Analysis of LncRNA Differential Expression in Mammary Tissue of Cows with Staphylococcus aureus Mastitis

doi:10.13560/j.cnki.biotech.bull.1985.2024-0159

Abstract

Abstract:

【Objective】Long non-coding RNA（lncRNA）is involved in inflammatory responses, however, their specific molecular regulatory mechanism in dairy mastitis remains unclear. In this study, differentially expressed lncRNA（DElncRNA）and its function in mammary tissues of cows with Staphylococcus aureus（S. aureus）-type mastitis were analysed to support subsequent in-depth studies.【Method】Transcriptome sequencing（RNA-Seq）technology and bioinformatics methods were used to carry out lncRNA sequencing, DElncRNA analysis, and GO and KEGG functional enrichment analysis of mammary tissues from healthy cows and cows with S. aureus-induced mastitis.【Result】A total of 1012 lncRNA are detected in the mammary tissues of both groups. Compared to the healthy dairy cows, total 75 lncRNA are up-regulated and 114 down-regulated in cows with S. aureus-induced mastitis. Further analysis reveals that DElncRNA TCONS_00042123, TCONS_00068055, TCONS_00108420 and TCONS_00076361 may be involved in the MAPK, NLR, Jak-STAT and TLR signaling pathways related to inflammation, regulating the occurrence and development of S.aureus-type mastitis in the dairy cows.【Conclusion】189 DElncRNA were obtained from mammary tissues of cows with S. aureus-induced inflammation, which may regulate the process of onset and progression of S. aureus-type cow mastitis through potential target genes.

Key words: dairy cow, mastitis, lncRNA, differential expression analysis, Staphylococcus aureus

ZHOU Ran, WANG Xing-ping, LI Yan-xia, LUORENG Zhuo-ma. Analysis of LncRNA Differential Expression in Mammary Tissue of Cows with Staphylococcus aureus Mastitis[J]. Biotechnology Bulletin, 2024, 40(8): 320-328.

