The Study Of The Expression Level And Clinical Significance Of Coding RNA CRIP1 In Acute Myeloid Leukemia

Purpose Numerious researches have reported that CRIP1 plays a crucial role in tumorigenesis,but its expression status and clinical significance in hematologic diseases remain to be elucidated.The purpose of this study was to evaluate the expression of CRIP1 in AML,the clinical significance of CRIP1 abnormalities in AML and the possible regulatory mechanisms involved.Methods The expression of CRIP1 in 138 AML patients and 38 normal controls was detected by real-time quantitative PCR(RQ-PCR).We collected clinical data from patients and evaluated their clinical significance.Methylation-specific PCR(MSP)and bisulfite sequencing PCR(BSP)were used to detect the methylation status of CRIP1 promoter in AML.The correlation between the expression level of CRIP1 in AML and its methylation was evaluated.Five available public AML datasets from The Cancer Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)were used to further validate our results.The microRNAs that may regulate the expression level of CRIP1 in AML were predicted through the bioinformatic analysis,and the correlation between the microRNAs screened and the target gene CRIP1 was analyzed in AML specimens.Results The whole cohort of AML patients was divided into two groups with low and high expression according to the median level of CRIP1 expression(1.330).The white blood cell(WBC)of patients with high expression in CRIP1 was significantly higher than that of CRIP1 low expression(P = 0.002).The hemoglobin count of patients with high CRIP1 expression was lower than that of CRIP1 low expression(P = 0.046).The CR rate of high CRIP1 expression group was significantly lower than that of low CRIP1 expression group(P = 0.020).Kaplan-Meier analysis showed that patients with high expression of CRIP1 had a shorter overall survival(OS)time than those patients with low expression of CRIP1 both in the whole-cohort AML and in CN-AML patients(P = 0.013 and 0.007,respectively).In Non-M3 AML patients,there was a trend that patients with high expression of CRIP1 had shorter OS than those with low expression of CRIP1(P = 0.057).In CN-AML patients,the leukemia-free survival(LFS)of CRIP1 high expression group was shorter than that of CRIP1 low expression group(P = 0.012).Cox regression analysis found that high expression of CRIP1 was an independent risk factor for adverse prognosis in patients with CN-AML(P = 0.005).These results were further verified in the TCGA dataset and the GSE12417 dataset.In the public database,patients with high expression of CRIP1 had shorter OS and LFS than patients with low expression of CRIP1 in CN-AML patients(P < 0.05).Moreover,the expression level of CRIP1 was significantly down-regulated in AML patients who obtained CR,and its expression was significantly up-regulated in AML patients relapsed(P < 0.05).Moreover,CRIP1 expression was analyzed in nine patients with serial samples,which also confirmed that CRIP1 expression significantly decreased after CR(P = 0.005).The methylation level of CRIP1 was examined by MSP,which showed that there was no significant difference between the control groups and AML patients(P = 0.535).Then,two controls and two AML patients were selected randomly to verify the MSP results by BSP.Consistent with the MSP results,the CRIP1 promoter was almost completely unmethylated not only in healthy donors but also in AML patients.Differential methylation analyses were performed in the dataset GSE63409(n = 74).Our analyses yielded 1628 significantly down-regulated genes(FDR < 0.05,log2 FC > 2)in controls versus AML patients,however,CRIP1 was not included.Spearman correlation analysis showed that there was no correlation between CRIP1 expression level and methylation level in AML patients(R =-0.027,P = 0.855).Furthermore,the data in the TCGA dataset also confirmed our results(R =-0.014,P = 0.858).Two microRNAs that could target CRIP1,miR-299-5p and miR-3173-5p,were screened through the website.We analyzed the expression of these two microRNAs AML patients.The results showed that there were no significant differences in gender,age,hemoglobin count,and platelet count between the miR-299-5p high and low expression groups(P > 0.05).However,the WBC count of patients with low expression of miR-299-5p was significantly higher than that of patients with high expression of miR-299-5p(P = 0.007).The bone marrow blast count of patients with miR-299-5p low expression group was significantly higher than that of miR-299-5p high expression group(P = 0.019).The CR rate of miR-299-5p low expression group was significantly lower than that of miR-299-5p high expression group(P = 0.031).In AML patients,the expression of miR-299-5p was correlated with cytogenetic risk groups and specific karyotypes(P = 0.002 and 0.011,respectively).Moreover,the mutation rate of NPM1 in miR-299-5p low expression group was significantly higher than that in miR-299-5p high expression group(P = 0.028).However,there were no significant differences between miR-3173-5p high and low expression groups in gender,age,WBC count,hemoglobin count,platelet count,bone marrow blast count,cytogenetic risk grouping,specific chromosome karyotype,and common gene mutations(P > 0.05).The CR rate of miR-299-5p low expression group was significantly lower than that of miR-299-5p high expression group.This is contrary to that the CR rate of CRIP1 high expression group was significantly lower than that of CRIP1 low expression group.There was no significant difference in CR rate between patients with low expression of miR-3173-5p and those with high expression of miR-3173-5p(P > 0.05).Spearman analysis further explored the correlation between miR-299-5p and miR-3173-5p and the expression level of CRIP1 in AML,which showed that CRIP1 expression in AML was not correlated with miR-299-5p(R =-0.050,P = 0.624)and miR-3173-5p(R =-0.111,P = 0.277).Conclusion High expression of CRIP1 is an independent risk factor that affects the prognosis of patients with CN-AML.CRIP1 expression could be used for disease monitoring.CRIP1 promoter was nearly fully unmethylated in AML as well as controls.The expression of CRIP1 may be not regulated by its methylation in AML.CRIP1 expression may be not regulated by miR-299-5p or miR-3173-5p in AML.