梅山猪和杜洛克猪卵泡中差异表达lncRNA克隆鉴定及与miRNAs相关性分析

doi:10.3864/j.issn.0578-1752.2024.09.014

摘要/Abstract

摘要：

【目的】通过克隆鉴定梅山猪和杜洛克猪卵泡期第4天M2卵泡中差异表达的lncRNA-ALDBSSCT0000005583，分析其在猪颗粒细胞与miRNAs表达量的相关性，为探究lncRNA调控miRNA在母猪卵泡发育过程中的作用提供理论依据。【方法】对课题组前期在梅山和杜洛克M2卵泡中筛选出的差异表达lncRNA-ALDBSSCT0000005583，进行RT-qPCR验证，并利用RACE克隆其全长序列；根据编码潜力评估工具CPAT、CPC对该lncRNA的编码能力进行预测，原核表达试验进一步鉴定其编码能力；核质分离试验对lncRNA-ALDBSSCT0000005583进行亚细胞定位，RT-qPCR检测其在各组织中的表达水平；利用miRBase网站查找猪的miRNA数据库，结合RNAhybrid、miRanda在线软件预测与lncRNA-ALDBSSCT0000005583有相互作用的物种间保守miRNA，使用TargetScan和miRanda预测与lncRNA-ALDBSSCT0000005583具有相互作用miRNA的靶基因，并对其靶基因进行GO富集和KEGG信号通路分析；通过过表达以及干扰lncRNA验证其对miRNA表达量的影响。【结果】lncRNA- ALDBSSCT0000005583在杜洛克猪M2卵泡中的表达量显著高于梅山猪，lncRNA 5'RACE和3'RACE片段大小为569 bp和546 bp，测序分析表明lncRNA-ALDBSSCT0000005583大小为588 bp。生物信息学预测其编码潜能较低，同时原核表达试验结果进一步证明其不编码蛋白质。组织表达谱分析表明，lncRNA-ALDBSSCT0000005583在肾上腺、脾肝和卵巢中表达量较高，在下丘脑和心脏中的表达量较低，亚细胞定位结果显示该lncRNA主要存在于细胞质中。生物信息学分析后共筛选出9个物种间保守的miRNA与lncRNA-ALDBSSCT0000005583具有潜在的相互作用，其中有两个与卵巢发育相关的miRNA：miR-193a-5p、miR-361-3p；KEGG和GO富集分析显示miR-193a-5p、miR-361-3p的靶基因与系统进程和细胞与细胞信号传导等生物学过程有关，并显著参与到催产素、Ras、NF-κB、促性腺激素释放激素分泌等通路。随后在颗粒细胞中过表达lncRNA-ALDBSSCT0000005583，经RT-qPCR发现miR-193a-5p、miR-361-3p的表达量均显著下调（P<0.05)，但干扰lncRNA-ALDBSSCT0000005583后无显著影响。【结论】lncRNA-ALDBSSCT0000005583是一个不具备编码蛋白质能力的lncRNA，在梅山与杜洛克猪中等卵泡间表达量有极显著差异，在肾上腺、脾肝和卵巢中表达量较高，主要存在于颗粒细胞的细胞质中，可能与miR-193a-5p、miR-361-3p相互作用，从而参与猪卵巢颗粒细胞的发育过程。

关键词: lncRNA-ALDBSSCT0000005583, 猪, 卵泡, 颗粒细胞, miR-193a-5p, miR-361-3p

Abstract:

