Effect of Interference with lncRNA RNF5-AS1 on the Inflammatory Response of Bovine Mammary Epithelial Cells

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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

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

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

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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