Study On Expression Of MiR-181b And The Relationship With Its Target Gene CARD11 In Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia(CLL)is the most common type of leukemia in western countries,characterized by malignant clonal proliferation and aggregation of mature and typical CD5+B lymphocytes in bone marrow,peripheral blood,lymph nodes and spleen.Its clinical manifestations,disease progression,response to therapy and prognosis is highly variable.Some patients with diseases characterized by inertia never require treatment and die from causes other than leukemia.In other patients with aggressive disease,treatment begins shortly after diagnosis but early recurrence may occur.Therefore,early detection of patient prognosis is crucial to CLL treatment and the choice of treatment regimen.Up to now,some studies have found that micro RNAs(mi RNAs)regulate the occurrence and development of CLL through a variety of mechanisms and are considered as important prognostic factors of diseases.In the previous study,our group found that mi R-181 b was specifically under-expressed in CLL patients.Through bioinformatics software analysis and combined with literature reports,CARD11 was predicted to be a potential target gene of mi R-181 b.However,the study on the mechanism of mi R-181 b regulating CARD11 in CLL has not been reported,which still needs to be further confirmed by experiments.Objective: To detect the expression level of mi R-181 b in CD19+ B lymphocytes of CLL,patients,analyze the correlation between its expression and the prognosis of CLL,explore its prognostic value and provide new biomarkers for the early prognosis of CLL.CARD11 was verified to be the target gene of mi R-181 b in vitro test and the relationship between mi R-181 b and target gene CARD11 was studied in CLL.That could provide a new target and theoretical basis for future targeted therapy of CLL.Methods: 1)84 cases of CLL patients and 20 cases of healthy controls were collected from June 2013 to June 2018 in the People’s Hospital of Xinjiang Uygur Autonomous Region.RNA was extracted from CD19+B lymphocytes of peripheral blood by magnetic bead sorting.2)The expression of mi R-181 b in 84 CLL patients was detected by RT-q PCR,and its expression differences in different CLL-IPI groups were analyzed to explore the correlation between the expression level of mi R-181 b and the prognosis of CLL patients.3)The double luciferase reporting system and western-blot were used to confirm the regulation of CARD11 by mi R-181 b.The expression level of CARD11 in CLL was detected by RT-q PCR,and its correlation with mi R-181 b expression was analyzed.Results: 1)The expression level of mi R-181 b in CLL patients was significantly lower than that in the control group(P<0.01);2)(1)The expression level of mi R-181 b in the low-risk group was higher than that in the high-risk group and the very high-risk group(p <0.05),but there was no difference between the low-risk group and the intermediate-risk group(p=1.00).The expression level of mi R-181 b in the intermediate-risk group was higher than that in the high-risk group and the very high-risk group(p <0.05),but there was no difference between the high-risk group and the extremely high-risk group(p=1.00).(2)ROC curve results showed that the area under the curve(AUC)was 0.792(P<0.01).When the expression level of mi R-181 b isat the threshold value of 0.279,it has a good sensitivity(62.9%)and specificity(91.8%).(3)The results of survival analysis showed that the CLL patients in the group with low mi R-181 b expression had worse prognosis than those in the group with high mi R-181 b expression(P=0.047).(4)Cox regression analysis suggested that Rai stage,abnormal p53,IGHV mutation and low expression level of mi R-181 b were independent prognostic factors affecting the survival of CLL patients(P<0.05).3)(1)The luciferase reporter assay verified that mi R-181 b could combine with the CARD11 gene 3’UTR region to inhibit luciferase activity in CLL cell lines.The results of RT-q PCR and western-blot showed that mi R-181 b overexpression reduced m RNA and protein expression levels of CARD11,while mi R-181 b underexpression increased the m RNA and protein expression levels of CARD11,proving that CARD11 was a direct target gene of mi R-181 b in CLL.(2)The results of correlation analysis suggested that the expression levels of mi R-181 b and CARD11 m RNA were negatively correlated(R=-0.8047,P<0.001).Conclusion:The expression of mi R-181 b is reduced in CLL patients,and it has a negative regulatory effect on the target gene CARD11.The low expression of mi R-181 b is related to the poor prognosis of CLL,and which can be used as a new index for predicting the prognosis of CLL in the early stage.