bta-miR-19b靶向BCL3的验证及其在牛初乳免疫调控中的功能分析OA
Validation of bta-miR-19b Targeting BCL3 and Its Functional Analysis in Bovine Colostrum Immune Regulation
[目的]旨在验证bta-miR-19b对B细胞白血病/淋巴瘤3(B-cell CLL/lymphoma 3,BCL3)基因的靶向关系,并解析bta-miR-19b介导牛初乳免疫调控的分子功能与作用机制.[方法]采用TargetScan和miRanda两个软件取交集筛选bta-miR-19b免疫相关靶基因,并通过RNAhybrid网站预测bta-miR-19b与免疫相关候选基因IL7R、EGFR、TNF、BCL3、NME7、MA PK8和MAP3K1的结合自由能(MFE).以BCL3基因3'UTR预测结合位点为模板合成野生型(WT)、突变型(MT)片段,构建pmirGLO荧光素酶报告载体;合成bta-miR-19b mimics(化学合成的双链RNA分子,模拟内源性miRNA功能)及NC对照(NC-mimics),与报告载体共转染293T细胞用于靶向验证.并通过生物信息学方法筛选bta-miR-19b-BCL3轴在牛初乳免疫调控过程中生物学过程和关键通路.[结果]bta-miR-19b与BCL3的结合自由能最小,提示BCL3存在潜在结合可能.双荧光素酶报告基因实验结果证实bta-miR-19b能够特异性靶向结合BCL3基因的3'UTR区.bta-miR-19b-BCL3调节轴共存在42个GO Term中,其中和免疫相关的生物学过程有24个.bta-miR-19b-BCL3调节轴参与的KEGG通路有肿瘤坏死因子TNF信号通路和C型凝集素受体信号通路.[结论]bta-miR-19b可靶向结合BCL3 3'UTR,鉴于BCL3是NF-κB关键调节因子,推测bta-miR-19b-BCL3-NF-κB-TNF调节轴可能是牛初乳发挥免疫功能的核心分子机制之一,具体机制有待功能实验验证.
[Objective]To validate the targeting relationship between bta-miR-19b and the B-cell CLL/lymphoma 3(BCL3)gene and to elucidate the molecular function and regulatory mechanism by which bta-miR-19b mediates immune regulation in bovine colostrum.[Methods]The intersection of TargetScan and miRanda predictions was used to screen the immune-related target genes of bta-miR-19b,and the minimum free energy(MFE)of binding between bta-miR-19b and immune-related candidate genes IL7R,EGFR,TNF,BCL3,NME7,MAPK8 and MAP3K1 was predicted using the RNAhybrid website.The wild-type(WT)and mutant(MT)fragments were synthesized using the predicted binding site of the 3'UTR of the BCL3 gene as template to construct pmirGLO luciferase reporter vectors.The bta-miR-19b mimics(chemically synthesized double-stranded RNA molecules that mimic endogenous miRNA functions)and NC control(NC-mimics)were synthesized and co-transfected with the reporter vector into 293T cells for target validation.The biological processes and key pathways of bta-miR-19b-BCL3 axis in the immune regulation of bovine colostrum were screened by bioinformatics methods.[Results]The binding free energy of bta-miR-19b with BCL3 was the lowest,suggesting that there was a potential binding of BCL3.The results of dual luciferase reporter gene assay confirmed that bta-miR-19b could specifically bind to the 3'UTR region of the BCL3 gene.The bta-miR-19b-BCL3 regulatory axis was enriched in 42 GO terms,of which 24 were immune-related biological processes.The KEGG pathways involving the bta-miR-19b-BCL3 regulatory axis included tumor necrosis factor TNF signaling pathway and C-type lectin receptor signaling pathway.[Conclusion]bta-miR-19b can target the 3'UTR of BCL3.Given that BCL3 is a regulatory factor of the NF-κB signaling pathway,it is speculated that the bta-miR-19b-BCL3-NF-κB-TNF regulatory axis may be one of the core molecular mechanisms for bovine colostrum to exert immune function,and the specific mechanism needs to be validated by functional experiments.
孔繁霞;王高峰;赵濛;吴美灵;王乐乐
乌兰察布医学高等专科学校,内蒙古 集宁区 012000乌兰察布市动物疫病预防控制中心,内蒙古 集宁区 012000内蒙古自治区农牧业科学院,内蒙古 呼和浩特 010031乌兰察布医学高等专科学校,内蒙古 集宁区 012000乌兰察布医学高等专科学校,内蒙古 集宁区 012000
农业科技
bta-miR-19bBCL3双荧光素酶报告基因实验牛初乳免疫调控
bta-miR-19bBCL3double luciferase reporter gene assaybovine colostrumimmune regulation
《畜牧与饲料科学》 2026 (3)
23-29,7
内蒙古自然科学基金项目(2025LHMS03065)
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