Transcriptome sequencing of transgelin-2 inhibiting high glucose induced microglia inflammation_Chinese Journal of Ocular Fundus Diseases

Authors：

Shi Pingling , Wei Yuanmeng , Huang Zixu , Lu Cong , Yang Qixiang , Li Pan , Wu Chengye ,  Song Zongming

Henan Provincial People's Hospital, Henan Eye Hospital (Henan Eye Institution), Zhengzhou University People's Hospital, Henan University People's Hospital, Zhengzhou 450003, China;

Corresponding author：

Song Zongming, Email: szmeyes@126.com

Keywords：

Transgelin-2; Retinal microglia; Transcriptome sequencing

DOI：

10.3760/cma.j.cn511434-20210716-00381

Video：

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Objective To analyze the change of differential genes and signaling pathways in high glucose induced BV2 cells, and to explore the mechanism of transgelin-2 (TAGLN2) regulating cellular inflammatory response and metabolic process. Methods An experimental study. The cultured BV2 cells were divided into mannitol treatment (Man) group, glucose treatment (Glu) group, overexpression control Glu treatment (Con) group, overexpression TAGLN2 Glu treatment group, silence control Glu treatment (shCon Glu) group, and silence TAGLN2 Glu treatment (shTAGLN2 Glu) group. Cells in the Man group were cultured in modified Eagle high glucose medium (DMEM) containing 25 mmol/L mannitol and 25 mmol/L glucose, cells in other groups (Glu group, Con Glu group, TAGLN2 Glu group, shCon Glu group and shTAGLN2 Glu group) were cultured in DMEM medium containing 50 mmol/L glucose. After 24 hours of cells culture, transcriptome sequencing of cells in each group were performed using high-throughput sequencing technology, and significantly differentially expressed genes (DEG) were screened. |log₂ (fold change)|≥1 and P≤0.05 were adopted as criteria to screen for DEG. Gene Ontology (GO) function enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network analysis were performed. Real-time polymerase chain reaction (RT-PCR) was used to detect the relative expression level of DEG mRNA. The data between groups were compared by independent sample t-test. Results When compared with Man group, a total of 517 differentially expressed genes were screened in Glu group, which including 277 up-regulated genes and 240 down-regulated genes. KEGG pathway enrichment analysis showed that the up-regulated genes were significantly enriched in immune system processes such as nuclear factor (NF)-κB signal pathway, Jak-signal transducers and activators of transcription (STAT) signal pathway, while down-regulated genes were significantly enriched in glycosaminoglycan degradation and glyceride metabolic pathway. Compared with Con Glu group, a total of 480 DEG were screened in TAGLN2 Glu group, among which 147 up-regulated and 333 down-regulated genes were detected. Up-regulated genes were significantly enriched in the metabolic processes of fatty acid, glyceride and pyruvate, while down-regulated genes were significantly enriched in immune system processes such as NF-κB signal pathway, Jak-STAT signal pathway and tumor necrosis factor (TNF) signal pathway. Compared with shCon Glu group, a total of 582 DEG were screened in shTAGLN2 Glu group, among which 423 up-regulated and 159 down-regulated genes were detected. Up-regulated DEG were significantly enriched in immune system processes such as TNF signal pathway and chemokine signal pathway, while down-regulated DEG were significantly enriched in pattern recognition receptor signal pathway. RT-PCR results showed that the relative expression levels of DEG mRNA Card11 (t=13.530), Icos (t=3.482), Chst3 (t=6.949), Kynu (t=5.399), interleukin (IL)-1β (t=2.960), TNF-α (t=5.800), IL-6(t=3.130), interferon-γ (t=7.690) and IL-17 (t=6.530) in the TAGLN2 Glu treatment group were decreased significantly compared with Con Glu group, and the difference was statistically significant. Conclusion TAGLN2 can inhibit glucose induced microglia inflammation by NF-κB and Jak-STAT signaling pathways, Card11, Icos, Chst3 and Kynu play an important role in the anti-inflammatory process of TAGLN2.

Citation： Shi Pingling, Wei Yuanmeng, Huang Zixu, Lu Cong, Yang Qixiang, Li Pan, Wu Chengye, Song Zongming. Transcriptome sequencing of transgelin-2 inhibiting high glucose induced microglia inflammation. Chinese Journal of Ocular Fundus Diseases, 2023, 39(2): 153-162. doi: 10.3760/cma.j.cn511434-20210716-00381 Copy

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10.	Kim HR, Park JS, Park JH, et al. Cell-permeable transgelin-2 as a potent therapeutic for dendritic cell-based cancer immunotherapy[J]. J Hematol Oncol, 2021, 14(1): 43. DOI: 10.1186/s13045-021-01058-6.
11.	杨琪翔, 史平玲, 卢聪, 等. 光感受器661细胞系早期低氧损伤转录组测序的生物信息学分析[J]. 中华眼底病杂志, 2021, 37(3): 214-223. DOI: 10.3760/cma.j.cn511434-20201009-00483.Yang QX, Shi PL, Lu C, et al. Bioinformatics analysis of transcriptome sequencing of early hypoxia damage in photoreceptor 661W cell line[J]. Chin J Ocul Fundus Dis, 2021, 37(3): 214-223. DOI: 10.3760/cma.j.cn511434-20201009-00483.
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19.	Maeda S, Fujimoto M, Matsushita T, et al. Inducible costimulator (ICOS) and ICOS ligand signaling has pivotal roles in skin wound healing via cytokine production[J]. Am J Pathol, 2011, 179(5): 2360-2369. DOI: 10.1016/j.ajpath.2011.07.048.
20.	Pires S, Jacquet R, Parker D. Inducible costimulator contributes to methicillin-resistant staphylococcus aureus pneumonia[J]. J Infect Dis, 2018, 218(4): 659-668. DOI: 10.1093/infdis/jix664.
21.	Kai Y, Tomoda K, Yoneyama H, et al. RNA interference targeting carbohydrate sulfotransferase 3 diminishes macrophage accumulation, inhibits MMP-9 expression and promotes lung recovery in murine pulmonary emphysema[J]. Respir Res, 2015, 16(1): 146. DOI: 10.1186/s12931-015-0310-7.
22.	Harden JL, Lewis SM, Lish SR, et al. The tryptophan metabolism enzyme L-kynureninase is a novel inflammatory factor in psoriasis and other inflammatory diseases[J]. J Allergy Clin Immunol, 2016, 137(6): 1830-1840. DOI: 10.1016/j.jaci.2015.09.055.
23.	O'farrell K, Fagan E, Connor TJ, et al. Inhibition of the kynurenine pathway protects against reactive microglial-associated reductions in the complexity of primary cortical neurons[J]. Eur J Pharmacol, 2017, 810(1): 163-173. DOI: 10.1016/j.ejphar.2017.07.008.

