lncRNA PFN1-AS1在奶牛乳腺上皮细胞炎症反应中的作用

doi:10.11843/j.issn.0366-6964.2025.11.043

摘要/Abstract

摘要：

长链非编码RNA(long non-coding RNA，lncRNA)在奶牛乳腺炎中存在差异表达，但其调控奶牛乳腺炎的分子机制尚不清楚。本研究旨在探究新发现的差异表达lncRNA TCONS_00062289在奶牛乳腺上皮细胞(bovine mammary epithelial cells，bMECs)炎症反应中的作用。因该lncRNA的序列与肌动蛋白单体结合蛋白反义链(profilin1 antisense，PFN1-AS1)相同，将其命名为lncRNA PFN1-AS1。采用RT-PCR技术克隆lncRNA PFN1-AS1，预测lncRNA PFN1-AS1的功能。采用脂多糖(lipopolysaccharides，LPS)诱导bMECs建立炎症细胞模型，探究在炎症模型中干扰lncRNA PFN1-AS1对细胞炎症、增殖和凋亡的影响。结果显示：lncRNA PFN1-AS1长度为811 bp，可能参与调控细胞凋亡和炎症过程。lncRNA PFN1-AS1主要分布于细胞质中。在LPS诱导的bMECs炎症模型中，lncRNA PFN1-AS1表达水平显著下降(P < 0.05)；干扰lncRNA PFN1-AS1后，促炎细胞因子白介素(interleukin，IL)6、IL-8和IL-1β的转录和分泌水平均显著增加(P < 0.05)。增殖基因细胞周期蛋白依赖激酶(cyclin dependent kinase，CDK)2、CDK4和增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)表达水平显著下降(P < 0.05)，细胞活力值和增殖率显著下降(P < 0.05)；凋亡基因半胱氨酸天冬氨酸蛋白酶(cysteinyl aspartate specific proteinase，CASP)3、CASP8和Bcl-2相关X蛋白(bcl-2-associated X protein，BAX)的表达水平显著升高(P < 0.05)，细胞凋亡率极显著增加(P < 0.01)。干扰lncRNA PFN1-AS1后促进了IL-6、IL-8和IL-1β的转录和蛋白分泌，促进了细胞凋亡水平，同时降低了细胞活力和增殖能力，这些结果揭示了lncRNA PFN1-AS1在bMECs炎症中的作用，为进一步探究其在奶牛乳腺炎中的作用提供了科学依据。

关键词: 奶牛乳腺上皮细胞, lncRNA PFN1-AS1, 脂多糖, 炎症反应

Abstract:

Studies have shown that long non-coding RNA (lncRNA) is differentially expressed in dairy cow mastitis, but the molecular mechanisms by which it regulates dairy cow mastitis are unknown. The aim of this study was to investigate the role of lncRNA TCONS_00062289 in the inflammatory response of bovine mammary epithelial cells (bMECs). The lncRNA was named lncRNA PFN1-AS1 because of its sequence identity with the actin monomer-binding protein antisense (PFN1-AS1). LncRNA PFN1-AS1 was cloned by RT-PCR and its function was predicted. Inflammatory cell modeling with lipopolysaccharides (LPS) induced bMECs, in order to explore the effect of interfering lncRNA PFN1-AS1 on cell inflammation, proliferation and apoptosis in the inflammatory model. The lncRNA PFN1-AS1 is 811 bp in length, may be involved in the regulation of apoptosis and inflammatory processes. LncRNA PFN1-AS1 mainly distributed in the cytoplasm. In the LPS-induced inflammation model of bMECs, the expression level of lncRNA PFN1-AS1 was significantly down-regulated (P < 0.05). After interfering with lncRNA PFN1-AS1, the mRNA expression and protein secretion level of pro-inflammatory cytokines interleukin (IL) -6, IL-8 and IL-1β were significantly up-regulated (P < 0.05). The expression level of cyclin dependent kinase2 (CDK2), CDK4 and proliferating cell nuclear antigen (PCNA) were significantly down-regulated (P < 0.05), and the cell viability value and proliferation rate were significantly decreased (P < 0.05); the expression level of cysteinyl aspartate specific proteinase (CASP3), CASP8 and bcl-2-associated X protein (BAX) were significantly up-regulated (P < 0.05), and the rate of apoptosis was highly significantly increased (P < 0.01). Interference with lncRNA PFN1-AS1 promoted the transcription and protein secretion of IL-6, IL-8, and IL-1β, promoted the level of apoptosis, and decreased the cell viability and proliferation. These results reveal the role of lncRNA PFN1-AS1 in the inflammation of bMECs, and provide a scientific basis for further investigation of its role in mastitis of dairy cows.

Key words: bovine mammary epithelial cells, lncRNA PFN1-AS1, lipopolysaccharides, inflammatory

中图分类号:

S857.26

虎喜敏, 罗仍卓么, 周冉, 李宇航, 王兴平. lncRNA PFN1-AS1在奶牛乳腺上皮细胞炎症反应中的作用[J]. 畜牧兽医学报, 2025, 56(11): 5875-5887.

HU Ximin, LUORENG Zhuoma, ZHOU Ran, LI Yuhang, WANG Xingping. Role of lncRNA PFN1-AS1 in the Inflammatory Response of Bovine Mammary Epithelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5875-5887.

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基因名 Gene name	基因登录号 GenBank ID	引物序列(5′→3′) Primer sequences	产物长度/bp Product length
lncRNAPFN1-AS1	PQ628053	F: GAATAAAGCCCGCCCTAG R: GAGTGCCTGCCAATCATACA	187
IL-6	NM_173923	F: CACTCCATTCGCTGTCT R: GTGTCTCCTTGCTGCTT	227
IL-8	NM_173925.2	F: ACACATTCCACACCTTTCCAC R: ACCTTCTGCACCCACTTTTC	149
IL-1β	NM_174093.1	F: CAACCGTACCTGAACCC R: GACACCACCTGCCTGAA	110
CDK2	NM_001014934	F: GATGGACGGAGCTTGTTATCG R: ATCACACTCACACTGGAGAAGA	182
CDK4	NM_001037594.2	F: CCTTCATGCCAACTGCATCG R: CCAGAGTGTAACAACCACAGGT	168
PCNA	NM_001034494.1	F: GAACCTCACCAGCATGTCCA R: ACGTGTCCGCGTTATCTTCA	86
CASP3	NM_001077840.1	F: AAGATTTAGTGCCGATGC R: GACCACCAAGTTCTAGGATA	175
CASP8	NM_001045970.2	F: TCACCCACGGAAACAAGG R: TCGGTCTCAACGGCTACAC	178
BAX	NM_173894.1	F: GCAAACTGGTGCTCAAGG R: GCACTCCAGCCACAAAGA	189
GAPDH	NM_001034034.2	F: GGCATCGTGGAGGGACTTATG R: CCAGTGAGCTTCCCGTTGAG	186
RPS18	NM_001033614.2	F: GTGGTGTTGAGGAAAGCAGACA R: TGATCACACGTTCCACCTCATC	79
U6	NM_001075477.2	F: GCTTCGGCAGCACATATACTAAAAT R: CGCTTCACGAATTTGCGTGTCAT	89

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