Figures/Tables 7

References 52

[1]	Doehring C, Sundrum A. The informative value of an overview on antibiotic consumption, treatment efficacy and cost of clinical mastitis at farm level[J]. Prev Vet Med, 2019, 165: 63-70. doi: S0167-5877(17)30827-9 pmid: 30851929
[2]	Royster E, Wagner S. Treatment of mastitis in cattle[J]. Vet Clin North Am Food Anim Pract, 2015, 31(1): 17-46. doi: 10.1016/j.cvfa.2014.11.010
[3]	Ruegg PL. A 100-Year Review: Mastitis detection, management, and prevention[J]. J Dairy Sci, 2017, 100(12): 10381-10397. doi: S0022-0302(17)31032-9 pmid: 29153171
[4]	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.
[5]	Chen Y, Yang J, Huang Z, et al. Vitexin mitigates Staphylococcus aureus-induced mastitis via regulation of ROS/ER stress/NF- κ B/MAPK pathway[J]. Oxid Med Cell Longev, 2022, 2022: 7977433.
[6]	Wu YH, He T, Fu YH, et al. Corynoline protects lipopolysaccharide-induced mastitis through regulating AKT/GSK3β/Nrf2 signaling pathway[J]. Environ Toxicol, 2021, 36(12): 2493-2499. doi: 10.1002/tox.23362 pmid: 34477289
[7]	Ran X, Yan Z, Yang YX, et al. Dioscin improves pyroptosis in LPS-induced mice mastitis by activating AMPK/Nrf2 and inhibiting the NF- κB signaling pathway[J]. Oxid Med Cell Longev, 2020, 2020: 8845521.
[8]	Mishra SK, Zhong ZM, Wang H. Hundreds of LncRNAs display circadian rhythmicity in zebrafish larvae[J]. Cells, 2021, 10(11): 3173.
[9]	Tang H, Yuan S, Chen T, et al. Development of an immune-related lncRNA-miRNA-mRNA network based on competing endogenous RNA in periodontitis[J]. J Clin Periodontol, 2021, 48(11): 1470-1479. doi: 10.1111/jcpe.13537 pmid: 34409632
[10]	Cao HL, Liu ZJ, Huang PL, et al. lncRNA-RMRP promotes proliferation, migration and invasion of bladder cancer via miR-206[J]. Eur Rev Med Pharmacol Sci, 2019, 23(3): 1012-1021.
[11]	Tian F, Wang JH, Zhang ZH, et al. LncRNA SNHG7/miR-34a-5p/SYVN1 axis plays a vital role in proliferation, apoptosis and autophagy in osteoarthritis[J]. Biol Res, 2020, 53(1): 9. doi: 10.1186/s40659-020-00275-6 pmid: 32066502
[12]	Lin CJ, Zhu YF, Hao ZY, et al. Genome-wide analysis of LncRNA in bovine mammary epithelial cell injuries induced by Escherichia coli and Staphylococcus aureus[J]. Int J Mol Sci, 2021, 22(18): 9719.
[13]	Chen Y, Jing HY, Chen MY, et al. Transcriptional profiling of exosomes derived from Staphylococcus aureus-infected bovine mammary epithelial cell line MAC-T by RNA-seq analysis[J]. Oxid Med Cell Longev, 2021, 2021: 8460355.
[14]	Tong C, Chen QL, Zhao LL, et al. Identification and characterization of long intergenic noncoding RNAs in bovine mammary glands[J]. BMC Genomics, 2017, 18(1): 468. doi: 10.1186/s12864-017-3858-4 pmid: 28629368
[15]	Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2[J]. Nat Methods, 2012, 9(4): 357-359. doi: 10.1038/nmeth.1923 pmid: 22388286
[16]	Kim D, Pertea G, Trapnell C, et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions[J]. Genome Biol, 2013, 14(4): R36.
[17]	Trapnell C, Roberts A, Goff L, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks[J]. Nat Protoc, 2012, 7(3): 562-578. doi: 10.1038/nprot.2012.016 pmid: 22383036
[18]	Bougarn S, Cunha P, Gilbert FB, et al. Technical note: validation of candidate reference genes for normalization of quantitative PCR in bovine mammary epithelial cells responding to inflammatory stimuli[J]. J Dairy Sci, 2011, 94(5): 2425-2430. doi: 10.3168/jds.2010-3859 pmid: 21524534
[19]	Tripurani SK, Xiao CD, Salem M, et al. Cloning and analysis of fetal ovary microRNAs in cattle[J]. Anim Reprod Sci, 2010, 120(1-4): 16-22. doi: 10.1016/j.anireprosci.2010.03.001 pmid: 20347535
[20]	Gil N, Ulitsky I. Regulation of gene expression by cis-acting long non-coding RNAs[J]. Nat Rev Genet, 2020, 21(2): 102-117. doi: 10.1038/s41576-019-0184-5 pmid: 31729473
[21]	Li JW, Ma W, Zeng P, et al. LncTar: a tool for predicting the RNA targets of long noncoding RNAs[J]. Brief Bioinform, 2015, 16(5): 806-812. doi: 10.1093/bib/bbu048 pmid: 25524864
[22]	Mao XZ, Cai T, Olyarchuk JG, et al. Automated genome annotation and pathway identification using the KEGG Orthology(KO)as a controlled vocabulary[J]. Bioinformatics, 2005, 21(19): 3787-3793.
[23]	Sun YJ, Zhao TQ, Ma YY, et al. Multiple roles of LncRNA-BMNCR on cell proliferation and apoptosis by targeting miR-145/CBFB axis in BMECs[J]. Vet Q, 2023, 43(1): 1-11.
[24]	Zhou M, Barkema HW, Gao J, et al. MicroRNA miR-223 modulates NLRP3 and Keap1, mitigating lipopolysaccharide-induced inflammation and oxidative stress in bovine mammary epithelial cells and murine mammary glands[J]. Vet Res, 2023, 54(1): 78. doi: 10.1186/s13567-023-01206-5 pmid: 37710276
[25]	Chen Z, Liang Y, Lu QY, et al. Cadmium promotes apoptosis and inflammation via the circ08409/miR-133a/TGFB2 axis in bovine mammary epithelial cells and mouse mammary gland[J]. Ecotoxicol Environ Saf, 2021, 222: 112477.
[26]	Chen Y, Yang J, Huang Z, et al. Exosomal lnc-AFTR as a novel translation regulator of FAS ameliorates Staphylococcus aureus-induced mastitis[J]. BioFactors, 2022, 48(1): 148-163.
[27]	Feng F, Li YX, Wang JP, et al. LncRNA CA12-AS1 targets miR-133a to promote LPS-induced inflammatory response in bovine mammary epithelial cells[J]. Int J Biol Macromol, 2024, 261(Pt 1): 129710.
