Study On The Regulation Of MiR-449a For LGR4 Gene In Dairy Goat Endometrial Stromal Cells

MicroRNAs(miRNAs) were small, evolutionarily conserved noncoding RNA molecules that acted as posttranslational regulators of gene expression in a sequence-specific manner. mi R-449 a induced cell differentiation or apoptosis in a mutually exclusive fashion. LGR4 regulated cell differentiation, it was thought to be involved in reproductive systems. Our previous Illumina Solexa sequencing results indicated the expression levels of mi R-449 a significantly increased in receptive endometrium compared with pre-receptive endometrium in dairy goat(P<0.01), the expression level of LGR4 was significantly reduced(P<0.05). Bioinformatics analysis showed LGR4 was a target of miR-449 a. So, it was important to study the target regulation of miR-449 a onLGR4 in endometrial stromal cells(ESC) for illuminating the molecular mechanism of dairy goat endometrial receptivity.The Saanen dairy goats were treated as the research subjects in this study, the expression levelsof miR-449 a were detected by stem-loop RT-qPCR in receptive endometrium and pre-receptive endometrium of dairy goat, and the expression levels of LGR4 were detected by RT-qPCR. The regulation of miR-449 a onLGR4 was validated by Dual luciferase report vector, RT-qPCR and Western blot, the effect of mi R-449 a on ESC was tested by MTT, cell apoptosis and cycle analysis, and the regulation of miR-449 a onP53, Bcl-2 and FAS were deceted by RT-qPCR and Western blot. The main results of this study were as follows:1. The expression levels of mi R-449 a and LGR4 in receptive endometrium and pre-receptive endometrium of dairy goat were respectively detected by stem-loop RT-qPCR and RT-qPCR. The expression level of miR-449 a was significantly higher in receptive endometrium compared with pre-receptive endometrium in dairy goat(P< 0.05), but the expression level of LGR4 mRNA significantly decreased(P< 0.05).2. The wild type psiCHECK2-LGR4-3′UTR-WT and the mutant type psiCHECK2-LGR4-3′UTR-Mut Dual-luciferase reporter vectors were constructed, and were co-transfected into HEK293 T cells with miR-449 a mimic, miR-449 a inhibitor, Negative control(NC) and Negative control inhibitor(NCH), respectively. The luciferase activities were measured with the Dual-Glo luciferase assay system according to the manufacturer′s instructions. The results showed that the luciferase activity of the mi R-449 a group was significantly lower than that of the NC group(P< 0.05), and the reduction was rescued in the mutation group. Thus, the results suggested that LGR4 was a target of miR-449 a in goat.3. ESC and HEK293 T cells were respectively transfected with miR-449 a mimic, mi R-449 a inhibitor, Negative control(NC) and Negative control inhibitor(NCH) using Lipofectamine 2000.The expression of LGR4 on transcriptional level and translational level were detectd using RT-qPCR and Western blot. This study found that miR-449 a inhibited expression of target gene LGR4 on transcriptional and protein levels. The results showed that mi R-449 a negativily regulated expression ofLGR4.4. The effect of miR-449 a on ESC was detected using MTT, cell apoptosis and cycle analysis. The results showed that mi R-449 a caused the inhibition of cells proliferation and induced ESC apoptosis. By cell cycle analysis, miR-449 a made ESC stagnating in S phase(39.09% Vs 36.13%). Further research showed that miR-449 a down-regulated the expression levels of p53 in ESC, and miR-449 a significantly decreased the mRNA levels of FAS(P< 0.05). No significant changes were found on the mRNA levels of Bcl-2 after the ESC were transfected with miR-449 a. These results suggested that miR-449 a induced ESC apoptosis though the FAS-mediated pathway.