lncRNA <i>TCONS_00143126</i>在奶牛乳腺上皮细胞炎症反应中的表达及生物信息学分析

doi:10.7668/hbnxb.20194818

摘要/Abstract

摘要：

为了深入探究lncRNA TCONS_00143126在E.coli型奶牛乳腺炎中的表达模式及其生物学功能,采用奶牛乳腺上皮细胞的cDNA为模板,运用PCR克隆及测序技术来确认lncRNA TCONS_00143126的存在,并进行lncRNA的亚细胞定位分析、预测可能靶向的miRNA及靶基因,并通过KEGG通路富集分析探讨其在奶牛乳腺炎中的潜在作用机制。此外,采用LPS诱导bMECs以构建奶牛乳腺炎体外模型,并通过RT-qPCR技术检测lncRNA TCONS_00143126在LPS诱导bMECs 6,12,24 h的表达情况。结果表明,lncRNA TCONS_00143126是真实存在的,在LPS诱导的bMECs中的表达量显著上调,且主要分布于细胞核中。靶基因预测及KEGG富集分析结果显示,lncRNA TCONS_00143126可能通过靶向的miRNA(bta-miR-133a、bta-miR-193a-5p和bta-miR-375等)和靶基因(IFNE、SLC2A10、MEX3B)来调节JAK-STAT、mTOR和MAPK等炎症信号通路,进而在奶牛乳腺上皮细胞炎症中发挥作用。

关键词: 奶牛, lncRNA TCONS_00143126, 乳腺上皮细胞, 炎症, 生物信息学分析

Abstract:

In order to investigate the expression pattern and biological function of lncRNA TCONS_00143126 in E.coli type mastitis of cows in depth.This study used cDNA from bovine mammary epithelial cells as a template,and confirmed the presence of lncRNA TCONS_00143126 using PCR cloning and sequencing techniques.Subcellular localization analysis of lncRNA was performed,and potential target miRNAs and genes were predicted.The potential mechanism of its action in bovine mastitis was explored through KEGG pathway enrichment analysis.In addition,LPS was used to induce bMECs to construct an in vitro model of bovine mastitis,and the expression of lncRNA TCONS_00143126 in LPS-induced bMECs at 6,12 and 24 h was detected by RT-qPCR.The results showed that lncRNA TCONS_00143126 was real,and its expression was significantly up-regulated in LPS-induced bMECs,and it was mainly distributed in the nucleus.The results of target gene prediction and KEGG enrichment analysis showed that lncRNA TCONS_00143126 might regulate inflammatory signaling pathways such as JAK-STAT,mTOR and MAPK by targeting miRNAs(bta-miR-133a,bta-miR-193a-5p and bta-miR-375,etc.)and target genes(IFNE,SLC2A10,MEX3B),and then play a role in the inflammation of bovine mammary epithelial cells.

Key words: Dairy cow, lncRNA TCONS_00143126, Mammary epithelial cell, Inflammation, Bioinformatics analysis

冯芬, 李彦霞, 王晋鹏, 董益闻, 罗仍卓么, 王兴平. lncRNA TCONS_00143126在奶牛乳腺上皮细胞炎症反应中的表达及生物信息学分析[J]. 华北农学报, 2025, 40(1): 199-206. doi: 10.7668/hbnxb.20194818.

FENG Fen, LI Yanxia, WANG Jinpeng, DONG Yiwen, LUORENG Zhuoma, WANG Xingping. Expression and Bioinformatics Analysis of lncRNA TCONS _ 00143126 in Inflammatory Response of Bovine Mammary Epithelial Cells[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(1): 199-206. doi: 10.7668/hbnxb.20194818.