[28]	Bai ZX, Wu YZ, Cai WD, et al. High-throughput analysis of lncRNA in cows with naturally infected Staphylococcus aureus mammary gland[J]. Anim Biotechnol, 2023, 34(7): 2166-2174.
[29]	Wang JP, Hu QC, Yang J, et al. Differential expression profiles of lncRNA following LPS-induced inflammation in bovine mammary epithelial cells[J]. Front Vet Sci, 2021, 8: 758488.
[30]	Jing HY, Chen Y, Qiu CW, et al. LncRNAs transcriptome analysis revealed potential mechanisms of selenium to mastitis in dairy cows[J]. Biol Trace Elem Res, 2022, 200(10): 4316-4324.
[31]	Zhang YH, Xu YQ, Chen BW, et al. Selenium deficiency promotes oxidative stress-induced mastitis via activating the NF-κB and MAPK pathways in dairy cow[J]. Biol Trace Elem Res, 2022, 200(6): 2716-2726.
[32]	Li JD, Yin P, Gong P, et al. 8-Methoxypsoralen protects bovine mammary epithelial cells against lipopolysaccharide-induced inflammatory injury via suppressing JAK/STAT and NF-κB pathway[J]. Microbiol Immunol, 2019, 63(10): 427-437. doi: 10.1111/1348-0421.12730 pmid: 31313848
[33]	Saxena M, Yeretssian G. NOD-like receptors: master regulators of inflammation and cancer[J]. Front Immunol, 2014, 5: 327. doi: 10.3389/fimmu.2014.00327 pmid: 25071785
[34]	Kiewiet MBG, Dekkers R, Gros M, et al. Toll-like receptor mediated activation is possibly involved in immunoregulating properties of cow's milk hydrolysates[J]. PLoS One, 2017, 12(6): e0178191.
[35]	Xu P, Xu XB, Fotina H, et al. Anti-inflammatory effects of chlorogenic acid from Taraxacum officinale on LTA-stimulated bovine mammary epithelial cells via the TLR2/NF-κB pathway[J]. PLoS One, 2023, 18(3): e0282343.
[36]	Cao FQ, Zhou W, Liu GH, et al. Staphylococcus aureus peptidoglycan promotes osteoclastogenesis via TLR2-mediated activation of the NF-κB/NFATc1 signaling pathway[J]. Am J Transl Res, 2017, 9(11): 5022-5030.
[37]	Li HW, Li Q, Guo T, et al. LncRNA CRNDE triggers inflammation through the TLR3-NF-κB-Cytokine signaling pathway[J]. Tumour Biol, 2017, 39(6): 1010428317703821.
[38]	Zhou CK, Gao J, Ji HY, et al. Benzoylaconine modulates LPS-induced responses through inhibition of toll-like receptor-mediated NF-κB and MAPK signaling in RAW_264.7 cells[J]. Inflammation, 2021, 44(5): 2018-2032.
[39]	Zhou HY, Simion V, Pierce JB, et al. LncRNA-MAP3K4 regulates vascular inflammation through the p38 MAPK signaling pathway and cis-modulation of MAP3K4[J]. FASEB J, 2021, 35(1): e21133.
[40]	Barrio L, Roman-Garcia S, Diaz-Mora E, et al. B cell development and T-dependent antibody response are regulated by p38γ and p38δ[J]. Front Cell Dev Biol, 2020, 8: 189. doi: 10.3389/fcell.2020.00189 pmid: 32266269
[41]	Sharma A, Tirpude NV, Kumari M, et al. Rutin prevents inflammation-associated colon damage via inhibiting the p38/MAPKAPK2 and PI3K/Akt/GSK3β/NF-κB signalling axes and enhancing splenic Tregs in DSS-induced murine chronic colitis[J]. Food Funct, 2021, 12(18): 8492-8506.
[42]	Ohto U. Activation and regulation mechanisms of NOD-like receptors based on structural biology[J]. Front Immunol, 2022, 13: 953530.
[43]	Broz P, Dixit VM. Inflammasomes: mechanism of assembly, regulation and signalling[J]. Nat Rev Immunol, 2016, 16(7): 407-420. doi: 10.1038/nri.2016.58 pmid: 27291964
[44]	Wang XZ, Liu MC, Geng N, et al. Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome[J]. Vet Res, 2022, 53(1): 10.
[45]	Ma MR, Pei YF, Wang XX, et al. LncRNA XIST mediates bovine mammary epithelial cell inflammatory response via NF-κB/NLRP3 inflammasome pathway[J]. Cell Prolif, 2019, 52(1): e12525.
[46]	Wang DH, Höing S, Patterson HC, et al. Inflammation in mice ectopically expressing human Pyogenic Arthritis, Pyoderma Gangrenosum, and Acne(PAPA)Syndrome-associated PSTPIP1 A230T mutant proteins[J]. J Biol Chem, 2013, 288(7): 4594-4601.
[47]	Huang W, Li YY, Zhang C, et al. IGF2BP3 facilitates cell proliferation and tumorigenesis via modulation of JAK/STAT signalling pathway in human bladder cancer[J]. J Cell Mol Med, 2020, 24(23): 13949-13960. doi: 10.1111/jcmm.16003 pmid: 33094561
[48]	Kumar N, Sharma N, Mehan S. Connection between JAK/STAT and PPARγ signaling during the progression of multiple sclerosis: insights into the modulation of T-cells and immune responses in the brain[J]. Curr Mol Pharmacol, 2021, 14(5): 823-837. doi: 10.2174/1874467214666210301121432 pmid: 33645493
[49]	Zhuo Q, Wei L, Yin XT, et al. LncRNA ZNF667-AS1 alleviates rheumatoid arthritis by sponging miR-523-3p and inactivating the JAK/STAT signalling pathway[J]. Autoimmunity, 2021, 54(7): 406-414.
[50]	Szydłowski M, Dębek S, Prochorec-Sobieszek M, et al. PIM kinases promote survival and immune escape in primary mediastinal large B-cell lymphoma through modulation of JAK-STAT and NF-κB activity[J]. Am J Pathol, 2021, 191(3): 567-574. doi: 10.1016/j.ajpath.2020.12.001 pmid: 33307035
[51]	Xu ZH, Gwin KA, Li YL, et al. Developmental stage-specific effects of Pim-1 dysregulation on murine bone marrow B cell development[J]. BMC Immunol, 2016, 17(1): 16. doi: 10.1186/s12865-016-0152-1 pmid: 27287229
[52]	de Vries M, Heijink IH, Gras R, et al. Pim1 kinase protects airway epithelial cells from cigarette smoke-induced damage and airway inflammation[J]. Am J Physiol Lung Cell Mol Physiol, 2014, 307(3): L240-L251.

