叶酸缺乏的小鼠胚胎千细胞ES-D3 中miR-302a、Bcl2Ll1 表达观察及其靶向关系预测和验证.

Objective To investigate the expression changes of miR-302a and Bcl2Ll 1 and to explore the target gene prediction and verification in folate deficiency mouse ES-D3 cell line. Methods Proper number of mouse ES-D3 cells were cultured in folate-deficient culture medium, and were collected at predetermined time points: 0, 24, 48, 72 h (groups A, B, C, D). MiR-302a and Bcl2111 expression changes (BimEL, BimL, BimS) were detected by real-time qPCR andW estern blotting.M ultiple target gene prediction software was used to predict the binding sites between Bcl2111 and miR-302a. The amplification primers were designed and constructed according to the 3 UTR sequence of Bcl2111. The fragment of the Bcl211 1-3 UTR sequence was cloned into the pmiR-RB-REPORT double luciferase reporter vector, and the wild vector was successfully constructed. Similarly, Bcl2111 mutation primers were designed to construct the mutation vector. M戊－302a mimics and/or negative control were co-transfected with Bcl211 13 UTR double reporter wild vector and/or mutant vector, respectively. Therefore, the following four groups were constructed: group 1 (Bcl21113 UTR wild vector and negative control), group 2 ( Bcl21113-UTR wild vector and miR-302a mimics), group 3 ( Bcl211 l3 -UTR mutation vector and negative control), and group 4 ( Bcl21113UTR mutation vector and miR-302a mimics) . Changes on the relative fluorescence value of the hRluc reporter gene were analyzed to further confirm the interaction between miR-302a and Bcl2111 Results The relative miR-302a mRNA expression in the groups A, B, C, and D was 1. 00 土0. 20, 0. 65 土0 . 32, 0. 50 ± 0. 22, 0.43 土。24, respectively. Compared with the group A, miR-302a expression was down-regulated in the group D (P < 0. 05) . The relative Bcl2111 mRNA expression in the groups A, B, C, and D was 1. 00 土0 . 00, 1 . 22 土0. 19, 1 . 82 ± 0. 37, and 3. 49 ±0. 45, respectively. Compared with the group A, Bcl2111 expression was up-regulated in the group D (P < 0. 05) . The relative BimEL expression in the groups A, B, C, and D was O. 86 士0. 02, 0. 87 士0. 03, 0 . 92 士0 . 02, and 1. 10 士0. 06, respectively. Compared with the group A, the BimEL expression was up-regulated in the groups C and D ( both P < 0. 05 ) . The relative BimL expression in the groups A, B, C, and D was O, 0. 09 土0. 02, 0. 16 士0. 04, and 0. 26 ± 0. 03, respectively. Compared with the group A, the BimL expression was up-regulated in the groups B, C and D ( all P < 0. 05) . The relative BimS expression in the groups A, B, C, and D was O, 0, 0. 11 ± 0 . 04, and 0. 18 ± 0. 04, respectively. Compared with the group A, the BimS expression was up-regulated in the groups C and D ( both P <0. 05) Double luciferase report experiment indicated that the luciferase activities in the groups 1, 2, 3, and 4 were 1 . 00 土0 . 07, 0. 64 ± 0. 02, 1 . 00 土0. 03, and 0. 95 ± 0. 06, respectively. Compared with the other groups, the luciferase activity decreased in the group 2 ( all P < 0. 05 ) . Conclusions The expression of miR-302a was down-regulated, while the expression of Bcl2111 RN A and protein levels were up-regulated in folate deficiency mouse ES-D3 cells. Bcl2111 was strongly interacted with miR-302a, and thus it could be considered as the target gene of miR-302a. [ABSTRACT FROM AUTHOR]