Placenta MicroRNA Expression Profile,Tanscriptome And Regulation Of MAPK Signaling Pathway By MicroRNA-424 In Amur Tiger

Placenta is an organ that connects the developing fetus to the uterine wall to allow nutrient uptake, to fight against internal infection and to produce hormones during pregnancy. After the release of genome data of the Amur tiger, epigenetics of Amur tiger has become a frontier research topic. MicroRNA (miRNA) is a small non-coding single-stranded RNA molecule with a length of about 18-25 nucleotides. It is mainly involved in posttranscriptional regulation. We applied high-throughput sequencing to miRNA expression profiling and transcriptome analysis of the Amur tiger placenta. The functions of placenta-specific miRNA were annotated, and the following conclusions are drawn:1. Small RNA sequencing was performed in the Amur tiger placenta using RNA-seq technique. After elimination of adapter sequences and low-quality sequences, the clean reads were aligned against the genome of the Amur tiger from http://www.ncbi.nlm.nih.gov/genome/?term=tiger, and annotated and analyzed. Thus the miRNA expression profile of Amur tiger placenta was obtained. The profile consisted of 465 miRNAs, which were divided into 41 clusters and 35 families by clustering analysis. Placenta-specific miRNAs were microRNA-424, microRNA-450, microRNA-495, microRNA-376a, microRNA-184, microRNA-369 and microRNA-503, which were further annotated. These miRNAs performed the functions of regulating fetal growth and development, angiogenesis, bone regeneration, muscle development, apoptosis and anti-apoptosis, cell proliferation and differentiation, granulocyte formation, immune response and hormone secretion.2. Transcriptome analysis was performed, including gene ontology (GO) analysis and clusters of orthologous groups of proteins (COG) analysis. In GO analysis 21103 genes were annotated by 45 GO terms, and 8462 genes were annotated and classified into COG categories. Using miRNA target gene prediction tools TargetScan and miRanda, the target genes were predicted throughout the transcriptome of the Amur tiger placenta. Moreover, the biological functions of these miRNA target genes were described and the miRNA regulatory network was mapped.3. The expressions of microRNA-424, the placenta-specific miRNA, and its target gene MAP2K1 in Amur tiger placenta and domestic cat placenta were detected using real-time PCR, tissue in situ hybridization and Western Blotting, respectively. The results showed that the expression of microRNA-424 in the Amur tiger placenta was significantly lower than that in the domestic cat placenta (**p<0.01); however, the expression of target genet MAP2K.1 in the Amur tiger placenta was significantly higher than that in the domestic cat placenta (**p<0.01).4. According to dual-luciferase reporter assay, there was no significant difference in the luciferase activity of the miRNA-424 locus targeting the 3’UTR of MAP2K1 gene after mutation. In contrast, the luciferase activity decreased significantly in the non-mutation group (**p<0.01). It was confirmed that miRNA-424 can directly bind to the 3’UTR of MAP2K1 gene. After miRNA-424 was overexpressed in the fibroblasts of the Amur tiger, Western Blotting showed that MAP2K1 expression was downregulated significantly (**p<0.01). To understand the biological functions of miRNA-424 in the fibroblasts of the Amur tiger, Western Blotting was performed to detect phosphorylation of ERK1/2, the downstream target of MAP2K1, and its expression level after the transfection of viral particles carrying miRNA-424. MTT assay was used to determine the proliferation of fibroblasts after transfection. The results showed that miRNA-424 not only directly regulated the transcription of MAP2K1, but also affected the expression of downstream ERK1/2, thus inhibiting cell proliferation and differentiation.To conclude, differential expressions of miRNA-424 and its target gene MAP2K1 in the Amur tiger placenta and the domestic cat placenta may be related to the size of the felines. We can show preliminarily that different size of Amur tiger and domestic cat is attributed to posttranscriptional regulation directed by miRNA-424. The specific regulatory mechanism will be elucidated by gene knockout experiment or mutation experiment in animals.