干扰lncRNA RNF5-AS1对奶牛乳腺上皮细胞炎症反应的影响

doi:10.13560/j.cnki.biotech.bull.1985.2024-1091

摘要/Abstract

摘要：

目的长链非编码RNA（long non-coding RNA, lncRNA）能够通过直接调控下游分子或竞争性结合微小RNA（microRNA, miRNA）参与奶牛乳房炎症过程。lncRNA RNF5-AS1是新发现的lncRNA，旨在分析lncRNA RNF5-AS1在奶牛乳腺上皮细胞（bovine mammary epithelial cells, bMECs）炎症反应中的作用。方法利用RT-PCR技术进行lncRNA RNF5-AS1的克隆，采用核质分离法探究其在bMECs中的亚细胞定位情况。采用脂多糖（lipopolysaccharides, LPS）诱导bMECs建立炎症模型，并利用RT-qPCR检测lncRNA RNF5-AS1在炎性bMECs中的表达水平；干扰lncRNA RNF5-AS1后，检测促炎细胞因子表达水平，并利用CCK-8法和EdU法分别评估细胞活力和增殖能力；通过流式细胞术分析细胞凋亡情况。进一步通过在线工具预测lncRNA RNF5-AS1的潜在靶基因。结果 lncRNA RNF5-AS1长度为1 125 bp，主要分布于细胞质中。在LPS诱导的bMECs炎症模型中，lncRNA RNF5-AS1表达水平显著上调；干扰lncRNA RNF5-AS1后，促炎因子IL-6和IL-8的表达水平显著下调，细胞活力和增殖能力显著增加，细胞凋亡水平极显著降低。KEGG结果表明，lncRNA RNF5-AS1可能通过靶向bta-miR-375、bta-miR-615、bta-miR-193a-5p、bta-miR-1291和bta-miR-1468而调控细胞炎症反应。结论 lncRNA RNF5-AS1在bMECs炎症反应中表达上调，干扰后会抑制促炎因子的表达和细胞凋亡，提升细胞活力和增殖能力，提示其参与奶牛乳房炎的调控。

关键词: 奶牛乳房炎, lncRNA RNF5-AS1, 乳腺上皮细胞, 炎症因子

Abstract:

Objective Long non-coding RNA (lncRNA) can participate in the inflammatory process of dairy cow mammary gland by directly regulating downstream molecules or competitively binding microRNA (miRNA). lncRNA RNF5-AS1 is a newly identified lncRNA and its role in dairy cow mastitis is unknown. The aim of this study is to analyze the role of lncRNA RNF5-AS1 in the inflammatory response of bovine mammary epithelial cells (bMECs). Method Cloning of lncRNA RNF5-AS1 was performed using RT-PCR, and its subcellular localization in bMECs was probed using nucleoplasmic separation. Lipopolysaccharides (LPS) were used to induce bMECs to establish an inflammation model, and the expression of lncRNA RNF5-AS1 in inflammatory bMECs was detected by RT-qPCR. After interfering with lncRNA RNF5-AS1, the expression of pro-inflammatory cytokines was detected, and CCK-8 and EdU methods were used to evaluate cell viability and proliferation, respectively. Apoptosis was analyzed by flow cytometry. Furthermore, the potential target genes of lncRNA RNF5-AS1 were predicted by online tools. Result The lncRNA RNF5-AS1 is 1 125 bp in length and is mainly distributed in the cytoplasm. In the LPS-induced inflammation model of bMECs, the expression of lncRNA RNF5-AS1 was significantly up-regulated. After interfering with lncRNA RNF5-AS1, the expression of pro-inflammatory factors IL-6 and IL-8 was down-regulated, cell viability and proliferation significantly increased, and the level of apoptosis significantly decreased. KEGG results suggest that lncRNA RNF5-AS1 may modulate cellular inflammatory responses by targeting bta-miR-375, bta-miR-615, bta-miR-193a-5p, bta-miR-1291, and bta-miR-1468. Conclusion The expression of lncRNA RNF5-AS1 is up-regulated in the inflammatory response of bMECs. Interference with lncRNA RNF5-AS1 inhibits the expression of pro-inflammatory factors and apoptosis, and elevates the cell viability and proliferative capacity, suggesting that it is involved in the regulation of mastitis in dairy cows.

Key words: mastitis in dairy cows, lncRNA RNF5-AS1, mammary epithelial cells, inflammatory cytokines

虎喜敏, 周冉, 王正兴, 李宇航, 罗仍卓么, 王兴平. 干扰lncRNA RNF5-AS1对奶牛乳腺上皮细胞炎症反应的影响[J]. 生物技术通报, 2025, 41(5): 333-342.

