miR-665在奶牛乳腺上皮细胞炎症中的表达及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1071

摘要/Abstract

摘要：

为探究miR-665在奶牛乳腺上皮细胞炎症中的表达及功能,利用脂多糖（LPS）诱导奶牛乳腺上皮细胞产生炎症反应,在LPS诱导0、3、6和12 h采用qPCR技术检测了miR-665及其潜在靶mRNA的表达水平,并采用生物信息学方法进行了miR-665保守性分析、靶基因预测及其KEGG和GO功能富集分析。结果表明,miR-665在牛、人和小鼠等物种间高度保守;与0 h相比,miR-665在LPS诱导乳腺上皮细胞产生炎症的3、6和12 h的表达量均显著上调,且与促进炎症相关的潜在靶基因（MAPK14、MAP3K2、SMAD2、SP1）的表达量呈相反趋势;miR-665的靶基因预测及其KEGG和GO功能富集结果显示,miR-665可能通过参与MAPK、Th17细胞分化、肿瘤坏死因子等信号通路发挥作用。推测miR-665可能在LPS诱导的乳腺上皮细胞炎症中具有抑制作用。

关键词: 奶牛, miR-665, 乳腺上皮细胞, 乳腺炎, 生物信息学分析

Abstract:

In order to explore the expression and function of miR-665 in bovine mammary epithelial cell inflammation,lipopolysaccharide（LPS）was used to induce bovine mammary epithelial cell inflammation,qPCR technology was applied to detect the expression levels of miR-665 and its potential target mRNA at 0,3,6 and 12 h of LPS-induced. Bioinformatics method was adapted to conduct the conserved miR-665,target gene prediction,KEGG and GO function enrichment analysis. The results showed that miR-665 was highly conserved in many species,such as Bos taurus,Homo sapiens and Mus musculus. Compared with 0 h,the expression levels of miR-665 were significantly up-regulated at 3,6 and 12 h of LPS-induced inflammation in the mammary epithelial cells,while the expression levels of MAPK14,MAP3K2,SMAD2,and SP1 genes related to inflammation promotion were in an opposite trend. The predicted target genes of miR-665 and the enrichment results of KEGG and GO functions showed that miR-665 may play its role by participating in MAPK and Th17 cell differentiation,tumor necrosis factor and other signaling pathways. It is speculated that miR-665 may have an inhibitory effect on the LPS-induced inflammation of mammary epithelial cells.

Key words: dairy cow, miR-665, mammary epithelial cell, mastitis, bioinformatics analysis

李宇航, 王兴平, 杨箭, 罗仍卓么, 任倩倩, 魏大为, 马云. miR-665在奶牛乳腺上皮细胞炎症中的表达及功能分析[J]. 生物技术通报, 2022, 38(5): 159-168.

LI Yu-hang, WANG Xing-ping, YANG Jian, LUORENG Zhuo-ma, REN Qian-qian, WEI Da-wei, MA Yun. Expression and Functional Analysis of miR-665 in Bovine Mammary Epithelial Cell Inflammation[J]. Biotechnology Bulletin, 2022, 38(5): 159-168.

图/表 10

表1 miR-665的逆转录及qPCR引物

Table 1 Reverse transcription and qPCR primers of miR-665

基因名称 Gene name	引物序列 Prime sequence（5'-3'）	产物大小 Product length/bp
miR-665	RT：GTCGTATCCAGTGCAGGGTCCGAGG TATTCGCACTGGATACGACAGGGGCCT	64
	F：GTCATGGAACCAGTAGGCCG
	R：CAGTGCAGGGTCCGAGGTAT
IL-1β	F：CAACCGTACCTGAACCC	193
IL-1β	R：GACACCACCTGCCTGAA	193
IL-8	F：ACACATTCCACACCTTTCCAC	149
IL-8	R：ACCTTCTGCACCCACTTTTC	149
GADPH	F：GGCATCGTGGAGGGACTTATG	186
GADPH	R：GCCAGTGAGCTTCCCGTTGAG	186
RPS18	F：GTGGTGTTGAGGAAAGCAGACA	79
RPS18	R：TGATCACACGTTCCACCTCATC	79

