BLV-miRNAs对牛乳过氧化物酶的影响

Effect of BLV-miRNAs on milk lactoperoxidase

为探究牛白血病病毒(bovine leukemia virus,BLV)编码的miRNAs(BLV-miRNAs)对奶牛乳腺上皮细胞(BMECs)乳过氧化物酶(LPO)的影响。首先通过ELISA测定高低不同载量BLV阳性奶牛牛乳中LPO的含量;随后体外培养BMECs,分别感染(1 MOI)BLV全长和缺失miRNAs的BLV(BLV-△miRNAs),通过荧光定量PCR、ELISA等方法检测各组BMECs LPO的表达水平;再通过StarMir等软件程序预测10种BLV-miRNAs是否靶向LPO;根据靶基因预测结果,进行BLV-miRNAs转染和靶基因初步验证。结果显示:BLV高载量阳性奶牛和低载量阳性奶牛牛乳中的LPO含量比阴性奶牛牛乳中的LPO分别降低了8.73%和9.68%;体外试验结果显示:与感染BLV全长病毒组相比,缺失BLV-miRNAs后BMECs中的LPO表达水平显著升高(P<0.05);靶位点预测结果显示BLV-miR-B1-5p、B3-3p和B4-5p可共同靶向LPO基因;转染BLV-miR-B1-5p到BMECs后LPO的基因表达显著下调(P<0.05),并随着转染剂量的增多,LPO的表达水平呈下降趋势;转染BLV-miR-B4-3p到BMECs后LPO的基因表达显著下调(P<0.05),但没有剂量依赖性。综上所述,BLV-miRNAs可以抑制BMECs中LPO的表达。

This study aims to investigate the effect of bovine leukemia virus encoded microRNAs(BLV-miRNAs)on lactoperoxidase(LPO)in bovine mammary epithelial cells(BMECs).Firstly,the LPO content in the milk of BLV-positive cows,categorized by different viral loads,was quantified by enzyme-linked immunosorbent assay(ELISA).Subsequently,BMECs were cultured in vitro and infected with(1MOI)full-length BLV and BLV lacking miRNAs(BLV-ΔmiRNAs).Various analytical techniques,including fluorescent quantitative PCR and ELISA,were used to assess LPO expression levels in different BMEC cohorts.Ten BLV-miRNAs were computationally predicted to target LPO using software tools such as StarMir.Based on these predictions,transfections of BLV-miRNAs were carried out and preliminary verification of their effects on target genes were performed.The results showed that compared to that of BLV-negative counterparts,the LPO levels in the milk of BLV-positive cows,stratified by high and low viral load,decreased by 8.73%and9.68%,respectively.In vitro experiments further corroborated these trends,revealing a significant increase(P<0.05)in LPO expression within BMECs following the deletion of BLV-miRNAs compared to the group infected with full-length BLV-infected group.Computational target site predictions implicated BLV-miR-B1-5p,B3-3p,and B4-5p in the collective regulation of the LPO gene.Transfection of BLV-miR-B1-5p into BMECs resulted in a significant downregulation of LPO gene expression(P<0.05),with the effect intensifying proportionally with the transfection dose.Similarly,the transfection of BLV-miR-B4-3p into BMECs significantly reduced LPO gene expression(P<0.05)without showing a dose-dependent behavior.In conclusion,this research indicates that BLV-miRNAs can suppress LPO expression in BMECs.