http://www.hbnxb.net/CN/Y2025/V40/I1/199

图/表 7

表1 引物信息

Tab.1 Primer information

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	产物长度/bp Product length	退火温度/℃ Annealing temperature
TCONS_00143126	F:GAGATCAGCCCTGGTTGTTT	919	Touch down PCR(58~50)
	R:TGGGCCTACCCTATGCAAAT
TCONS_00143126	F:GGAGCTATTTCCTCTGATTC	217	60
	R:GTGCTGTTTGTTCTGTCTGA
IL-6	F:CACTCCATTCGCTGTCT	227	60
	R:GTGTCTCCTTGCTGCTT
IL-8	F:ACACATTCCACACCTTTCCAC	149	60
	R:ACCTTCTGCACCCACTTTTC
GAPDH	F:GGCATCGTGGAGGGACTTATG	89	60
	R:CCAGTGAGCTTCCCGTTGAG
RPS18	F:GTGGTGTTGAGGAAAGCAGACA	186	60
	R:TGATCACACGTTCCACCTCATC
U6	F:GCTTCGGCAGCACATATACTAAAAT	79	60
	R:CGCTTCACGAATTTGCGTGTCAT

图1 lncRNA TCONS_00143126的PCR扩增与测序结果

Fig.1 PCR amplification and sequencing results of lncRNA TCONS-00143126

图2 IL-6、IL-8和lncRNA TCONS_00143126在LPS诱导的bMECs中的表达模式不同小写字母表示在P<0.05水平差异显著。

Fig.2 The mRNA expression patterns of IL-6,IL-8 and lncRNA TCONS_00143126 in LPS-induced bMECs The different lowercase letters represent significant differences(P<0.05).

表2 lncRNA TCONS_00143126在bMECs中的亚细胞定位预测结果

Tab.2 The prediction results of subcellular localization of lncRNA TCONS _ 00143126 in bMECs

亚细胞定位 Subcellular locations	预测分数 Prediction score
细胞核 Nucleus	0.312 1
细胞质 Cytoplasm	0.276 9
核糖体 Ribosome	0.128 2
细胞溶质 Cytosol	0.121 4
外泌体 Exosome	0.161 4
定位预测 Location prediction	细胞核

图3 lncRNA TCONS_00143126的核质定位结果

Fig.3 Nucleoplasmic localization of lncRNA TCONS_00143126

图4 lncRNA TCONS_00143126的KEGG功能注释分析 1.RIG-I样受体信号通路;2.JAK-STAT信号通路;3.细胞因子受体相互作用;4.甲状腺癌;5.癌症的中心碳代谢;6.非小细胞肺癌癌症;7.自噬动物;8.mTOR信号通路;9.钙信号通路;10.单纯疱疹病毒感染。

Fig.4 KEGG functional annotation analysis of lncRNA TCONS_00143126 1.RIG-I-like receptor signaling pathway;2.JAK-STAT signaling pathway;3.Cytokine-cytokine receptor interaction;4.Thyroid cancer;5.Central carbon metabolism in cancer;6.Non-small cell lung cancer;7.Autophagy-animal;8.mTOR signaling pathway;9.Calcium signaling pathway;10. Herpes simplex virus infection.