表2 miR-665潜在关键靶基因的qPCR引物

Table 2 qPCR primers for potential key target genes of miR-665

基因名称 Gene name	引物序列 Prime sequence（5'-3'）	产物大小 Product length/bp
MAPK14	F：ATCTTGGATTTCGGACTGGC	264
MAPK14	R：ATGGCTTGGCATCCTGTTA	264
MAP3K2	F：CGGAAATACACCCGTCAGA	324
MAP3K2	R：AGCCATCGCTTCAAACTCG	324
SMAD2	F：GAAGGCAGACGGTAACAAGC	114
SMAD2	R：TTGAGCAACGCACTGAAGG	114
SP1	F：CGGTGGGCAGTATGTTGT	237
SP1	R：CCTCCACTTCCTCGGTTT	237

图1 主要炎症因子在LPS诱导的乳腺上皮细胞炎症中的表达 *表示P < 0.05;**表示P < 0.01,下同

Fig.1 Expression of major inflammatory factors in the LPS-induced inflammation of mammary epithelial cells * refers to P < 0.05;** refers to P < 0.01,the same below

图2 miR-665在LPS诱导的乳腺上皮细胞中的表达量检测

Fig.2 Expression detection of miR-665 in the LPS-induced mammary epithelial cells

图3 牛与人、小鼠miR-665的序列对比图

Fig.3 Sequence comparison of miR-665 from bovine,hu-man and mouse

图4 miR-665差异表达的潜在靶基因筛选的韦恩图

Fig.4 Venn diagram for screening potential target genes differentially expressed by miR-665

图5 miR-665潜在靶基因的GO功能注释结果

Fig.5 GO functional annotation results of miR-665 potential target genes

图6 miR-665潜在靶基因的KEGG通路分析结果

Fig.6 Results of KEGG pathway analysis of miR-665 potential target genes

图7 miR-665相关基因在奶牛乳腺上皮细胞炎症中的表达量

Fig.7 Expression of miR-665 related genes in the bovine mammary epithelial cell inflammation

表3 牛与人、小鼠相关基因的同源性分析结果

Table 3 Homology analysis of related genes in the bovine,human and mouse

基因名称Gene name	牛Bos taurus	人Homo sapiens		小鼠Mus musculus
基因名称Gene name	GenBank ID	GenBank ID	同源性Homology/%	GenBank ID	同源性Homology/%
MAPK14	NM_001102174.1	NM_001315.3	84.34	NM_011951.3	84.88
MAP3K2	XM_002685232.6	NM_006609.5	85.91	NM_011946.3	85.14
SMAD2	NM_001046218.1	NM_005901.6	93.96	NM_010754.5	88.70
SP1	NM_001078027.1	NM_138473.3	91.03	NM_013672.2	88.99