长链非编码RNA lncRNA	引物序列 Primer sequence（5'-3'）	产物长度 Product length/bp
TCONS_00018117	F: TGCCAGGACAAGGAGAAA R: TCAGCACCTAAACCACAAGA	100
TCONS_00096253	F: GGAGCGTAAGTAGGAAGCG R: AATGGTCCTGAATAGGGGTT	92
TCONS_00013236	F: ATGGAAAAACGGACTCGG R: CGGTCTGCCTGAGTCTTG	88
TCONS_00026262	F: AATGCCAGGGCTCCAGTT R: CTCAGATGGACAGTATTCCCTTT	89
TCONS_00018115	F: GCCAGGACAAGGAGAAACA R: TCAGCACCTAAACCACAAGAC	100
TCONS_00113348	F: GGTTCTATTGGATACTGGACAT R: CCTTGCTCTGCTCTTCTTTA	100
TCONS_00120135	F: GCCTTTCATTTTCAAGAGCAT R: TGTCAAGGGAAGGGTGTCTG	90
TCONS_00044738	F: AGGGCTGTTTACCTGCTTA R: GGTGATTTCGGTTCCTCTA	93
TCONS_00032949	F: CAAGACGGCGACTTAGAAA R: ATGCCATCAAGTGGAACAA	94
TCONS_00124429	F: GTCCACCTCCACCCTACA R: ATGCTCACCGAAGTCAAAG	100
TCONS_00117470	F: TGTGCTGGACATAAATCGTG R: AGGTTCTGGGATTGTCTGC	93
TCONS_00088875	F: GATGGTGCTGCTTCAGGATG R: TGGAGGCTACGCCAGGTT	94
GAPDH^a	F: GGCATCGTGGAGGGACTTATG R: CCAGTGAGCTTCCCGTTGAG	185
RPS18^b	F: GTGGTGTTGAGGAAAGCAGACA R: TGATCACACGTTCCACCTCATC	79

