奶牛乳腺炎差异表达lncRNA BCL2对炎症及凋亡相关mRNA表达的影响

doi:10.11843/j.issn.0366-6964.2022.07.013

摘要/Abstract

摘要： 本研究旨在探讨长链非编码RNA BCL2(lncRNA BCL2)对奶牛乳腺上皮细胞炎症及凋亡中的调控作用。利用RNAhybrid、Targetscan和miRwalk软件分析lncRNA BCL2海绵吸附的miRNAs及其所靶向的mRNAs，并对这些靶基因进行KEGG通路富集分析；采用qPCR和lncRNA超表达技术研究了lncRNA BCL2在乳腺组织和乳腺上皮细胞炎症中的表达情况及其对乳腺上皮细胞炎症和凋亡相关基因表达的影响。结果发现，与对照组相比，lncRNA BCL2在临床乳腺炎奶牛的乳腺组织和LPS诱导炎症的乳腺上皮细胞中的表达量均显著下调(P<0.05)；在lncRNA BCL2超表达的情况下，乳腺上皮细胞内炎症因子IL-1β与IL-8及炎症信号通路关键基因NF-κB(p65/p50)的表达量均显著上调(P<0.05)；细胞凋亡相关的CASP3和CASP9基因的表达显著下降(P<0.05)，BCL2表达量显著上调(P<0.001)，且BCL2与BAX比值大于2。该结果与生物信息学分析结果相符，表明lncRNA BCL2可能通过lncRNA BCL2-miRNA-BCL2-(NLRP1，CASP1，NLRP3)网络调控奶牛乳腺上皮细胞的炎症与凋亡。综上，奶牛乳腺炎过程中，lncRNA BCL2的表达量下调，减弱了其对IL-1β、IL-8和NF-κB表达上调的作用，从而可能缓解了乳腺炎症；同时，lncRNA BCL2表达量下降减弱了其下调CASP3和CASP9基因表达和上调BCL2基因表达的作用，从而可能促进了奶牛乳腺上皮细胞凋亡。

关键词: lncRNA BCL2, 乳腺上皮细胞, 细胞炎症, 细胞凋亡, 乳腺炎

Abstract: The aim of this study was to investigate the role of long non-coding RNA BCL2 (lncRNA BCL2) in inflammation and apoptosis of dairy cow mammary epithelial cells. The lncRNA BCL2 sponge adsorbed miRNAs and the mRNAs they targeted were analyzed using RNAhybrid, Targetscan and miRwalk softwares, and KEGG pathway enrichment analysis was performed on these target genes; lncRNA BCL2 expression in mammary tissue and mammary epithelial cell inflammation and its effect on the expression of genes involved in inflammation and apoptosis in mammary epithelial cells were studied using qPCR and lncRNA super expression technology. The results showed that, compared with control group, the lncRNA BCL2 was significantly down-regulated (P<0.05) in mammary gland tissues of clinical mastitis cow and inflammatory mammary epithelial cells induced by LPS. In the presence of lncRNA BCL2 overexpression, the expression of the inflammatory cytokines IL-1βand IL-8 and the key gene NF-κB (p65/p50) of the inflammatory signaling pathway were significantly up-regulated (P<0.05). The expression of CASP3 and CASP9 genes related to apoptosis were significantly decreased (P<0.05), BCL2 expression was significantly up-regulated (P<0.001), and the ratio of BCL2 to BAX was greater than 2. The above results were consistent with the results of bioinformatics analysis, which indicated that lncRNA BCL2 might regulate inflammation and apoptosis of bovine mammary epithelial cells through lncRNA BCL2-miRNA-BCL2 (NLRP1, CASP1, NLRP3) network. It can conclude from the above results that in the process of cow mastitis, the expression of lncRNA BCL2 is down-regulated, which weakens its effect on increasing the expression of IL-1β, IL-8, and NF-κB, subsequently alleviates mammary inflammation. At the same time, the decrease of lncRNA BCL2 expression attenuates its role in down-regulating CASP3 and CASP9 gene expression and up-regulating BCL2 gene expression, which might promote the apoptosis of cow mammary epithelial cells.

Key words: lncRNA BCL2, mammary epithelial cells, cellular inflammation, apoptosis, mastitis

中图分类号:

焦鹏, 王兴平, 汪书哲, 王晋鹏, 罗仍卓么, 贾立, 魏大为, 马云. 奶牛乳腺炎差异表达lncRNA BCL2对炎症及凋亡相关mRNA表达的影响[J]. 畜牧兽医学报, 2022, 53(7): 2160-2171.

