牛下丘脑CART转录后调控关键miRNA的筛选与鉴定

Screening and identification of key miRNAs in post-transcriptional regulation of CART in the bovine hypothalamus

摘要:

本研究旨在探究microRNA(miRNA)对牛下丘脑可卡因-苯丙胺调节转录肽(cocaine- and amphetamine-regulated transcript peptide, CART)的转录后调控作用，并筛选调控关键miRNA。通过生物信息学分析来预测与牛CART 3ʹ非翻译区(3ʹ untranslated regions, 3ʹUTR)结合的潜在miRNA，发现CART 3ʹUTR存在7个miRNA结合位点，即bta-miR-377、bta-miR-331-3p、bta-miR-491、bta-miR-493、bta-miR-758、bta-miR-877和bta-miR-381结合位点；使用逆转录聚合酶链式反应(reverse transcription-PCR, RT-PCR)技术检测CART基因与靶miRNA在牛下丘脑内源性表达情况，发现它们均在牛下丘脑组织内表达；通过双荧光素酶报告实验验证CART 3ʹUTR与靶细胞miRNA结合位点，证明了CART 3ʹUTR与7个miRNA均具有靶向结合关系；通过细胞实验检测靶miRNA对外源性CART基因与CART蛋白表达的影响并筛选调控关键miRNA，结果表明，7个miRNA均能抑制外源CART信使RNA (messenger RNA, mRNA)的表达，其中以bta-miR-491的抑制作用最强，bta-miR-377、bta-miR-331-3p、bta-miR-491、bta-miR-493及bta-miR-381均能抑制外源性CART蛋白表达，其中以bta-miR-381的抑制作用最强；通过动物实验研究关键miRNA对下丘脑CART基因、CART蛋白表达及血清CART浓度的影响，结果表明，miR-491和miR-381可结合CART 3ʹUTR位点，调节下丘脑CART内源性表达并影响其血清浓度。本研究的结果表明，miR-491和miR-381是调节牛下丘脑CART表达的主要miRNA，可通过调节CART内源性表达进而影响血清CART浓度。

Abstract:

This study aimed to explore the roles of microRNAs (miRNAs) in the post-transcriptional regulation of cocaine- and amphetamine-regulated transcript (CART) peptide in the bovine hypothalamus and to screen key regulatory miRNAs. Targetscan was used to predict the potential miRNAs binding to CART 3ʹ untranslated regions (3ʹUTR). Bioinformatics analysis predicted 7 miRNA binding sites in the bovine CART 3ʹUTR, which were bta-miR-377, bta-miR-331-3p, bta-miR-491, bta-miR-493, bta-miR-758, bta-miR-877, and bta-miR-381, respectively. Reverse transcription-PCR (RT-PCR) was carried out to determine the endogenous expression of CART and target miRNAs in the bovine hypothalamus. All the 7 target miRNAs and CART were endogenously expressed in the bovine hypothalamus. The dual-luciferase reporter gene assay was employed to detect the targeted binding relationship between CART 3ʹUTR and target miRNAs obtained from bioinformatics analysis. The dual-luciferase reporter gene assay confirmed that the 3ʹUTR of CART had a targeted binding relationship with the 7 target miRNAs. Cell experiments were conducted to examine the effects of target miRNAs on the messenger RNA (mRNA) and protein levels of exogenous CART and screen for key regulatory miRNAs. The results of cell experiments showed that the 7 miRNAs downregulated the mRNA level of CART, with bta-miR-491 demonstrating the strongest downregulating effect. Bta-miR-377, bta-miR-331-3p, bta-miR-491, bta-miR-493, and bta-miR-381 downregulated the protein level of CART, with bta-miR-381 exerting the strongest downregulating effect. Animal experiments were conducted to explore the effects of key regulatory miRNAs on the mRNA and protein levels of CART in the hypothalamus and the CART concentration in the serum. The results from animal experiments showed that miR-491 and miR-381 regulated the endogenous expression of CART in the hypothalamus and the concentration in the serum by binding to the CART 3ʹUTR. These results suggest that miR-491 and miR-381 are the main miRNAs regulating CART expression in the bovine hypothalamus, which can affect serum CART concentration by modulating endogenous CART expression.