长链非编码RNA lncRNA	引物序列 Primer sequence（5'-3'）	产物长度 Product length/bp
TCONS_00018117	F: TGCCAGGACAAGGAGAAA R: TCAGCACCTAAACCACAAGA	100
TCONS_00096253	F: GGAGCGTAAGTAGGAAGCG R: AATGGTCCTGAATAGGGGTT	92
TCONS_00013236	F: ATGGAAAAACGGACTCGG R: CGGTCTGCCTGAGTCTTG	88
TCONS_00026262	F: AATGCCAGGGCTCCAGTT R: CTCAGATGGACAGTATTCCCTTT	89
TCONS_00018115	F: GCCAGGACAAGGAGAAACA R: TCAGCACCTAAACCACAAGAC	100
TCONS_00113348	F: GGTTCTATTGGATACTGGACAT R: CCTTGCTCTGCTCTTCTTTA	100
TCONS_00120135	F: GCCTTTCATTTTCAAGAGCAT R: TGTCAAGGGAAGGGTGTCTG	90
TCONS_00044738	F: AGGGCTGTTTACCTGCTTA R: GGTGATTTCGGTTCCTCTA	93
TCONS_00032949	F: CAAGACGGCGACTTAGAAA R: ATGCCATCAAGTGGAACAA	94
TCONS_00124429	F: GTCCACCTCCACCCTACA R: ATGCTCACCGAAGTCAAAG	100
TCONS_00117470	F: TGTGCTGGACATAAATCGTG R: AGGTTCTGGGATTGTCTGC	93
TCONS_00088875	F: GATGGTGCTGCTTCAGGATG R: TGGAGGCTACGCCAGGTT	94
GAPDH^a	F: GGCATCGTGGAGGGACTTATG R: CCAGTGAGCTTCCCGTTGAG	185
RPS18^b	F: GTGGTGTTGAGGAAAGCAGACA R: TGATCACACGTTCCACCTCATC	79

样品 Sample	质控后的读段数 Clean reads	对比到参考基因组的读段数 Mapped reads	参考基因组上有唯一比对位置的读段数 Uniq mapped reads	参考基因组上有多个比对位置的读段数 Multiple mapped reads	GC /%	Q30 /%
M.C	128 303 002	108 631 698 （84.67%）	97 000 181 （89.29%）	11 631 517 （10.71%）	52.30	85.60
M.S	108 776 484	91 300 177 （83.93%）	84 635 626 （92.70%）	6 664 551 （7.30%）	50.84	85.16

样品 Sample	质控后的读段数 Clean reads	对比到参考基因组的读段数 Mapped reads	参考基因组上有唯一比对位置的读段数 Uniq mapped reads	参考基因组上有多个比对位置的读段数 Multiple mapped reads	GC /%	Q30 /%
M.C	128 303 002	108 631 698 （84.67%）	97 000 181 （89.29%）	11 631 517 （10.71%）	52.30	85.60
M.S	108 776 484	91 300 177 （83.93%）	84 635 626 （92.70%）	6 664 551 （7.30%）	50.84	85.16