lncRNA RRAS2-AS1在LPS诱导奶牛乳腺上皮细胞炎症中的功能

doi:10.3864/j.issn.0578-1752.2024.14.014

摘要/Abstract

摘要：

【背景】奶牛乳腺炎是奶牛中危害最严重的疾病之一，严重影响乳品质，不仅造成经济损失，甚至危及人类健康。已有研究表明，lncRNAs广泛参与人和动物的炎症和免疫调节。lncRNA RRAS2-AS1是我们在前期研究中新发现的差异表达lncRNA，其表达模式和功能尚不清楚。【目的】通过探究lncRNA RRAS2-AS1在奶牛乳腺上皮细胞（bovine mammary epithelial cells, bMECs）炎症反应中的作用机制，为奶牛乳腺炎的分子调控机制解析和抗乳腺炎分子选育提供理论依据。【方法】利用RT-PCR和RACE等技术进行了lncRNA RRAS2-AS1的克隆，并通过生物信息学方法进行lncRNA RRAS2-AS1的靶基因预测及功能富集分析；利用细胞免疫荧光技术鉴定bMECs，利用细胞核质分离及半定量PCR技术检测了lncRNA RRAS2-AS1的亚细胞定位情况；采用qRT-PCR检测了lncRNA RRAS2-AS1在脂多糖（lipopolysaccharide, LPS）诱导bMECs炎症反应和乳腺炎奶牛乳腺组织中的表达模式；利用LPS诱导bMECs构建了炎症细胞模型，在此基础上，通过lncRNA超表达、qRT-PCR和ELISA等技术研究了lncRNA RRAS2-AS1对炎性bMECs内的促炎细胞因子基因、增殖相关基因和凋亡相关基因在mRNA和（或）蛋白质水平表达的影响，同时采用EdU、CCK-8和流式细胞术进一步验证了lncRNA RRAS2-AS1对bMECs增殖、活力和凋亡的影响。【结果】 lncRNA RRAS2-AS1基因的长度为363 bp，主要定位于细胞质中。表达量检测结果表明，与对照组相比，lncRNA RRAS2-AS1在LPS诱导炎症的bMECs和乳腺炎组织中的表达量显著下调（P<0.05）；lncRNA RRAS2-AS1超表达试验结果表明，与对照组相比，lncRNA RRAS2-AS1超表达组的炎症信号通路关键基因（TLR4和NF-κB1）、促炎细胞因子基因（IL-1β、IL-6和IL-8）、促凋亡基因（BAD、CASP3和BAX等）的表达量显著下调（P<0.01），而增殖标志基因（CDK2、CDK4和PCNA）的表达量显著上调（P<0.05）。此外，lncRNA RRAS2-AS1超表达组的细胞活力显著增加（P<0.05），而bMECs的凋亡率极显著降低（P<0.01）。【结论】bbbblncRNA RRAS2-AS1在LPS诱导bMECs的炎症反应和乳腺炎奶牛的乳腺组织中均显著下调；超表达lncRNA RRAS2-AS1可下调促炎细胞因子IL-6、IL-8和IL-1β在mRNA和蛋白质水平的表达，并促进细胞活力和增殖、抑制细胞凋亡，从而减轻LPS诱导的bMECs炎症反应，该结果可为解析奶牛乳腺炎的分子调控网络奠定了基础。

关键词: 奶牛, 乳腺炎, lncRNA RRAS2-AS1, 细胞增殖, 细胞凋亡

Abstract:

【Background】 Bovine mastitis is one of the most serious diseases in dairy cows, which seriously affects milk quality and not only causes economic losses, but also even jeopardizes human health. Previous studies have shown that lncRNAs were widely involved in inflammation and immune regulation in humans and animals. lncRNA RRAS2-AS1 was a newly identified differentially expressed lncRNA in our previous study, and its expression pattern and function are still unclear. 【Objective】 The aim of this study was intended to investigate the mechanism of lncRNA RRAS2-AS1 in the inflammatory response of bovine mammary epithelial cells (bMECs) in dairy cows, so as to provide a theoretical basis for the resolution of molecular regulatory mechanisms of bovine mastitis and molecular breeding for anti-mastitis.【Method】 The cloning of lncRNA RRAS2-AS1 was performed by RT-PCR and RACE, and the target genes prediction and functional enrichment analysis of lncRNA RRAS2-AS1 were performed by bioinformatics methods. The bMECs were identified by immunofluorescence staining, and the subcellular localization of lncRNA RRAS2-AS1 was detected by cytoplasmic isolation and semi-quantitative PCR. The expression patterns of lncRNA RRAS2-AS1 in lipopolysaccharide (LPS) induced bMECs inflammatory response and in bovine mammary tissues with mastitis were detected using qRT-PCR. An inflammatory cell model was constructed using LPS-induced bMECs, on this basis, the effects of lncRNA RRAS2-AS1 on the expression of pro-inflammatory cytokine genes, proliferation-related genes and apoptosis-related genes at the mRNA and/or protein levels in inflammatory bMECs were studied using lncRNA overexpression, qRT-PCR and ELISA techniques. At the same time, the effects of lncRNA RRAS2-AS1 on the proliferation, viability, and apoptosis of bMECs were further verified using EdU, CCK-8 and flow cytometry.【Result】 The length of lncRNA RRAS2-AS1 was 363 bp, which was mainly localized in the cytoplasm. The expression assay results showed that, compared with the control group, the expression of lncRNA RRAS2-AS1 was significantly down-regulated in LPS induced bMECs inflammation and in bovine mammary tissues with mastitis (P<0.05). The results of lncRNA RRAS2-AS1 overexpression assay showed that, compared with the control group, the expression of key genes the inflammatory signaling pathway (TLR4 and NF-κB1), pro-inflammatory cytokine genes (IL-1β, IL-6 and IL-8), pro-apoptotic genes (BAD, CASP3, BAX, etc.) were significantly down-regulated (P<0.01) in the lncRNA RRAS2-AS1 overexpression group, while the expression levels of proliferation marker genes (CDK2, CDK4, and PCNA) were significantly up-regulated (P<0.05). In addition, the cell viability of the lncRNA RRAS2-AS1 overexpression group was significantly increased (P<0.05), while the apoptosis rate of bMECs was significantly reduced (P<0.01).【Conclusion】 lncRNA RRAS2-AS1 was significantly down-regulated in LPS induced bMECs inflammation and in bovine mammary tissues with mastitis. Overexpression of lncRNA RRAS2-AS1 decreased the expression of pro-inflammatory cytokines IL-6, IL-8, and IL-1β at mRNA and protein levels, and promoted cell viability and proliferation and inhibited apoptosis, which attenuated the inflammatory response of LPS-induced bMECs. These and the results laid the foundation for analyzing the molecular regulatory network of mastitis in dairy cows.

