found that the expression of PRR34-AS1 gene was significantly up-regulated in AML patients compared with the control group(P < 0.001).Subsequently,according to the cutoff value determined by the median expression level of PRR34-AS1,AML patients were divided into two groups with PRR34-AS1 low expression and PRR34-AS1 high expression for comparison.The onset age of patients in the PRR34-AS1 high expression group was significantly older than that of the PRR34-AS1 low expression group(median 60 years vs 48 years,P = 0.001).In addition,the white blood cell(WBC)and platelet count(PLT)of patients in the PRR34-AS1 high expression group were significantly higher than those in the PRR34-AS1 low expression group(P = 0.041 and P = 0.004).In FAB subtypes,compared with PRR34-AS1 low expression group,PRR34-AS1 high expression group had a lower frequency of M3 type(7.1% and 26.8%,P = 0.020).The frequency of favorable karyotypes in PRR34-AS1 high expression group was lower(14.3% and 31.7%,P =0.059),while the frequency of intermediate karyotype was higher(76.2% and 58.5%,P = 0.069).AML patients who received induction chemotherapy showed that the complete remission(CR)rate of PRR34-AS1 high expression group was significantly lower than that of PRR34-AS1 low expression group(P = 0.004).Cox regression multivariate analyses demonstrated that PRR34-AS1 expression could serve as an independent factor affecting CR in patients with whole AML,non-acute promyelocytic leukemia AML(non-APL AML),and cytogenetically normal AML(CN-AML;P = 0.032,0.039,and 0.033,respectively).Kaplan-Meier survival analysis showed that high PRR34-AS1 expressers presented a significantly shorter overall survival(OS)than low PRR34-AS1 expressers in AML(P = 0.002).Bioinformatics analysis of GSE6891 chip data analysis confirmed that that the OS time of PRR34-AS1 high expression group was significantly shorter than that of PRR34-AS1 low expression group in overall AML patients(P < 0.001).2.DNA methylation of PRR34-AS1 gene in AMLCompared with the control,the methylation level of PRR34-AS1 promoter in AML patients showed a trend of hypomethylation(P = 0.120).According to the cutoff value determined by the receiver operating characteristic curve(ROC)curve,AML patients were divided into two groups: patients with hypomethylation and patients with hypermethylation in PRR34-AS1 promoter region.Patients with hypomethylated PRR34-AS1 had a higher WBC count than those with hypermethylated PRR34-AS1(P= 0.006).In karyotype classification,PRR34-AS1 hypomethylated group had a lower proportion of favorable karyotypes,while patients with intermediate karyotypes accounted for a higher proportion(P = 0.071).In the whole-cohort AML and non-APL AML,OS of hypomethylated PRR34-AS1 patients showed a trend of obvious shortening than patients with PRR34-AS1 hypermethylated(P = 0.010 and P= 0.032,respectively).Cox regression multivariate analysis was used to evaluate the prognostic value of PRR34-AS1 methylation.The results showed that PRR34-AS1 hypomethylation was an independent risk factor for OS in both AML patients and non-APL AML patients(P = 0.057 and P = 0.018,respectively).By analyzing the TCGA dataset,the results also confirmed that the OS of patients in the PRR34-AS1 hypomethylated group was significantly shorter than that of the PRR34-AS1 hypermethylated group(P < 0.001).Spearman correlation analysis showed that the expression level of PRR34-AS1 was negatively correlated with the methylation level of its promoter region(R =-0.236,P = 0.027).3.Study on the biological function of PRR34-AS1 in vitroAfter using siRNA to interfere with PRR34-AS1 gene in the leukemia cell line THP-1,the proliferation rate of THP-1 si PRR34-AS1 in the interference group was significantly slower than that of THP-1 si NC cells(P < 0.05),and apoptotic rate increased significantly(P = 0.002).Conclusion:1.High expression of PRR34-AS1 is a common molecular event in AML patients.PRR34-AS1 high expression is an independent risk factor for complete remission in AML patients.The overall survival time of AML patients with high expression of PRR34-AS1 is significantly shortened.The high expression of PRR34-AS1 may be related to the poor clinical prognosis of AML patients.2.PRR34-AS1 expression level depend on the regulation of the methylation status of its promoter region.PRR34-AS1 hypomethylation is significantly related to the overall survival time of AML patients,and PRR34-AS1 hypomethylation serves as an independent poor prognostic factor among whole-AML and non-APL AML patients.3.Silencing PRR34-AS1 expression can inhibit the proliferation of leukemia cells and promote the apoptosis of leukemia cells,suggesting that PRR34-AS1 may be associated with the occurrence and development of leukemia. |
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