HU Xi-min, ZHOU Ran, WANG Zheng-xing, LI Yu-hang, LUORENG Zhuo-ma, WANG Xing-ping. Effect of Interference with lncRNA RNF5-AS1 on the Inflammatory Response of Bovine Mammary Epithelial Cells[J]. Biotechnology Bulletin, 2025, 41(5): 333-342.

图/表 7

表1 RT-qPCR 引物序列

Table 1 Primer sequences used for RT-qPCR

基因 Gene	基因ID Gene ID	引物序列 Primer sequence（5′-3′）	产物长度 Product length （bp）
lncRNARNF5-AS1	PQ567897	F： GAATAAAGCCCGCCCTAG R： GAGTGCCTGCCAATCATACA	187
IL-6	280826	F： CACTCCATTCGCTGTCT R： GTGTCTCCTTGCTGCTT	227
IL-8	280828	F： ACACATTCCACACCTTTCCAC R： ACCTTCTGCACCCACTTTTC	149
IL-1β	281251	F： CAACCGTACCTGAACCC R： GACACCACCTGCCTGAA	110
GAPDH	281181	F： GGCATCGTGGAGGGACTTATG R： CCAGTGAGCTTCCCGTTGAG	186
RPS18	326602	F： GTGGTGTTGAGGAAAGCAGACA R： TGATCACACGTTCCACCTCATC	79
U6	613630	F： GCTTCGGCAGCACATATACTAAAAT R： CGCTTCACGAATTTGCGTGTCAT	89

图1 lncRNA RNF5-AS1的序列特征与亚细胞定位A：lncRNA RNF5-AS1 PCR 产物的琼脂糖凝胶电泳；M：DL2000 DNA marker；B：编码能力预测结果；C：亚细胞定位预测结果；D：亚细胞定位检测结果；M：DL500 DNA marker；1：细胞核；2：细胞质；3：全细胞

Fig. 1 Sequence features and subcellular localization of lncRNA RNF5-AS1A: Agarose gel electrophoresis of PCR product of lncRNA RNF5-AS1; M: DL2000 DNA marker. B: Predicted result of coding ability. C: Predicted result of subcellular localization. D: Predicted results of subcellular localization assay. M: DL500 DNA marker; 1: nucleus; 2: cytoplasm; 3: whole cell

图2 lncRNA RNF5-AS1在bMECs炎症反应中的表达水平A-C：主要炎症因子在bMECs炎症反应中的表达量；D：lncRNA RNF5-AS1在bMECs炎症反应中的表达量；* P <0.05，** P <0.01，ns P >0.05，下同

Fig. 2 Expressions of lncRNA RNF5-AS1 in the inflammatory response of bMECsA-C: The expression of inflammatory factors in bMECs inflammation. D: The expression of lncRNA RNF5-AS1 in bMECs inflammation; * P <0.05, ** P <0.01, ns P >0.05, the same below

图3 干扰lncRNA RNF5-AS1对bMECs炎症反应的影响A：lncRNA RNF5-AS1干扰效率；B-C：炎症因子mRNA 水平

Fig. 3 Effects of interference with lncRNA RNF5-AS1 on the inflammatory response of bMECsA: Interference efficiency of lncRNA RNF5-AS1. B-C: mRNA levels of inflammatory factors

图4 CCK8检测干扰lncRNA RNF5-AS1 对炎性bMECs活力的影响

Fig. 4 Effects of interference with lncRNA RNF5-AS1 on inflammatory bMECs viability by CCK-8

图5 干扰lncRNA RNF5-AS1 对炎性bMECs的影响A：EdU检测细胞增殖（红色荧光为EdU标记的增殖细胞；蓝色荧光为Hoechst标记的全部细胞）；B：流式细胞术检测细胞凋亡

Fig. 5 Effect of interference with lncRNA RNF5-AS1 on inflammatory bMECsA: EdU assay of cell proliferation(Red fluorescence is EdU-labeled proliferating cells; blue fluorescence is Hoechst-labeled all cells). B: Flow cytometry assay of apoptosis

图6 lncRNA RNF5-AS1的靶基因预测A：靶基因预测结果；B：bta-miR-375的靶基因富集的信号通路；C：bta-miR-615的靶基因富集的信号通路；D：bta-miR-193a-5p的靶基因富集的信号通路；E：bta-miR-1291的靶基因富集的信号通路；F：bta-miR-1468的靶基因富集的信号通路

Fig. 6 Prediction of target genes of lncRNA RNF5-AS1A: Prediction results of target genes; B: signaling pathway enriched for the target genes of bta-miR-375; C: signaling pathway enriched for the target genes of bta-miR-615; D: signaling pathway enriched for the target genes of bta-miR-193a-5p; E: signaling pathway enriched for the target genes of bta-miR-1291; F: signaling pathway enriched for the target genes of bta-miR-1468

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