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

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

作　　者：蔡小艳 李雅辉 鲍正潘 陈秋萍[2] 李胜[2] 周宇[2] 邓凯[2] 石德顺[2] 刘庆友[2] CAI Xiao-yan;LI Ya-hui;BAO Zheng-pan;CHEN Qiu-ping;LI Sheng;ZHOU Yu;DENG Kai;SHI De-shun;LIU Qing-you(Agricultural Colleg,Ningxia University/Key Laboratory of Ruminant Molecular and Cellular Breeding,Yinchuan 750021,China;Guangxi University/National Key Laboratory for Protection and Utilization of Subtropical Agricultural Biological Resources,Nanning 530004,China)

机构地区：[1]宁夏大学农学院/宁夏反刍动物分子细胞育种重点实验室,银川750021 [2]广西大学/亚热带农业生物资源保护与利用国家重点实验室,南宁530004

出　　处：《南方农业学报》2021年第2期269-279,共11页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31960680);宁夏重点研发计划项目(2018BEB04031)。

摘　　要：【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。【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 inhib

关 键 词：水牛 Novel-miR-57 靶基因 BMECs细胞 Bcap-37细胞 调控作用 

分 类 号：S823.83[农业科学—畜牧学]