【Objective】The objective of this study was to clone and identify the differentially expressed lncRNA-ALDBSSCT0000005583 in M2 follicles on the fourth day of follicular stage in Meishan pigs and Duroc pigs, and to analyze the correlation between the expression of miRNAs in porcine granulosa cells, so as to provide a theoretical basis for exploring the role of lncRNAs in the development of follicles in sows by regulating miRNAs. 【Method】Based on the differentially expressed lncRNA- ALDBSSCT0000005583 screened in Meishan and Duroc M2 follicles in our early research, the full-length sequence of ALDBSSCT0000005583 was verified by RT-qPCR and cloned by RACE; the coding ability of this lncRNA was predicted by the coding potential assessment tool of CAPT and CPC, which was further identified by the primary expression test; the coding ability of this lncRNA was identified by the primary expression test; the subcellular coding ability of NA-ALDBSSCT0000005583 was identified by the nucleoplasmic separation experiment and tested to identify its coding ability; the subcellular localization of lncRNA-ALDBSSCT0000005583 by nucleoplasmic isolation assay and its expression level in various tissues were detected by RT-qPCR; miRBase website was used to locate the miRNA database of pigs, and the combination of RNAhybrid and miRanda online software was used to predicte the relationship with the lncRNA-ALDBSSCT0000005583. The inter-species conserved miRNAs that interacted with lncRNA-ALDBSSCT0000005583 were predicted by TargetScan and miRanda, the target genes that interacted with lncRNA-ALDBSSCT0000005583 were predicted by TargetScan and miRanda, and their target genes were subjected to GO enrichment and KEGG signaling pathway analyses; the effects of target genes on miRNA expression were verified by overexpression as well as interference with lncRNA. 【Result】The expression level of lncRNA-ALDBSSCT0000005583 in M2 follicles of Duroc pigs was significantly higher than that in Meishan pigs, and the size of lncRNA 5′RACE and 3′RACE fragments was 569 bp and 546 bp, respectively, and the sequencing analysis showed that the size of lncRNA-ALDBSSCT0000005583 was 588 bp. Bioinformatics predicted that the encoding potential was low, and the results of prokaryotic expression assay further proved that it did not code for proteins. Tissue expression profiling showed that lncRNA-ALDBSSCT0000005583 was expressed in the adrenal gland, spleen, liver and ovaries, and low in the hypothalamus and heart, while the subcellular localization results showed that the lncRNA was mainly present in the cytoplasm. After bioinformatics analysis, a total of 9 conserved miRNAs were screened for potential interaction with lncRNA-ALDBSSCT0000005583, including two miRNAs related to ovarian development: miR-193a-5p and miR-361-3p. KEGG and GO enrichment analysis showed that the target genes of miR-193a-5p and miR-361-3p were related to phylogenetic processes and biological processes such as cell-to-cell signaling. It was also significantly involved in oxytocin, Ras, NF-κB gonadotropin- releasing hormone secretion and other pathways. Subsequently, lncRNA-ALDBSSCT0000005583 was overexpressed in granulosa cells, and the expressions of miR-193a-5p and miR-361-3p were significantly down-regulated by RT-qPCR (P<0.05), but there was no significant effect after interference with lncRNA-ALDBSSCT0000005583. 【Conclusion】 lncRNA-ALDBSSCT0000005583 was a lncRNA that did not have the ability to code for proteins. There was a significant difference in the expression level between the medium follicles of Meishan and Duroc pigs, and the expression level was higher in the adrenal glands, spleen, liver and ovaries, which is mainly found in the cytoplasm of granulosa cells, and might be involved in the development of porcine ovarian granulosa cells by interacting with miR-193a-5p and miR-361-3p.

Key words: lncRNA-ALDBSSCT0000005583, pig, follicle, granulosa cells, miR-193a-5p, miR-361-3p

张化鹏, 张庆泽, 何凡, 祁梦凡, 符彬彬, 李清春, 李梦寻, 马力鹏, 刘乙, 黄涛. 梅山猪和杜洛克猪卵泡中差异表达lncRNA克隆鉴定及与miRNAs相关性分析[J]. 中国农业科学, 2024, 57(9): 1807-1819.

ZHANG HuaPeng, ZHANG QingZe, HE Fan, QI MengFan, FU BinBin, LI QingChun, LI MengXun, MA LiPeng, LIU Yi, HUANG Tao. Cloning and Identification of Differentially Expressed lncRNAs in Follicles of Meishan Pigs and Duroc Pigs with Their Correlation Analysis with miRNAs[J]. Scientia Agricultura Sinica, 2024, 57(9): 1807-1819.

