摘要
为了分析血液外胞体miRNA对延边黄牛垂体细胞中生长激素(GH)分泌的影响,试验选择在延边黄牛和韩延牛血液外胞体中存在显著差异表达的miR-23b-3p,分析其对延边黄牛垂体细胞中GH分泌的影响机制。首先进行延边黄牛垂体细胞的原代培养,然后将miR-23b-3p的mimics(miR-23b-3p-mi组)、mimics对照品(NC组)、inhibitor(miR-23b-3p-in组)、inhibitor对照品(iNC组)转染至已建立的垂体原代细胞中,48 h后收集细胞,提取总mRNA和总蛋白,利用实时荧光定量PCR(qPCR)和Western-blot技术分别检测GH mRNA转录和蛋白的表达情况;利用targetscan和RNAhybrid分析软件对miR-23b-3p的靶基因进行预测,再利用双荧光素酶报告基因系统对miR-23b-3p的靶关系进行了验证;最后利用实时荧光定量PCR和Western-blot技术分别检测miR-23b-3p靶基因的mRNA转录和蛋白表达情况。结果表明:miR-23b-3p-mi组的延边黄牛垂体细胞中GH mRNA转录和蛋白表达水平极显著低于NC组(P<0.01),而miR-23b-3p-in组的延边黄牛垂体细胞中GH mRNA转录和蛋白表达水平显著高于iNC组(P<0.05);miR-23b-3p靶向了垂体特异性转录因子(POU1F1)的3′UTR;miR-23b-3p-mi组的POU1F1 mRNA转录和蛋白表达水平极显著低于NC组(P<0.01),而miR-23b-3p-in组的POU1F1 mRNA转录和蛋白表达水平极显著高于iNC组(P<0.01)。说明miR-23b-3p可通过调节POU1F1基因的表达来调控延边黄牛垂体细胞GH的分泌,进而调控延边黄牛的生长发育。
In order to analyze the effect of blood exosome miRNAs on growth hormone(GH)secretion in pituitary cells of Yanbian yellow cattle,miR-23 b-3 p,which was significantly differentially expressed in the blood exosome of Yanbian yellow cattle and Han Yan cattle,was selected to analyze the mechanism of its effect on GH secretion in pituitary cells of Yanbian yellow cattle.The primary culture of Yanbian yellow cattle pituitary cells was carried out,and mimics(miR-23 b-3 p-mi group),mimics reference substance(NC group),inhibitor(miR-23 b-3 p-in group)and inhibitor reference substance(iNC group)of miR-23 b-3 p were transfected into the established pituitary primary cells.After 48 h,the cells were collected and total RNA and protein were extracted.The GH mRNA transcription and protein expression level were detected by real time fluorescence quantitative PCR(qPCR)and Western blot assays,respectively.The target genes of miRNA-23 b-3 p were predicted by targetscan and RNA hybridization analysis software,and the target relationship of miRNA-23 b-3 p was validated by double luciferase reporter gene system.Finally,the mRNA transcription and protein expression level of miRNA-23 b-3 p target genes were detected by qPCR and Western blot assays,respectively.The results showed that the transcription and protein expression levels of GH in pituitary cells of Yanbian yellow cattle in miR-23 b-3 p-mi group were significantly lower than those in NC group(P<0.01),while the transcription and protein expression levels of GH in pituitary cells of Yanbian yellow cattle in miR-23 b-3 p-in group were significantly higher than those in iNC group(P<0.05);miR-23 b-3 p targeted 3′UTR of pituitary specific transcription factor(POU1 F1).The transcription and protein expression levels of POU1 F1 in miR-23 b-3 p-mi group were significantly lower than those in NC group(P<0.01),while the transcription and protein expression levels of POU1 F1 in miR-23 b-3 p-in group were significantly higher than those in iNC group(P<0.01).These results demonstrated tha
作者
张睿
季久秀
相思宇
金太花
陈莹莹
娄安钢
崔成都
于龙政
关立增
ZHANG Rui;JI Jiuxiu;XIANG Siyu;JIN Taihua;CHEN Yingying;LOU Angang;CUI Chengdu;YU Longzheng;GUAN Lizeng(College of Agriculture,Yanbian University,Yanji 133002,China;College of Agriculture and Forestry Sciences,Linyi University,Linyi 276005,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2020年第11期1-6,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金项目(31660614)。