JIAO Peng, WANG Xingping, WANG Shuzhe, WANG Jinpeng, LUORENG Zhuoma, JIA Li, WEI Dawei, MA Yun. Effect of Differentially Expressed lncRNA BCL2 in Dairy Cow with Mastitis on the Expression of Inflammation and Apoptosis Related mRNA[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2160-2171.

参考文献 39

[1]	HUGHES K,WATSON C J.The mammary microenvironment in mastitis in humans,dairy ruminants,rabbits and rodents:a one health focus[J].J Mammary Gland Biol Neoplasia,2018,23(1-2):27-41.
[2]	SAH K,KARKI P,SHRESTHA R D,et al.MILK Symposium review:improving control of mastitis in dairy animals in Nepal[J].J Dairy Sci,2020,103(11):9740-9747.
[3]	AKHTAR M,GUO S,GUO Y F,et al.Upregulated-gene expression of pro-inflammatory cytokines (TNF-α,IL-1β and IL-6) via TLRs following NF-κB and MAPKs in bovine mastitis[J].Acta Trop,2020,207:105458.
[4]	WU J,LI L,SUN Y,et al.Altered molecular expression of the TLR4/NF-κB signaling pathway in mammary tissue of Chinese Holstein cattle with mastitis[J].PLoS One,2015,10(2):e0118458.
[5]	CHEN J,AO L F,YANG J.Long non-coding RNAs in diseases related to inflammation and immunity[J].Ann Transl Med,2019,7(18):494.
[6]	HADJICHARALAMBOUS M R,LINDSAY M A.Long non-coding RNAs and the innate immune response[J].Noncoding RNA,2019,5(2):34.
[7]	SCACALOSSI K R,VAN SOLINGEN C,MOORE K J.Long non-coding RNAs regulating macrophage functions in homeostasis and disease[J].Vascul Pharmacol,2019,114:122-130.
[8]	LI Y,CHEN D,GAO X,et al.LncRNA NEAT1 regulates cell viability and invasion in esophageal squamous cell carcinoma through the miR-129/CTBP2 Axis[J].Dis Markers,2017,2017:5314649.
[9]	LI Y H,ZHANG S S,ZHANG C X,et al.LncRNA MEG3 inhibits the inflammatory response of ankylosing spondylitis by targeting miR-146a[J].Mol Cell Biochem,2020,466(1-2):17-24.
[10]	NITSCHE A,STADLER P F.Evolutionary clues in lncRNAs[J].Wiley Interdiscip Rev RNA,2017,8(1):e1376,doi:10.1002/wrna.1376.
[11]	PARDINI B,DRAGOMIR M P.SCIRT lncRNA blocks the shot of breast cancer cells self-renewal mechanism[J].Cancer Res,2021,81(3):535-536.
[12]	CHEN Y,JING H Y,CHEN M Y,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.
[13]	ÖZDEMIR S,ALTUN S.Genome-wide analysis of mRNAs and lncRNAs in Mycoplasma bovis infected and non-infected bovine mammary gland tissues[J].Mol Cell Probes,2020,50:101512.
[14]	YANG B,JIAO B L,GE W,et al.Transcriptome sequencing to detect the potential role of long non-coding RNAs in bovine mammary gland during the dry and lactation period[J].BMC Genomics,2018,19(1):605.
[15]	杨兵.奶牛乳腺差异表达长链非编码RNA的筛选、鉴定及其功能研究[D].杨凌:西北农林科技大学,2019.YANG B.Screening,identification and functional studies of long non-coding RNAs differentially expressed in mammary gland of dairy cows[D].Yangling:Northwest A&F University,2019.(in Chinese)
[16]	MA M R,PEI Y F,WANG X X,et al.LncRNA XIST mediates bovine mammary epithelial cell inflammatory response via NF-κB/NLRP3 inflammasome pathway[J].Cell Prolif,2019,52(1):e12525.
[17]	WANG H,WANG X X,LI X R,et al.A novel long non-coding RNA regulates the immune response in MAC-T cells and contributes to bovine mastitis[J].FEBS J,2019,286(9):1780-1795.
[18]	WANG X X,WANG H,ZHANG R Q,et al.LRRC75A antisense lncRNA1 knockout attenuates inflammatory responses of bovine mammary epithelial cells[J].Int J Biol Sci,2020,16(2):251-263.
[19]	YANG W,LI X Z,QI S P,et al.lncRNA H19 is involved in TGF-β1-induced epithelial to mesenchymal transition in bovine epithelial cells through PI3K/AKT signaling pathway[J].PeerJ,2017,5:e3950.
[20]	HUYNH H T,ROBITAILLE G,TURNER J D.Establishment of bovine mammary epithelial cells (MAC-T):an in vitro model for bovine lactation[J].Exp Cell Res,1991,197(2):191-199.
