miR-93对延边黄牛垂体细胞GH分泌的影响  

Effect of miR-93 on GH Secretion in Pituitary Cells of Yanbian Yellow Cattle

作　　者：娄安钢[1] 崔长艳 相思宇 金太花 陈莹莹[1] 张睿[1] 关立增[1,2] LOU Angang;CUI Changyan;XIANG Siyu;JIN Taihua;CHEN Yingying;ZHANG Rui;GUAN Lizeng(College of Agriculture,Yanbian University,Yanji 133002,China;College of Agriculture and Forestry Sciences,Linyi University,Linyi 276005,China)

机构地区：[1]延边大学农学院,延吉133002 [2]临沂大学农林科学学院,临沂276005

出　　处：《中国畜牧兽医》2020年第1期116-123,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(31660614)

摘　　要：为了分析血液外泌体miRNA对延边黄牛垂体细胞生长激素(GH)分泌的影响,试验选择在延边黄牛和韩延牛血液外泌体中存在显著差异表达的miR-93,分析其对延边黄牛垂体细胞GH分泌的影响机制。试验首先进行延边黄牛垂体细胞的原代培养,之后将miR-93的mimics(miR-93-mi组)、mimics对照品(NC对照组)、inhibitor(miR-93-in组)、inhibitor对照品(iNC组)转染给已建立的垂体原代细胞,48 h后收集细胞,提取总mRNA和总蛋白。试验利用targetscan和RNAhybrid分析软件对miR-93的靶基因进行预测,并利用双荧光素酶报告基因系统对miR-93的靶关系进行验证;利用实时荧光定量PCR和Western blotting技术分别检测靶基因mRNA转录和蛋白的表达情况,结果表明,miR-93靶向了生长激素释放激素受体(GHRHR)的3′UTR;与NC对照组比较,miR-93-mi组的延边黄牛垂体细胞中GH mRNA转录和蛋白表达均极显著低于NC对照组(P<0.01);miR-93-mi组的GHRH mRNA转录和蛋白表达均极显著低于NC对照组(P<0.01),而miR-93-in组的GHRHR蛋白表达显著高于iNC对照组(P<0.05)。说明miR-93可通过调节GHRHR的表达而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 miRNAs on growth hormone(GH)secretion in pituitary cells of Yanbian Yellow cattle,the differently expressed miR-93 in the blood of Yanbian Yellow cattle and Hanyan cattle was selected to analyze the effects of miR-93 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-93-mi group),mimics reference substance(NC control group),inhibitor(miR-93-in group)and inhibitor reference substance(iNC group)of miR-93 were transfected into the established pituitary primary cells.After 48 h,the cells were collected and the total RNA and protein were extracted.The target genes of miR-93 were predicted by targetscan and RNA hybrid analysis software,and the target relationship of miR-93 was validated by double luciferase reporter gene system.The GH mRNA transcription and protein expression were detected by Real-time PCR and Western blotting,respectively.The results showed that miR-93 targeted 3′UTR of growth hormone releasing hormone receptor(GHRHR);The transcription and protein expression of GH in pituitary cells of Yanbian Yellow cattle in miR-93-mi group were extremely significantly lower than those of NC control group(P<0.01);The mRNA transcription and protein expression of GHRHR in miR-93-mi group were extremely significantly lower than those of NC control group(P<0.01),while the protein expression of GHRHR in miR-93-in group were significantly higher than those of iNC group(P<0.05).The above results demonstrated that miR-93 could regulate GH secretion in pituitary cells by regulating the expression of GHRHR gene and further regulated the growth and development of Yanbian Yellow cattle.

关 键 词：miR-93 延边黄牛 垂体细胞 生长激素(GH) 生长激素释放激素受体(GHRHR) 

分 类 号：S823.8[农业科学—畜牧学]