MiR-199a-5p regulates sirtuin1 and PI3K in the rat hippocampus with intrauterine growth restriction

Abstract

In humans, malnutrition during pregnancy results in intrauterine growth restriction (IUGR) and an increased risk of neurological morbidities; altered miRNA characteristics have been suggested to contribute to IUGR neurological pathogenesis. A miRNA microarray was used to identify differentially expressed miRNA molecules in the hippocampi of rats with IUGR. Five of the molecules in question were selectively validated using real-time PCR in rats with IUGR. We then investigated the role of miR-199a-5p in hippocampal pathology. Bioinformatics analysis results suggested that TNF-α, caspase-3 and SIRT1 were potential targets of miR-199a-5p. Changes in PI3K, SIRT1 and caspase-3 protein expressions levels in the hippocampus were confirmed by Western blot analysis (all P < 0.05). Studies using the pheochromocytoma cell line PC12 cells and primary neurons demonstrated that miR-199a-5p modulated PI3K, caspase-3 and SIRT1 expression. Additionally, there was an inverse correlation between miR-199a-5p and caspase-3 expression, though dual-luciferase reporter assays showed that caspase-3 is not a target of miR-199a-5p. We conclude that IUGR affects hippocampal miRNAs characteristics. Our results also indicated that aberrantly high expression levels of miR-199a-5p may play an important role in the pathogenesis of IUGR by regulating SIRT1 and PI3K.

Figure 1

MiR-199a-3p, miR-199a-5p, miR-204,miR-34C and miR-219 expression levels in the hippocampi in the two groups on DOL 1 and DOL7 as measured via qRT-PCR. (a) miR-199a-3p, miR-199a-5p, miR-204, miR-34C and miR-219 expression levels between the control and IUGR groups on DOL7 (all groups, n = 8 rats). (b) The fold differences between the control and IUGR groups on DOL1. The values are expressed as the mean ± SD. *P < 0.05, **P < 0.01.

Figure 2

KEGG pathway analysis of differentially expressed miRNAs.

Figure 3

Hippocampal Caspase-3, PI3K, SIRT1 and TNF-α protein and mRNA expression levels on DOL1. (a) Representative Western blots for caspase-3, PI3K, SIRT1, TNF-α and β-actin (all groups, n = 8 rats). (full-length blots/gels are presented in Supplementary Fig. S5). (b) Representative Western blotting results for PI3K, TNF-α, SIRT1, pro-caspase3, cleaved-Casp3 (19KD) and cleaved-Casp3 (17KD) in control and IUGR groups. 35KD is pro-Casp3, 17KD and 19KD are cleaved Casp3. (c) Representative Casp3, SIRT1, PI3K and TNF-α mRNA expression levels as determined via RT-PCR in two groups. The values are expressed as the mean ± SD. *P < 0.05, **P < 0.01.

Figure 4

MiR-199a-5p inhibitors PI3K, TNF-α and SIRT1. (a,b) The alignment between Mus musculus miR-199a-5p and the 3-UTR of TNF-α and SIRT1, identified by TargetScanS software. (c–e) qRT-PCR analysis of PI3K (c), TNF-α (d) and SIRT1(e) mRNA expression levels in the PC12 cells following transfection. (g–i) Western blot analysis of PI3K (g), TNF-α (h) and SIRT1 (i) protein expression in PC12 cells following transfection. (full-length blots/gels are presented in Supplementary Fig. S7). The values are expressed as the mean ± SD. *P < 0.05, **P < 0.01.

Figure 5

Casp3 isn’t target of miR-199a-5p. (a) The alignment between Mus musculus miR-199a-5p and the 3-UTR of Casp3, identified by TargetScanS software. (b) qRT-PCR analysis of Casp3 mRNA expression levels in the PC12 cells following transfection. (c,d) Western blot analysis of Casp3 protein expression in PC12 cells following transfection. (full-length blots/gels are presented in Supplementary Fig. S8). (e) Comparison of luciferase activity in the NC and miR-199a-5p mimic groups. The values are expressed as the mean ± SD. *P < 0.05, **P < 0.01.