Regulating effects of Novel-miR-57 in buffalo to DOK4 gene on Bcap-37 and BMECs cells

【Objective】In order to provide scientific basis for revealing the differentiation mechanism of buffalo mammary epithelial cells(BMECs), the regulatory target gene of Novel-miR-57 was screened to clarify its regulatory function and biological function on target genes.【Method】MiRscan was used to predict the secondary structure of Novel-miR-57.The target gene of Novel-miR-57 was predicted by Miranda(v3.3a) using buffalo mRNA truncated 3-untranslated region(3'-UTR) annotated by Ensembl(v80) as prediction database.Key target genes were screened by real-time fluorescence quantitative PCR.To verify the correlation between Novel-miR-57 and target gene expression, chemically synthesized mimics and inhibitor were transfected into human breast cancer cells(Bcap-37) and BMECs cells, respectively.【Result】The precursor sequence of Novel-miR-57 formed seven stem-loops, and the mature sequence was located between the first, second and third stem-loops, and its binding free energy was-53.70 kcal/mol.With the binding free energy lower than-20.00 kcal/mol as the standard, 34 possible target genes were finally screened out, which were associated with 42 KEGG signaling pathways.The enriched signaling pathways mainly included metabolic pathway(ID:bta01100), PI3KAkt(ID:bta04151), MAPK signaling pathway(ID:bta04010) and cytokine-cytokine receptor interaction(ID:bta04060).The real-time fluorescence quantitative PCR showed that the relative expression of seven target genes, DLX3, CANCNG3, DOK4, NFKBID, C17orf53, RTN1 and FBXO10, were significantly higher in non-lactation period than in lactation period(P< 0.01), and the difference between them was more than 100.0 times, which were negatively correlated with the relative expression of Novel-miR-57.Only the expression of DOK4 gene was correlated with the expression of Novel-miR-57 among the seven target genes.Transfection of B-cap37 cells with 200 nmol/L inhibitor could significantly increase the expression of DOK4 gene(P< 0.05, the same below), while addition of 100 nmol/L mimics could significantly inhibit the expression of DOK4 gene.The relative expression of Novel-miR-57 and DOK4 gene was significantly increased when BMECs cells were transfected with 100 nmol/L mimics.The expression of Novel-miR-57 and DOK4 gene were significantly inhibited when BMECs cells were transfected with 200 nmol/L inhibitor.【Conclusion】Novel-miR-57 contains seven stem-loops, and its mature sequence locates on the first to the third stem rings.Overexpression of Novel-miR-57 can down-regulate DOK4 gene expression in Bcap-37 cells or up-regulate DOK4 gene expression in BMECs cells, that is, Novel-miR-57 has different regulating effects on target genes due to different physiological states of breast cells.