图5 lncRNA TCONS_00143126互作miRNA的靶基因KEGG注释 A.bta-miR-133a:1.AMPK信号通路,2.心肌细胞中的肾上腺素能信号通路,3.胆汁分泌,4.丁酸代谢,5.细胞黏附分子,6.昼夜节律,7.多巴胺能突触,8.GABA能突触,9.磷酸肌醇代谢,10.胰岛素分泌,11.长寿调节途径-哺乳动物,12.神经活性配体-受体的相互作用,13.甲状旁腺激素的合成、分泌和活性,14.肌动蛋白细胞骨架调控,15.肾素分泌,16.肾素-血管紧张素系统,17.志贺氏菌病,18.鞘脂类代谢,19.萜类骨架生物合成,20.β-丙氨酸代谢;B.bta-miR-193a-5p:1.细胞黏附分子,2.补体和凝血级联反应,3.糖基磷脂酰肌醇(GPI)-锚定生物合成,4.造血细胞系,5.单纯疱疹感染,6.甲型H1N1流感,7.利氏曼病,8.MAPK信号通路,9.癌症中的MicroRNAs,10.NOD样受体信号通路,11.新霉素、卡那霉素和庆大霉素的生物合成,12.破骨细胞分化,13.过氧物酶体,14.苯丙氨酸代谢,15.苯丙氨酸、酪氨酸和色氨酸的生物合成,16.磷酸盐和磷酸盐的代谢,17.前列腺癌,18.Rap1信号通路,19.弓形虫病,20.泛素介导的蛋白质水解;C.bta-miR-375:1.氨酰基-tRNA生物合成,2.胆汁分泌,3.昼夜节律,4.扩张型心肌病,5.脂肪酸延长,6.流体剪切应力和动脉粥样硬化,7.胰高血糖素信号通路,8.造血细胞系,9.肥厚性心肌病,10.长寿调节途径-多物种,11.MAPK信号通路,12.磷脂酰肌醇信号传导系统,13.磷脂酶D信号通路,14.黄体酮介导的卵母细胞成熟,15.内质网中的蛋白质加工,16.硫磺继电器系统,17.紧密连接,18.Toll和Imd信号通路,19.泛素介导的蛋白质水解,20.cAMP信号通路。

Fig.5 KEGG annotation of miRNAs target genes absorbed by lncRNA TCONS_00143126 A.bta-miR-133a:1.AMPK signaling pathway,2.Adrenergic signaling in cardiomyocytes,3.Bile secretion,4.Butanoate metabolism,5.Cell adhesion molecules(CAMs),6.Circadian rhythm,7.Dopaminergic synapse,8.GABAergic synapse,9.Inositol phosphate metabolism,10.Insulin secretion,11.Longevity regulating pathway-mammal,12.Neuroactive ligand-receptor interaction,13.Parathyroid hormone synthesis,secretion and action,14.Regulation of actin cytoskeleton,15.Renin secretion,16.Renin-angiotensin system,17.Shigellosis,18.Sphingolipid metabolism,19.Terpenoid backbone biosynthesis,20.beta-Alanine metabolism;B.bta-miR-193a-5p:1.Cell adhesion molecules(CAMs),2.Complement and coagulation cascades,3.Glycosylphosphatidylinositol(GPI)-anchor biosynthe,4.Hematopoietic cell lineage,5.Herpes simplex infection,6.Influenza A,7.Leishmaniasis,8.MAPK signaling pathway,9.MicroRNAs in cancer,10.NOD-like receptor signaling pathway,11.Neomycin,kanamycin and gentamicin biosynthesis,12.Osteoclast differentiation,13.Peroxisome,14.Phenylalanine metabolism,15.Phenylalanine,tyrosine and tryptophan biosynthesis,16.Phosphonate and phosphinate metabolism,17.Prostate cancer,18.Rap1 signaling pathway,19.Toxoplasmosis,20.Ubiquitin mediated proteolysis;C.bta-miR-375:1.Aminoacyl-tRNA biosynthesis,2.Bile secretion,3.Circadian rhythm,4.Dilated cardiomyopathy(DCM),5.Fatty acid elongation,6.Fluid shear stress and atherosclerosis,7.Glucagon signaling pathway,8.Hematopoietic cell lineage,9.Hypertrophic cardiomyopathy(HCM),10.Longevity regulating pathway-multiple species,11.MAPK signaling pathway,12.Phosphatidylinositol signaling system,13.Phospholipase D signaling pathway,14.Progesterone-mediated oocyte maturation,15.Protein processing in endoplasmic reticulum,16.Sulfur relay system,17.Tight junction,18.Toll and Imd signaling pathway,19.Ubiquitin mediated proteolysis,20.cAMP signaling pathway.

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lncRNA TCONS_00143126在奶牛乳腺上皮细胞炎症反应中的表达及生物信息学分析

Expression and Bioinformatics Analysis of lncRNA TCONS _ 00143126 in Inflammatory Response of Bovine Mammary Epithelial Cells

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