ABSTRACT
Objective To verify the targeting regulatory relationship between microRNA -939 (miR -939) and CD2 -associated protein (CD2AP).Methods The online RegRNA software was used to predict the human CD2AP promoter for potential binding sites complementary to miR -939.HEK -293T cells were cotransfected with hu-man CD2AP promoter plasmid pGL3 -2K and microRNA negative control (miR -NC)or miR -939 mimics,and the relative luciferase activity(RLA)was detected at 24 h post -transfection.HEK -293T cells were transfected with miR -NC or miR -939 mimics for 48 h,and the CD2AP mRNA expression level was detected by adopting reverse tran-script and real -time fluorescence quantification -PCR,while the CD2AP protein expression level was detected by using Western blot.Results (1 )There were 2 miR -939 binding sites at CD2AP promoter region,located at -468 to -491 and -654 to -677 upstream of initiation codon ATG (marked as +1 )relatively.(2)At 30 nmol/L,50 nmol/L,the RLA in miR -NC group and miR -939 group were 6.81 ±0.88 vs 6.07 ±2.24,5.88 ±1 .44 vs 3.94 ± 0.79 relatively,and there were no significant differences between the 2 groups (t =3.04,2.06,all P >0.05),while the RLA between the 2 groups were 5.58 ±0.58 vs 3.29 ±0.64 at 1 00 nmol/L,and the difference was significant between the 2 groups(t =4.07,P <0.05).(3)At 30 nmol/L,50 nmol/L and 100 nmol/L,the relative CD2AP mRNA expression in miR -NC group and miR -939 group were 1 .00 ±0.01 vs 0.80 ±0.08,1 .00 ±0.00 vs 0.80 ±0.1 3 and 1 .00 ± 0.00 vs 0.72 ±0.07 relatively,while the CD2AP mRNA expression was decreased by 20% -30% at each concentration level,and there were significant differences between the 2 groups (t =4.44,2.93,6.84,all P <0.05).(4)At 50 nmol/L, the relative CD2AP protein expression in miR -NC group and miR -939 group were 0.48 ±0.09 vs 0.19 ±0.12,and the CD2AP protein expression was decreased,and the difference was significant (t =3.36,P <0.05).Conclusions CD2AP is the target gene of miR -939,and miR -939 can down -regulate the expression of CD2AP both in mRNA and protein levels by targeting its promoter region,which indicates that miR -939 may mediate the podocyte injury.
ABSTRACT
Objective To study the mRNA expression levels and clinical significance of omsomucoid 1-like protein 3 (ORMDL3) gene in the peripheral blood of recurrent wheeze children under 3 years old.Methods Peripheral blood specimens of 25 recurrent wheeze children including 14 non-atopy patients (group A) and 11 atopy patients (group B) that were registered in the First Affiliated Hospital of Nanjing Medical University,from Sep.2010 to Sep.2012 were enrolled based on the inclusion criteria and 24 non-allergic controls(the children with food allergy,drug allergy or ectema was excepted).The mRNA expression levels of ORMDL3 gene were detected by reverse transcription (RT)-PCR and clinical features were analyzed.Results The expression levels of ORMDL3 were up-regulated in the peripheral blood specimens of group B compared with group A (t =14.12,P ＜ 0.01).Compared with peripheral blood specimens from normal subjects,mRNA expression of ORMDL3 were significantly increased in recurrent wheeze children(t =10.29,5.73,P ＜0.01).The incidence of wheeze groups exist gender differences,male ＞ female.Wheeze usually with a high incidence in winter and spring.Conclusions The increase of ORMDL3 gene expression levels were correlated with recurrent wheeze under 3 years old especially in atopy group and may be involved in the pathogenesis of recurrent wheeze.