The Expression And Clinical Significance Of Nanog Foxo3 And Circ-Foxo3 In Acute Myeloid Leukemia

Aim: Many researchers believed that Nanog gene was an oncogene which can promote the occurrence and development of tumors.Therefore,we will study the expression of Nanog in acute myeloid leukemia(AML)patients and its diagnosis value of Nanog in AML.In addition,we wonder whether there was a tumor suppressor gene which can inhibit the progression of the tumor and affect the patient's overall prognosis.Foxo3 was considered by most researchers as a longevity gene and tumor suppressor gene.So in this study,we tested the expression of Foxo3 and and its circular RNA(circ-Foxo3).Besides,we did a research on the diagnostic value of Foxo3 and and circ-Foxo3 to AML.Moreover,the correlation between Foxo3 and circ-Foxo3 was conducted on Pearson correlation analysis.At last,we study the affect of Foxo3 and circ-Foxo3 on prognosis of AML patients.Methods: 1.We collected a large number of AML patients' bone marrow specimens.We used Ficoll to separate AML patients and healthy donors of mononuclear cells.Then,we used Trizol reagent to separate total RNA from mononuclear cells,and use random primers to reverse transcription of total RNA to form c DNA.The Nanog expression of 146 de novo AML patients and 31 healthy people was conducted on Q-PCR.ROC was used to determine the diagnosis of AML by Nanog.We used ROC curve to distinguish AML patients from healthy controls.Kaplan-Meier survival analysis was performed to study the effect of Nanog on the prognosis of AML patients.2.The same method as above was used to detect the expression of Foxo3 in 122 de novo.AML samples,30 healthy control samples and five different cell lines;circ-Foxo3 expression was detected in 117 de novo.AML samples,24 healthy control samples,and five different cell lines.Resutlt: In our research group,we took on heavy experinment task,and we test the Nanog,Foxo3 and circ-Foxo3 in differnet periods.Moreover,the number of samples of the specimen library was in dynamic fluctuation.So,we tested these molecular abnormalities in different amount of samples respectly.The results of the two research was as follows:1.Compared with 31 healthy controls,the expression of Nanog in 146 AML patients was significant up-regulated(P=0.01).Through ROC curve analysis,Nanog has diagnostic value for AML patients with an area of 0.644(95% CI: 0.556 ~0.731;P=0.012).sensitivity and specificity are 46.9% and 87.1%,respectively.Based on the cut-off value of 0.324,we divided AML patients into Nanog high and Nanog low group groups.There was no significant difference among gender,age,WBC,PLT,Blast,FAB classification and multiple gene mutations between Nanog high and Nanog low group.High expression of Nanog was mainly found in non-favorable patients(P=0.027).After COX multivariate analysis,low expression of Nanog and WBC are two independent risk factors for AML patients.The median of overall survival time in AML patients was 7 months.The Nanog high group patients survived longer than that Nanog low group(P=0.484).In non-M3 patients,the survival time of Nanog low group was lower than in the Nanog high group(P=0.126);in the non-favorable group,Nanog high group patients had longer survival time than in Nanog low group(P=0.031).2.(1)compared with 30 healthy controls,the expression of Foxo3 in 122 de novo.AML patients was significantly up-regulated(P<0.01).There was no statistically significance between Foxo3 high and Foxo3 low groups on sex,age,WBC,PLT,Blast,WHO classification,chromosome type and seven gene mutations.Through ROC curve analysis,Foxo3 has diagnostic value for AML patients with an area of 0.655(95%CI:0.556~0.753;P=0.009),the sensitivity and specificity are 44.7% and 87.7% respectively.Based on the cut-off value of 0.856,we divided AML patients into Foxo3 high and Foxo3 low group groups.After COX multivariate analysis,the high expression of Foxo3 in AML patients is an independent protective factor in AML patients.Meanwhile,chromosome risk classification and age were independent risk factors in AML patients.The median value for overall survival time of AML patients was 10 months.The Foxo3 high group patients lived longer than Foxo3 low group(P=0.192).In the non-M3 group,non-favorable and normal chromosome group,Foxo3 high group patients survived longer than Foxo3 low group.The P values were 0.002,0.004,and 0.034,respectively.The results were statistically significant.(2)The expression of circ-Foxo3 was lower in 117 de novo.AML patients than in 24 healthy controls(P=0.04).There was no statistically significance between Foxo3 high and Foxo3 low groups on sex,age,WBC,PLT,Blast,WHO classification,chromosome type and seven gene mutations.Through ROC curve analysis,Foxo3 has diagnostic value for AML patients with an area of 0.633(95%CI: 0.523 to 0.742;P=0.041),sensitivity and specificity of 62.1% and 75%,respectively.Similarly,the cut-off value of circ-Foxo3 was 0.233,and we used this value to divide AML patients into Foxo3 high and Foxo3 low group.COX multivariate analysis resulted that high expression of circ-Foxo3 in AML patients was not an independent factor in AML patients.Meanwhile,chromosome risk classification,age,WBC,PLT,etc.were also not independent risk factors in AML patients.So we conclude that circ-Foxo3 was not an indispensable factor in predicting prognosis of AML.(3)We performed Pearson correlation analysis on expression of Foxo3 and circ-Foxo3 in the five cell lines.Results showed that the expression of Foxo3 and circ-Foxo3 in cell lines was positive correlation(R=0.98,P<0.0021).The expression of Foxo3 and circ-Foxo3 in AML patients were also positive correlation(R=0.63,P<0.0001).Conclusion: 1.Overexpression of Nanog was a common molecular event in AML,and had a significant impact on prognosis of non-favorable AML patients.It was a new biomarker in AML.2.The abnormal expression of circ-Foxo3 and Foxo3 was a common molecular event in AML,and circ-Foxo3 could down-regulated Foxo3 gene and leads to a better prognosis of acute myeloid leukemia patients.