MicroRNA-939 regulating CD2-associated protein expression by targeting promoter

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 human 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 transcript 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 100 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±0.13 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. Key words: MicroRNA-939; CD2-associated protein; Podocyte; Promoter; Gene expression regulation