[21]	ZHANG Y N,ZHU Y Y,GAO G S,et al.Knockdown XIST alleviates LPS-induced WI-38 cell apoptosis and inflammation injury via targeting miR-370-3p/TLR4 in acute pneumonia[J].Cell Biochem Funct,2019,37(5):348-358.
[22]	YUE P J,JING L J,ZHAO X Y,et al.Down-regulation of taurine-up-regulated gene 1 attenuates inflammation by sponging miR-9-5p via targeting NF-κB1/p50 in multiple sclerosis[J].Life Sci,2019,233:116731.
[23]	ZHU W,MEN X L.Negative feedback of NF-κB signaling by long noncoding RNA MALAT1 controls lipopolysaccharide-induced inflammation injury in human lung fibroblasts WI-38[J].J Cell Biochem,2020,121(2):1945-1952.
[24]	LI X Z,WANG H,ZHANG Y F,et al.Overexpression of lncRNA H19 changes basic characteristics and affects immune response of bovine mammary epithelial cells[J].PeerJ,2019,7:e6715.
[25]	CHAN C,LI L W,MCCALl C E,et al.Endotoxin tolerance disrupts chromatin remodeling and NF-κB transactivation at the IL-1β promoter[J].J Immunol,2005,175(1):461-468.
[26]	GIRIDHARAN S,SRINIVASAN M.Mechanisms of NF-κB p65 and strategies for therapeutic manipulation[J].J Inflamm Res,2018,11:407-419.
[27]	ROLOVA T,DHUNGANA H,KORHONEN P,et al.Deletion of nuclear factor kappa B p50 subunit decreases inflammatory response and mildly protects neurons from transient forebrain ischemia-induced damage[J].Aging Dis,2016,7(4):450-465.
[28]	LIU S Z,SHI X M,BAUER I,et al.Lingual antimicrobial peptide and IL-8 expression are oppositely regulated by the antagonistic effects of NF-κB p65 and C/EBPβ in mammary epithelial cells[J].Mol Immunol,2011,48(6-7):895-908.
[29]	LIU X F,YE F,XIONG H B,et al.IL-1β induces IL-6 production in retinal Müller cells predominantly through the activation of p38 MAPK/NF-κB signaling pathway[J].Exp Cell Res,2015,331(1):223-231.
[30]	CHEN L,YANG W J,GUO Y J,et al.Exosomal lncRNA GAS5 regulates the apoptosis of macrophages and vascular endothelial cells in atherosclerosis[J].PLoS One,2017,12(9):e0185406.
[31]	LI T H,CHEN Y,ZHANG J J,et al.LncRNA TUG1 promotes cells proliferation and inhibits cells apoptosis through regulating AURKA in epithelial ovarian cancer cells[J].Medicine (Baltimore),2018,97(36):e12131.
[32]	WU X G,ZHENG X H,CHENG J,et al.LncRNA TUG1 regulates proliferation and apoptosis by regulating miR-148b/IGF2 axis in ox-LDL-stimulated VSMC and HUVEC[J].Life Sci,2020,243:117287.
[33]	ROBERTS A W.Therapeutic development and current uses of BCL-2 inhibition[J].Hematology Am Soc Hematol Educ Program,2020,2020(1):1-9.
[34]	SIDDIQUI W A,AHAD A,AHSAN H.The mystery of BCL2 family:Bcl-2 proteins and apoptosis:an update[J].Arch Toxicol,2015,89(3):289-317.
[35]	LI S F,LI H,ZHANG Y L,et al.SFTSV infection induces BAK/BAX-dependent mitochondrial DNA release to trigger NLRP3 inflammasome activation[J].Cell Rep,2020,30(13):4370-4385.e7.
[36]	FAUSTIN B,CHEN Y,ZHAI D Y,et al.Mechanism of Bcl-2 and Bcl-XL inhibition of NLRP1 inflammasome:loop domain-dependent suppression of ATP binding and oligomerization[J].Proc Natl Acad Sci U S A,2009,106(10):3935-3940.
[37]	LIU X M,ZHAO H B,JIN Q,et al.Resveratrol induces apoptosis and inhibits adipogenesis by stimulating the SIRT1-AMPKα-FOXO1 signalling pathway in bovine intramuscular adipocytes[J].Mol Cell Biochem,2018,439(1-2):213-223.
[38]	HU Q L,CUI X J,TAO L,et al.Staphylococcus aureus induces apoptosis in primary bovine mammary epithelial cells through Fas-FADD death receptor-linked caspase-8 signaling[J].DNA Cell Biol,2014,33(6):388-397.
[39]	FU Y R,JIN Y C,ZHAO Y,et al.Zearalenone induces apoptosis in bovine mammary epithelial cells by activating endoplasmic reticulum stress[J].J Dairy Sci,2019,102(11):10543-10553.