BLV-miR-B1-5p靶向牛MUC1基因的靶位点预测及验证  

Target Site Prediction and Validation of BLV-miR-B1-5p Targeting Bovine MUC1

作　　者：李宵 廖献茂 刘伟琦 连帅[1] 王建发[1] Li Xiao;Liao Xianmao;Liu Weiqi;Lian Shuai;Wang Jianfa(College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing163319)

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319

出　　处：《黑龙江八一农垦大学学报》2024年第5期40-46,111,共8页journal of heilongjiang bayi agricultural university

基　　金：国家自然科学基金(32002247,32272968);黑龙江省大学生创新创业训练计划项目(S202210223036);黑龙江省创新人才资助项目(UNPYSCT-2020038)。

摘　　要：牛白血病病毒(bovine leukemia virus,BLV)可经血液和淋巴系统下行持续感染奶牛乳腺上皮细胞,可导致细胞抗菌能力下降,而BLV编码的mi RNAs(BLV-mi RNAs)是BLV复制和致病所必需的功能元件。为探究BLV编码的BLV-mi R-B1-5p是否通过靶向牛黏蛋白1(MUC1)基因参与抑制乳腺上皮细胞的抗菌防御屏障,在研究中首先通过STar Mir软件进行靶位点预测,再通过细胞转染、双荧光素酶报告试验、qRT-PCR和Western Blot进行验证。靶位点预测结果显示,BLV-mi R-B1-5p与MUC13′UTR有完全结合位点,结合的自由度为-35.6 kcal·mol^(-1);双荧光素酶报告试验结果显示:BLV-mi R-B1-5p可显著下调MUC1-3UTR-WT的荧光值(P<0.001);且通过mi RNA细胞转染、qRT-PCR和Western Blot体外验证结果显示,BLV-mi R-B1-5p可抑制奶牛乳腺上皮细胞MUC1 m RNA和蛋白的表达。综合以上结果表明,BLV-mi R-B1-5p可靶向并抑制牛MUC1,从而参与抑制乳腺上皮细胞的抗菌防御屏障。研究为BLV影响乳腺上皮细胞的防御能力提供了数据参考。Bovine leukemia virus(BLV)can persistently infect the mammary epithelial cells of dairy cows downstream through the blood and lymphatic systems,which can lead to a decrease in cellular antimicrobial capacity.BLV-encoded miRNAs(BLV-miRNAs)are functional components necessary for BLV replication and pathogenesis.In order to explore whether BLV-miR-B1-5p encoded by BLV participates in inhibiting the antibacterial defense barrier of breast epithelial cells by targeting bovine mucin 1(MUC1)gene,in this study,target sites were first predicted by the software of StarMir,and then verified by cell transfection,dual luciferase report experiments,qRT-PCR and Western Blot.The predicted target sites showed that BLV-miR-B1-5p had a complete binding site with MUC13′UTR,with a degree of freedom of-35.6 kcal·mol^(-1).The results of dual luciferase report experiment showed that BLV-miR-B1-5p could significantly reduce the fluorescence value of MUC1-3UTR-WT(P<0.001);And the results of miRNA cell transfection,qRT-PCR,and Western Blot in vitro validation showed that BLV-miR-B1-5p can inhibit the expression of MUC1 mRNA and protein in bovine mammary epithelial cells.Based on the above results,BLV-miR-B1-5p can target and inhibit bovine MUC1,and this conclusion provides data support for BLV promote pathogen adhesion on breast epithelial cells.

关 键 词：牛白血病病毒 黏蛋白1 奶牛乳腺上皮细胞 miRNA 

分 类 号：S854.4[农业科学—临床兽医学]