水牛Novel-miR-57对Bcap-37和BMECs细胞DOK4基因的调控作用

【目的】筛选Novel-miR-57调控靶基因，并明确其对靶基因的调控作用及生物功能，为揭示水牛乳腺上皮细胞（BMECs）的分化机理提供科学依据。【方法】利用MiRscan预测Novel-miR-57二级结构；以自编软件Ensembl（v80）注释的水牛mRNA截取3'-非翻译区（3'-UTR）作为预测数据库，采用Miranda（v3.3a）对Novel-miR-57进行靶基因预测；运用实时荧光定量PCR筛选重点靶基因。以化学合成的Novel-miR-57模拟物Mimics和抑制剂Inhibitor，分别转染人类乳腺癌细胞（Bcap-37）及BMECs细胞，以验证Novel-miR-57与靶基因的表达相关性。【结果】Novel-miR-57前体序列形成7个茎环结构，成熟序列位于第1、2和3个茎环结构间，其结合自由能为-53.70 kcal/mol。以结合自由能低于-20.00 kcal/mol为标准，最终筛选出34个可能的靶基因，共与42条KEGG信号通路存在关联，其富集的信号通路主要有代谢通路（ID:bta01100）、PI3K-Akt（信号通路（ID:bta04151）、MAPK信号通路（ID:bta04010）和细胞因子—细胞因子受体相互作用（ID:bta04060）等；经实时荧光定量PCR检测分析发现DLX3、CANCNG3、DOK4、NFKBID、C17orf53、RTN1和FBXO10等7个靶基因在非泌乳期的相对表达量极显著高于泌乳期（P< 0.01），二者间相差100.0倍以上，且与NovelmiR-57的相对表达量呈负相关。7个靶基因中仅DOK4基因与Novel-miR-57的表达具相关性，以200 nmol/L Inhibitor转染B-cap37细胞能显著提高DOK4基因表达（P< 0.05，下同），添加100 nmol/L Mimics则显著抑制DOK4基因表达。以100 nmol/L Mimics转染BMECs细胞，Novel-miR-57和DOK4基因的相对表达量显著提高；而以200 nmol/L Inhibitor转染BMECs细胞，Novel-miR-57和DOK4基因的表达均受到显著抑制。【结论】Novel-miR-57含有7个茎环结构，且其成熟序列位于第1～3个茎环上。Novel-miR-57过表达可下调Bcap-37细胞DOK4基因表达或上调BMECs细胞DOK4基因表达，即Novel-miR-57对靶基因的调控作用因乳腺细胞生理状态不同而存在差异。

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.