牛下丘脑lncRNA SNHG3的筛选及其调控CART表达的功能鉴定

Screening of the lncRNA SNHG3 regulating CART expression in the bovine hypothalamus

摘要:

本研究旨在筛选调控牛下丘脑可卡因-苯丙胺调节转录肽(cocaine and amphetamine regulated transcript peptide, CART)表达的长链非编码RNA (long non-coding RNA, lncRNA)小核仁RNA宿主基因3 (small nucleolar RNA host gene 3, SNHG3)，明确lncRNA SNHG3调控牛下丘脑CART表达的作用机制。利用StarBase v2.0、NCBI、DIANA tools数据库预测分别与极显著抑制CART表达的miR-381、miR-491存在靶向关系的lncRNAs，分析其结合位点；选取3头健康成年西门塔尔母牛，提取其下丘脑组织总RNA，通过半定量RT-PCR技术鉴定所筛选的lncRNAs内源表达情况；双荧光素酶报告基因技术检测miR-381/491与lncRNAs间的靶向结合关系；构建lncRNAs、CART过表达载体及miR-381/491 mimics，分别转染至293T细胞，分析lncRNAs对CART基因表达的调控作用机制；利用动物活体实验对在细胞水平调控作用效果最强的lncRNA进行功能分析。lncRNA TUG1、SNHG3与miR-381存在结合位点，lncRNA H19、SNHG12、DANCR与miR-491存在结合位点，且lncRNA TUG1、SNHG3、H19、SNHG12、DANCR均在牛下丘脑中有表达；双荧光素酶检测结果显示，miR-381显著抑制TUG1-WT重组荧光质粒的相对荧光活性(P<0.05)，极显著抑制SNHG3-WT重组荧光质粒的相对荧光活性(P<0.01)；miR-491显著抑制DANCR-WT和H19-WT的荧光素酶活性表达(P<0.05)，极显著抑制SNHG12-WT荧光素酶活性表达(P<0.01)；在细胞水平，lncRNA SNHG3通过特异性结合miR-381极显著提高CART表达(P<0.001)，lncRNA SNHG12通过特异性结合miR-491极显著提高CART表达(P<0.01)，其中，lncRNA SNHG3调控CART表达效果最强；动物实验结果表明，lncRNA SNHG3通过特异性结合miR-381显著提高CART mRNA和蛋白表达。本研究证实了lncRNA SNHG3作为miR-381的竞争性内源RNA (competing endogenous RNAs, ceRNA)，在转录和转录后水平均显著上调CART表达，为进一步研究牛下丘脑CART的分子网络调控机制奠定了基础。

Abstract:

This study aimed to screen for the long non-coding RNA (lncRNA) small nucleolar RNA host gene 3 (SNHG3) capable of regulating the expression of cocaine- and amphetamine-regulated transcriptional peptide (CART) in the bovine hypothalamus and elucidate the underlying mechanism. StarBase v2.0, NCBI, and DIANA tools were used to predict the lncRNAs targeting miR-381 and miR-491, which were responsible for inhibiting CART expression. The binding sites were analyzed, and the endogenous expression of the selected lncRNAs was determined by semi-quantitative RT-PCR of the hypothalamus tissue from three healthy adult Simmental cows. The dual-luciferase reporter gene assay was employed to detect the targeted binding relationship between miR-381/491 and lncRNAs. The over-expression vectors of lncRNAs, CART, and miR-381/491 mimics were constructed and transfected into 293T cells to reveal the mechanism of lncRNAs in regulating the CART expression. Animal experiments were conducted to analyze the regulatory function of the strongest lncRNA at the cellular level. The results showed that lncRNAs TUG1, SNHG3, H19, SNHG12, and DANCR were expressed in the bovine hypothalamus. The lncRNAs TUG1 and SNHG3 had binding sites for miR-381, and H19, SNHG12, and DANCR had binding sites for miR-491. The dual-luciferase reporter gene assay showed that miR-381 inhibited the relative luciferase activities of TUG1-WT (P<0.05) and SNHG3-WT (P<0.01), and miR-491 inhibited the luciferase activities of DANCR-WT (P<0.05), H19-WT (P<0.05), and SNHG12-WT (P<0.01). SNHG3 and SNHG12 up-regulated the CART expression by specifically binding to miR-381 (P<0.001) and miR-491 (P<0.01), respectively, and SNHG3 had the strongest effect of regulating CART expression. The results from animal experiments showed that SNHG3 significantly up-regulated the mRNA and protein levels of CART by specifically binding to miR-381. This study confirmed that the lncRNA SNHG3, acting as a competing endogenous RNA of miR-381, significantly up-regulated CART expression at the transcriptional and post-transcriptional levels, laying a foundation for deciphering the mechanism of the molecular network regulation of CART in the bovine hypothalamus.