参考文献 48

[1]	He W, Ma S, Lei L, et al. Prevalence, etiology, and economic impact of clinical mastitis on large dairy farms in China[J]. Vet Microbiol, 2020, 242:108570. doi: 10.1016/j.vetmic.2019.108570 URL
[2]	Royster E, Wagner S. Treatment of mastitis in cattle[J]. Vet Clin North Am Food Anim Pract, 2015, 31(1):17-46, v. doi: 10.1016/j.cvfa.2014.11.010 URL
[3]	Gorji AE, Roudbari Z, Sadeghi B, et al. Transcriptomic analysis on the promoter regions discover gene networks involving mastitis in cattle[J]. Microb Pathog, 2019, 137:103801. doi: 10.1016/j.micpath.2019.103801 URL
[4]	Asselstine V, Miglior F, Suárez-Vega A, et al. Genetic mechanisms regulating the host response during mastitis[J]. J Dairy Sci, 2019, 102(10):9043-9059. doi: S0022-0302(19)30701-5 pmid: 31421890
[5]	Saliminejad K, Khorram Khorshid HR, Soleymani Fard S, et al. An overview of microRNAs:Biology, functions, therapeutics, and analysis methods[J]. J Cell Physiol, 2019, 234(5):5451-5465. doi: 10.1002/jcp.27486 pmid: 30471116
[6]	Correia de Sousa M, Gjorgjieva M, Dolicka D, et al. Deciphering miRNAs’ action through miRNA Editing[J]. Int J Mol Sci, 2019, 20(24):6249. doi: 10.3390/ijms20246249 URL
[7]	Rupaimoole R, Slack FJ. MicroRNA therapeutics:towards a new era for the management of cancer and other diseases[J]. Nat Rev Drug Discov, 2017, 16(3):203-222. doi: 10.1038/nrd.2016.246 pmid: 28209991
[8]	Luoreng ZM, Wang XP, Mei CG, et al. Comparison of microRNA profiles between bovine mammary glands infected with Staphylococcus aureus and Escherichia coli[J]. Int J Biol Sci, 2018, 14(1):87-99. doi: 10.7150/ijbs.22498 URL
[9]	Li R, Zhang CL, Liao XX, et al. Transcriptome MicroRNA profiling of bovine mammary glands infected with Staphylococcus aureus[J]. Int J Mol Sci, 2015, 16(12):4997-5013. doi: 10.3390/ijms16034997 URL
[10]	Luoreng ZM, Yang J, Wang XP, et al. Expression profiling of microRNA from peripheral blood of dairy cows in response to Staphylococcus aureus-infected mastitis[J]. Front Vet Sci, 2021, 8:691196. doi: 10.3389/fvets.2021.691196 URL
[11]	Luoreng ZM, Wang XP, Mei CG, et al. Expression profiling of peripheral blood miRNA using RNAseq technology in dairy cows with Escherichia coli-induced mastitis[J]. Sci Rep, 2018, 8(1):12693. doi: 10.1038/s41598-018-30518-2 URL
[12]	Wang XP, Luoreng ZM, Zan LS, et al. Bovine miR-146a regulates inflammatory cytokines of bovine mammary epithelial cells via targeting the TRAF6 gene[J]. J Dairy Sci, 2017, 100(9):7648-7658. doi: 10.3168/jds.2017-12630 URL
[13]	Cai MC, Fan WQ, Li XY, et al. The regulation of Staphylococcus aureus-induced inflammatory responses in bovine mammary epithelial cells[J]. Front Vet Sci, 2021, 8:683886. doi: 10.3389/fvets.2021.683886 URL
[14]	Li M, Zhang S, Qiu Y, et al. Upregulation of miR-665 promotes apoptosis and colitis in inflammatory bowel disease by repressing the endoplasmic Reticulum stress components XBP₁ and ORMDL3[J]. Cell Death Dis, 2017, 8(3):e2699. doi: 10.1038/cddis.2017.76 URL
[15]	Liu S, Li XM, Yuan JB, et al. MiR-665 inhibits inflammatory response in microglia following spinal cord injury by targeting TREM2[J]. Eur Rev Med Pharmacol Sci, 2021, 25(1):65-70.
[16]	Guo Q, Lin Y, Hu J. Inhibition of miR-665-3p enhances autophagy and alleviates inflammation in Fusarium solani-induced keratitis[J]. Invest Ophthalmol Vis Sci, 2021, 62(1):24.
