miR-93对延边黄牛垂体细胞GH分泌的影响

doi:10.16431/j.cnki.1671-7236.2020.01.014

摘要/Abstract

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。

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)

Abstract: 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.

Key words: miR-93; Yanbian Yellow cattle; pituitary cells; GH; GHRHR

中图分类号:

S823.8

娄安钢, 崔长艳, 相思宇, 金太花, 陈莹莹, 张睿, 关立增. miR-93对延边黄牛垂体细胞GH分泌的影响[J]. 中国畜牧兽医, 2020, 47(1): 116-123.

LOU Angang, CUI Changyan, XIANG Siyu, JIN Taihua, CHEN Yingying, ZHANG Rui, GUAN Lizeng. Effect of miR-93 on GH Secretion in Pituitary Cells of Yanbian Yellow Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(1): 116-123.

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