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基因名称 Gene name	引物序列Primer sequence（5′-3′）	用途 Usage
lncRNA-ALDBSSCT0000005583	DL-F:ACGTGTACAGGGCCTGACTCG	RT-qPCR
lncRNA-ALDBSSCT0000005583	DL-R:TTCTCGCATCCACGTACCTG	RT-qPCR
pcDNA3.1-lncRNA-5583	F:GG G AATTCGTAGTGACGCAGGCGCGAGACTG	载体构建 Vector construction
pcDNA3.1-lncRNA-5583	R:GGG CTCGAGCCAAATTTGAAACTGTTTTATTAAA	载体构建 Vector construction
GAPDH	F:AACATCATCCCTGCTTCTACCG	RT-qPCR
GAPDH	R:GGTCAGATCCACAACCG	RT-qPCR
U6	F: CGCTTCGGCAGCACATATACTA	RT-qPCR
U6	R: ATGGAACGCTTCACGAATTTGC	RT-qPCR
miR-193a-5p	F: AACAAGTGGGTCTTTGCGGGC	RT-qPCR
miR-361-3p	F:AACAAGCCCCCAGGTGTGATTCTG	RT-qPCR
miRNAs通用引物 Universal primers for miRNAs	R:AGTGCAGGGTCCGAGGTATT	RT-qPCR
5′ RACE特异性引物 5′ RACE-specific primers	GSP:GATTACGCCAAGCTTCCCACTTGTCATTCCGATGTCAGAGGGG	RACE克隆 Rapid-amplification of cDNA ends clone
3′ RACE特异性引物 3′ RACE-specific primers	GSP:GATTACGCCAAGCTTGCGGCTATCCAAGACCATTGAGGGTCC	RACE克隆 Rapid-amplification of cDNA ends clone
通用引物（UPM） Universal primers	TAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT	RACE克隆 Rapid-amplification of cDNA ends clone
siRNA-lncRNA sense（5′-3′）	CAGAUCUCCUGUAAGAUGUTT	lncRNA干扰序列 lncRNA interference sequence
siRNA-lncRNA antisense（5′-3′）	ACAUCUUACAGGAGAUCUGTT	lncRNA干扰序列 lncRNA interference sequence

基因名称 Gene name	编码/非编码 Encoding/Non-coding	编码潜能 Coding potential
lncRNA-ALDBSSCT0000005583	非编码Non-coding	0.0122981
HG19	编码 Encoding	0.9998311
HOTAIR	非编码Non-coding	0.1257913

基因名称 Gene name	编码/非编码 Encoding/Non-coding	编码潜能 Coding potential
lncRNA-ALDBSSCT0000005583	非编码Non-coding	0.01716
HG19	编码Encoding	0.99987
HOTAIR	非编码Non-coding	0.18488

GO条目 GO term	基因数目 Gene count	富集倍数 Fold enrichment	P值 P value
系统进程System process	13	1.90	0.018140
细胞酰胺代谢过程Cellular amide metabolic process	11	1.91	0.027469
有机氮化合物分解代谢过程Organonitrogen compound catabolic process	11	1.86	0.033325
细胞-细胞信号转导Cell-cell signaling	11	1.74	0.048981
神经系统过程Nervous system process	8	2.15	0.032080
循环系统过程Circulatory system process	7	3.62	0.003088
细胞因子产生Cytokine production	7	2.17	0.041634
硫化合物代谢过程Sulfur compound metabolic process	6	4.46	0.002150
血液循环Blood circulation	6	3.25	0.010149
系统过程调节Regulation of system process	6	2.84	0.018724

信号通路条目 Pathway term	基因数目 Gene count	富集倍数 Fold enrichment	P值 P value
多巴胺能突触Dopaminergic synapse	6	3.77	0.00510
Ras信号通路Ras signaling pathway	8	2.76	0.00841
钙信号通路Calcium signaling pathway	8	2.68	0.00993
酪氨酸代谢Tyrosine metabolism	3	6.63	0.01022
嘌呤代谢Purine metabolism	5	3.17	0.02076
肥厚型心肌病Hypertrophic cardiomyopathy	4	3.63	0.02423
催产素信号通路Oxytocin signaling pathway	5	2.76	0.03482
心肌细胞中的肾上腺素能信号转导Adrenergic signaling in cardiomyocytes	5	2.74	0.03569
NF-κB信号通路NF-kappa B signaling pathway	4	3.06	0.04188
促性腺激素释放激素分泌GnRH secretion	3	3.83	0.04330