[17]	Pareek R, Wellnitz O, Van Dorp R, et al. Immunorelevant gene expression in LPS-challenged bovine mammary epithelial cells[J]. J Appl Genet, 2005, 46(2):171-177. pmid: 15876684
[18]	Strandberg Y, Gray C, Vuocolo T, et al. Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells[J]. Cytokine, 2005, 31(1):72-86. pmid: 15882946
[19]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J]. Methods, 2001, 25(4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[20]	Sharun K, Dhama K, Tiwari R, et al. Advances in therapeutic and managemental approaches of bovine mastitis:a comprehensive review[J]. Vet Q, 2021, 41(1):107-136. doi: 10.1080/01652176.2021.1882713 pmid: 33509059
[21]	Dalanezi FM, Joaquim SF, Guimarães FF, et al. Influence of pathogens causing clinical mastitis on reproductive variables of dairy cows[J]. J Dairy Sci, 2020, 103(4):3648-3655. doi: S0022-0302(20)30114-4 pmid: 32089296
[22]	Xu H, Zhang T, Hu X, et al. Exosomal miR-193b-5p as a regulator of LPS-induced inflammation in dairy cow mammary epithelial cells[J]. In Vitro Cell Dev Biol Anim, 2021, 57(7):695-703. doi: 10.1007/s11626-021-00596-0 URL
[23]	Chen Z, Xu X, Tan T, et al. MicroRNA-145 regulates immune cytokines via targeting FSCN1 in Staphylococcus aureus-induced mastitis in dairy cows[J]. Reprod Domest Anim, 2019, 54(6):882-891. doi: 10.1111/rda.13438 pmid: 30974481
[24]	Zhang M, Wang S, Yi A, et al. microRNA-665 is down-regulated in gastric cancer and inhibits proliferation, invasion, and EMT by targeting PPP2R2A[J]. Cell Biochem Funct, 2020, 38(4):409-418. doi: 10.1002/cbf.3485 URL
[25]	Liu J, Jiang Y, Wan Y, et al. MicroRNA-665 suppresses the growth and migration of ovarian cancer cells by targeting HOXA10[J]. Mol Med Rep, 2018, 18(3):2661-2668.
[26]	Zhao XG, Hu JY, Tang J, et al. miR-665 expression predicts poor survival and promotes tumor metastasis by targeting NR4A3 in breast cancer[J]. Cell Death Dis, 2019, 10(7):479. doi: 10.1038/s41419-019-1705-z URL
[27]	Damasceno LEA, Prado DS, Veras FP, et al. PKM2 promotes Th17 cell differentiation and autoimmune inflammation by fine-tuning STAT3 activation[J]. J Exp Med, 2020, 217(10):e20190613. DOI: 10.1084/jem,20190613. doi: 10.1084/jem,20190613 URL
[28]	Bougarne N, Weyers B, Desmet SJ, et al. Molecular actions of PPARα in lipid metabolism and inflammation[J]. Endocr Rev, 2018, 39(5):760-802. doi: 10.1210/er.2018-00064 pmid: 30020428
[29]	Rahman A, Henry KM, Herman KD, et al. Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation[J]. Elife, 2019, 8:e50990. doi: 10.7554/eLife.50990 URL
[30]	Clarke DC, Lauffenburger DA. Multi-pathway network analysis of mammalian epithelial cell responses in inflammatory environments[J]. Biochem Soc Trans, 2012, 40(1):133-138. doi: 10.1042/BST20110633 URL
[31]	Madkour MM, Anbar HS, El-Gamal MI. Current status and future prospects of p38α/MAPK14 kinase and its inhibitors[J]. Eur J Med Chem, 2021, 213:113216. doi: 10.1016/j.ejmech.2021.113216 URL
[32]	Pan W, Wei N, Xu W, et al. MicroRNA-124 alleviates the lung injury in mice with septic shock through inhibiting the activation of the MAPK signaling pathway by downregulating MAPK14[J]. Int Immunopharmacol, 2019, 76:105835. doi: 10.1016/j.intimp.2019.105835 URL
[33]	Zhang L, Han B, Liu H, et al. Circular RNA circACSL1 aggravated myocardial inflammation and myocardial injury by sponging miR-8055 and regulating MAPK14 expression[J]. Cell Death Dis, 2021, 12(5):487. doi: 10.1038/s41419-021-03777-7 pmid: 33986259
