幼龄和成年太行山羊附睾头差异竞争性内源RNAs机制分析

doi:10.11843/j.issn.0366-6964.2021.05.015

摘要/Abstract

摘要： 旨在筛选出幼龄和成年太行山羊附睾头中差异表达的长链非编码RNAs（lncRNAs）、微小RNAs（miRNAs）和mRNAs，构建太行山羊附睾头中免疫相关基因调控的竞争性内源RNAs（ceRNAs）网络。本研究选取健康状况良好、体重相近的幼龄（2月龄）和成年太行山羊（2周岁）公羊各3只，去势采集其附睾头组织进行全转录组测序，用DESeq2软件筛选出幼龄和成年太行山羊附睾头差异mRNAs、lncRNAs和miRNAs。利用miRanda软件和R-package（reshape2、dplyr、tidyr），基于ceRNA-score原理分析得到差异ceRNAs表达谱，对预测所得具有ceRNAs关系的mRNAs进行GO和KEGG富集分析，并绘制得到太行山羊附睾头免疫相关ceRNAs网络。最后，各随机挑选8个mRNAs、miRNAs和lncRNAs，并通过qRT-PCR验证全转录组测序结果的准确性。根据分析结果，以幼龄太行山羊附睾头为对照，成年山羊附睾头差异表达mRNAs有6 461个，其中上调2 997个、下调3 464个；差异表达lncRNAs共有1 147个，其中703个上调、444个下调；差异表达miRNAs共有182个，其中81个上调、101个下调。共得到具有ceRNAs调控关系的lncRNAs 366个，其中上调213个，下调153个；mRNAs有3 131个，其中1 253个上调，1 878个下调；miRNAs有140个，其中48个上调，92个下调。分析具有ceRNAs机制的基因发现，表达量显著上调的与免疫相关的mRNAs：淋巴细胞抗原6复合位点蛋白G5B（LY6G5B）、脂质运载蛋白9（LCN9）、解整合素金属蛋白酶28（ADAM28）和粘蛋白15（MUC15）基因在成年太行山羊附睾头表达量高，且极显著高于幼龄太行山羊（P<0.01）。GO和KEGG富集分析表明，具有ceRNAs机制的差异表达基因富集在内质网蛋白加工通路、蛋白质输出通路、粘蛋白型O-聚糖生物合成通路、细胞外基质受体相互作用通路等。qRT-PCR验证结果表明，除chi-miR-320-3p外，其余差异表达的mRNAs、lncRNAs和miRNAs表达趋势与全转录组测序结果一致。附睾头免疫相关ceRNAs网络分析表明，lncRNA-MSTRG.22929.11、lncRNA-MSTRG.57822.5、lncRNA-MSTRG.26758.1、lncRNA-MSTRG.12113.3、lncRNA-MSTRG.59930.2等lncRNAs作为ceRNAs可以调控附睾头免疫相关基因表达。本研究筛选出了幼龄和成年太行山羊附睾头差异ceRNAs，挖掘并绘制了免疫相关的关键ceRNAs网络，这些lncRNAs作为ceRNAs可为太行山羊附睾头免疫调控机制研究提供参考依据。

关键词: ceRNA, 太行山羊, 附睾头, 免疫

Abstract: The aim of this study was to explore and screen the differentially expressed long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and mRNAs in caput epididymis between Taihang goat kids and adult Taihang goats, and to construct a competitive endogenous RNAs (ceRNAs) network associated with the regulatory mechanism of immune-related genes in caput epididymis of Taihang goats. Three 2-month-old healthy Taihang goat kids with similar body weight and three 2-year-old healthy adult Taihang goats with similar body weight were selected in this study. After castration, the caput epididymis were collected for whole transcriptome sequencing. The differentially expressed mRNAs, lncRNAs and miRNAs in caput epididymis were screened between goat kids and adult goats using DESeq2. Based on the principle of ceRNA-score, the differentially expressed ceRNAs was obtained by using miRanda software and R-package (reshape2, dplyr, tidyr). GO and KEGG enrichment analysis were used to study the function of mRNAs in the ceRNAs mechanism. The ceRNAs network related with immune function was drawn in caput epididymis of Taihang goats. Finally, 8 mRNAs, 8 miRNAs and 8 lncRNAs were randomly selected to verify the accuracy of the whole transcriptome sequencing results by qRT-PCR. According to the analysis results, compared with caput epididymis of goat kids, there were 6 461 differentially expressed mRNAs in caput epididymis of adult Taihang goats， including 2 997 up-regulated mRNAs and 3 464 down-regulated mRNAs; There were 1 147 differentially expressed lncRNAs, including 703 up-regulated lncRNAs and 444 down-regulated lncRNAs; There were 182 differentially expressed miRNAs, including 81 up-regulated miRNAs and 101 down-re-gulated miRNAs. Three hundred and sixty-six lncRNAs with ceRNAs regulatory relationship were obtained, including 213 up-regulated lncRNAs and 153 down-regulated lncRNAs; There were 3 131 mRNAs with ceRNAs regulatory relationship, including 1 253 up-regulated mRNAs and 1 878 down-regulated mRNAs; There were 140 miRNAs with ceRNAs regulatory relationship, including 48 up-regulated miRNAs and 92 down-regulated miRNAs. Among the genes in the ceRNAs mechanism, lymphocyte antigen 6 complex locus protein G5B (LY6G5B)，epididymal-specific lipocalin-9 (LCN9), adisintegrinand metalloproteinase 28 (ADAM28) and Mucin 15 (MUC15) were the significantly up-regulated genes related with immune function, which expression in adult goats were higher than those in goat kids (P<0.01). GO and KEGG analysis of the DEGs in the ceRNAs mechanism showed that of differentially expressed genes were involved in protein processing in endoplasmic reticulum, protein export, mucin type O-Glycan biosynthesis, ECM-receptor interaction and other pathways related to cell protein synthesis, secretion and transport. The results of qRT-PCR showed that the expression trends of the differentially expressed mRNAs, lncRNAs and miRNAs selected randomly were consistent with the whole transcriptome sequencing results, except for chi-miR-320-3p. In this study, the analysis results of the immune-related ceRNAs network in caput epididymis showed that lncRNA-MSTRG.22929.11, lncRNA-MSTRG.57822.5, lncRNA-MSTRG.26758.1, lncRNA-MSTRG.12113.3, lncRNA-MSTRG.59930.2 and other lncRNAs were related to immunity and sperm maturation in caput epididymis. In this study, the differentially expressed ceRNAs were screened in caput epididymais between Taihang goat kids and adult Taihang goats. The ceRNAs network related to immunity will provides a foundation for the study of lncRNAs as ceRNAs in the regulation mechanism of epididymal immune function in Taihang goats.

Key words: ceRNA, Taihang goat, caput epididymis, immune

中图分类号:

郭相前, 董复成, 黄鑫芸, 刘文忠, 乔利英, 张春香, 任有蛇. 幼龄和成年太行山羊附睾头差异竞争性内源RNAs机制分析[J]. 畜牧兽医学报, 2021, 52(5): 1293-1306.

GUO Xiangqian, DONG Fucheng, HUANG Xinyun, LIU Wenzhong, QIAO Liying, ZHANG Chunxiang, REN Youshe. Analysis of the Differentially Expressed ceRNAs Mechanism in Caput Epididymis between Goat Kids and Adult Goats[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1293-1306.

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