Key words: cow, mastitis, lncRNA RRAS2-AS1, cell proliferation, cell apoptosis

王晋鹏, 罗仍卓么, 李彦霞, 冯芬, 王正兴, 潘传英, 蓝贤勇, 王兴平. lncRNA RRAS2-AS1在LPS诱导奶牛乳腺上皮细胞炎症中的功能[J]. 中国农业科学, 2024, 57(14): 2874-2888.

WANG JinPeng, LUORENG ZhuoMa, LI YanXia, FENG Fen, WANG ZhengXing, PAN ChuanYing, LAN XianYong, WANG XingPing. The Function of lncRNA RRAS2-AS1 in LPS Induced Bovine Mammary Epithelial Cells Inflammation[J]. Scientia Agricultura Sinica, 2024, 57(14): 2874-2888.

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https://www.chinaagrisci.com/CN/Y2024/V57/I14/2874

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循环步骤 Circulation procedure	温度 Temperature	时间 Time	循环数 Number of cycles
预变性 Pre-denaturation	95 ℃	5 min	1
变性 Denaturation	95 ℃	30 sec	35
退火 Annealing	64 ℃-58 ℃	30 sec
延伸 Extension	72 ℃	1000 bp/ min
彻底延伸 Final extension	72 ℃	5 min	1
保温 Hold	4 ℃	∞	1

基因名 Gene name	登录号 GenBank ID	引物序列（5′-3′） Primer sequences（5′-3′）	产物长度 Product length（bp）
lncRNA RRAS2-AS1	/	F: CGAAGCGCGGAGAAGCTA	80
lncRNA RRAS2-AS1	/	R: TGCGAAGTCGTGTGGAGGA	80
IL-6	NM_173923.2	F: CACTCCATTCGCTGTCT	227
IL-6	NM_173923.2	R: GTGTCTCCTTGCTGCTT	227
IL-8	NM_173925.2	F: ACACATTCCACACCTTTCCAC	149
IL-8	NM_173925.2	R: ACCTTCTGCACCCACTTTTC	149
IL-1β	NM_174093.1	F: ATGAAGAGCTGCATCCAACACCTG	110
IL-1β	NM_174093.1	R: ACCGACACCACCTGCCTGAAG	110
NF-κB1	NM_001076409.1	F: ACGGGGAAGGTCTGAATG	220
NF-κB1	NM_001076409.1	R: GTAGTCCCGTCATAAGTGG	220
TLR4	NM_174198.6	F: GCCGTAAGGTTATTGTCGTG	125
TLR4	NM_174198.6	R: CAGGACGATGAAGATGATGC	125
CDK2	NM_001014934.1	F: GATGGACGGAGCTTGTTATCG	182
CDK2	NM_001014934.1	R: ATCACACTCACACTGGAGAAGA	182
CDK4	NM_001037594.2	F: CCTTCATGCCAACTGCATCG	148
CDK4	NM_001037594.2	R: CCAGAGTGTAACAACCACAGGT	148
PCNA	NM_001034494.1	F: GAACCTCACCAGCATGTCCA	86
PCNA	NM_001034494.1	R: ACGTGTCCGCGTTATCTTCA	86
BAD	NM_001035459.2	F: TCAGCAAGCACTGGCTAACA	268
BAD	NM_001035459.2	R: TGAAACTCGTCGCTCATCCT	268
CASP3	NM_001077840.1	F: AAGATTTAGTGCCGATGC	175
CASP3	NM_001077840.1	R: GACCACCAAGTTCTAGGATA	175
CASP9	NM_001205504.2	F: CCTGCCTTACCATTCACC	205
CASP9	NM_001205504.2	R: GCATTCTGCTCCTCCTCC	205
BAX	NM_173894.1	F: GCAAACTGGTGCTCAAGG	189
BAX	NM_173894.1	R: GCACTCCAGCCACAAAGA	189
BCL2	NM_001166486.1	F: GATGACCGAGTACCTGAACCG	120
BCL2	NM_001166486.1	R: GACAGCCAGGAGAAATCAAACA	120
GAPDH	NM_001034034.2	F: GGCATCGTGGAGGGACTTATG	186
GAPDH	NM_001034034.2	R: GCCAGTGAGCTTCCCGTTGAG	186
RPS18	NM_001033614.2	F: GTGGTGTTGAGGAAAGCAGACA	79
RPS18	NM_001033614.2	R: TGATCACACGTTCCACCTCATC	79