[34]	Schmidt C, Peng B, Li Z, et al. Mechanisms of proinflammatory cytokine-induced biphasic NF-kappaB activation[J]. Mol Cell, 2003, 12(5):1287-1300. doi: 10.1016/S1097-2765(03)00390-3 URL
[35]	Wang AD, Dai LF, Yang L, et al. Upregulation of miR-335 reduces myocardial injury following myocardial infarction via targeting MAP3K2[J]. Eur Rev Med Pharmacol Sci, 2021, 25(1):344-352.
[36]	Wu N, Chen D, Sun H, et al. MAP3K2 augments Th1 cell differentiation via IL-18 to promote T cell-mediated colitis[J]. Sci China Life Sci, 2021, 64(3):389-403. doi: 10.1007/s11427-020-1720-9 URL
[37]	Martinez GJ, Zhang Z, Reynolds JM, et al. Smad2 positively regulates the generation of Th17 cells[J]. J Biol Chem, 2010, 285(38):29039-29043. doi: 10.1074/jbc.C110.155820 pmid: 20667820
[38]	Kao YH, Chen PH, Wu TY, et al. Lipopolysaccharides induce Smad2 phosphorylation through PI3K/Akt and MAPK cascades in HSC-T6 hepatic stellate cells[J]. Life Sci, 2017, 184:37-46. doi: 10.1016/j.lfs.2017.07.004 URL
[39]	Zhou YX, Han WW, Song DD, et al. Effect of miR-10a on Sepsis-induced liver injury in rats through TGF-β1/Smad signaling pathway[J]. Eur Rev Med Pharmacol Sci, 2020, 24(2):862-869.
[40]	Jia L, Sun P, Gao H, et al. Mangiferin attenuates bleomycin-induced pulmonary fibrosis in mice through inhibiting TLR4/p65 and TGF-β1/Smad2/3 pathway[J]. J Pharm Pharmacol, 2019, 71(6):1017-1028. doi: 10.1111/jphp.13077 pmid: 30847938
[41]	Chen L, Yang T, Lu DW, et al. Central role of dysregulation of TGF-β/Smad in CKD progression and potential targets of its treatment[J]. Biomed Pharmacother, 2018, 101:670-681. doi: S0753-3322(17)36823-3 pmid: 29518614
[42]	Yang Q, Ren GL, Wei B, et al. Conditional knockout of TGF-βRII /Smad2 signals protects against acute renal injury by alleviating cell necroptosis, apoptosis and inflammation[J]. Theranostics, 2019, 9(26):8277-8293. doi: 10.7150/thno.35686 pmid: 31754396
[43]	Cai F, Chen L, Sun Y, et al. MiR-539 inhibits the malignant behavior of breast cancer cells by targeting SP1[J]. Biochem Cell Biol, 2020, 98(3):426-433. doi: 10.1139/bcb-2019-0111 URL
[44]	Peng XB, Wu MH, Liu WX, et al. miR-502-5p inhibits the proliferation, migration and invasion of gastric cancer cells by targeting SP1[J]. Oncol Lett, 2020, 20(3):2757-2762. doi: 10.3892/ol.2020.11808 URL
[45]	Fu J, Li T, Jiang X, et al. MicroRNA-199-3p targets Sp1 transcription factor to regulate proliferation and epithelial to mesenchymal transition of human lung cancer cells[J]. 3 Biotech, 2021, 11(7):352. doi: 10.1007/s13205-021-02881-x URL
[46]	Wang R, Yang Y, Wang H, et al. MiR-29c protects against inflammation and apoptosis in Parkinson’s disease model in vivo and in vitro by targeting SP1[J]. Clin Exp Pharmacol Physiol, 2020, 47(3):372-382. doi: 10.1111/1440-1681.13212 URL
[47]	Lee WR, Kim KH, An HJ, et al. Effects of chimeric decoy oligodeoxynucleotide in the regulation of transcription factors NF-κB and Sp1 in an animal model of atherosclerosis[J]. Basic Clin Pharmacol Toxicol, 2013, 112(4):236-243. doi: 10.1111/bcpt.12029 URL
[48]	Zheng H, Dong X, Liu N, et al. Regulation and mechanism of mouse miR-130a/b in metabolism-related inflammation[J]. Int J Biochem Cell Biol, 2016, 74:72-83. doi: 10.1016/j.biocel.2016.02.021 URL