1. Chen M, Xu H. Parainflammation, chronic inflammation, and age-related macular degeneration[J]. J Leukoc Biol, 2015, 98(5): 713-725. DOI: 10.1189/jlb.3RI0615-239R.
2. Wu H, Wang M, Li X, et al. The metaflammatory and immunometabolic role of macrophages and microglia in diabetic retinopathy[J]. Human Cell, 2021, 34(6): 1617-1628. DOI: 10.1007/s13577-021-00580-6.
3. Jo Dh, Yun Jh, Cho Cs, et al. Interaction between microglia and retinal pigment epithelial cells determines the integrity of outer blood-retinal barrier in diabetic retinopathy[J]. Glia, 2019, 67(2): 321-331. DOI: 10.1002/glia.23542.
4. Zhang T, Ouyang H, Mei X, et al. Erianin alleviates diabetic retinopathy by reducing retinal inflammation initiated by microglial cells inhibiting hyperglycemia-mediated ERK1/2-NF-κB signaling pathway[J]. FASEB J, 2019, 33(11): 11776-11790. DOI: 10.1096/fj.201802614RRR.
5. Jo S, Kim Hr, Mun Y, et al. Transgelin-2 in immunity: its implication in cell therapy[J]. J Leukoc Biol, 2018, 104(5): 903-910. DOI: 10.1002/jlb.mr1117-470r.
6. Na BR, Kim HR, Piragyte I, et al. TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse[J]. J Cell Biol, 2015, 209(1): 143-162. DOI: 10.1083/jcb.201407130.
7. Tsuji-Tamura K, Morino-Koga S, Suzuki S, et al. The canonical smooth muscle cell marker TAGLN is present in endothelial cells and is involved in angiogenesis[J/OL]. J Cell Sci, 2021, 134(15): jcs254920[2021-08-01]. https://pubmed.ncbi.nlm.nih.gov/34338296/. DOI: 10.1242/jcs.254920.
8. Na BR, Kwon MS, Chae MW, et al. Transgelin-2 in B-cells controls T-cell activation by stabilizing T cell - B cell conjugates[J/OL]. PLoS One, 2016, 11(5): e0156429[2016-05-27]. https://pubmed.ncbi.nlm.nih.gov/27232882/. DOI: 10.1371/journal.pone.0156429.
9. Kim HR, Lee HS, Lee KS, et al. An essential role for TAGLN2 in phagocytosis of lipopolysaccharide-activated macrophages[J/OL]. Sci Rep, 2017, 7(1): 8731[2017-08-18]. https://pubmed.ncbi.nlm.nih.gov/28821818/. DOI: 10.1038/s41598-017-09144-x.
10. Kim HR, Park JS, Park JH, et al. Cell-permeable transgelin-2 as a potent therapeutic for dendritic cell-based cancer immunotherapy[J]. J Hematol Oncol, 2021, 14(1): 43. DOI: 10.1186/s13045-021-01058-6.
11. 杨琪翔, 史平玲, 卢聪, 等. 光感受器661细胞系早期低氧损伤转录组测序的生物信息学分析[J]. 中华眼底病杂志, 2021, 37(3): 214-223. DOI: 10.3760/cma.j.cn511434-20201009-00483.Yang QX, Shi PL, Lu C, et al. Bioinformatics analysis of transcriptome sequencing of early hypoxia damage in photoreceptor 661W cell line[J]. Chin J Ocul Fundus Dis, 2021, 37(3): 214-223. DOI: 10.3760/cma.j.cn511434-20201009-00483.
12. Liu Y, Xiao J, Zhao Y, et al. microRNA-216a protects against human retinal microvascular endothelial cell injury in diabetic retinopathy by suppressing the NOS2/JAK/STAT axis[J/OL]. Exp Mol Pathol, 2020, 115(1): 104445[2020-04-23]. https://pubmed.ncbi.nlm.nih.gov/32335083/. DOI: 10.1016/j.yexmp.2020.104445.
13. Chen M, Lv H, Gan J, et al. Tang Wang Ming Mu Granule attenuates diabetic retinopathy in type 2 diabetes rats[J/OL]. Front Physiol, 2017, 8(1): 1065[2017-12-19]. https://pubmed.ncbi.nlm.nih.gov/29311988/. DOI: 10.3389/fphys.2017.01065.
14. Dai X, Thiagarajan D, Fang J, et al. SM22α suppresses cytokine-induced inflammation and the transcription of NF-κB inducing kinase (Nik) by modulating SRF transcriptional activity in vascular smooth muscle cells[J/OL]. PloS One, 2017, 12(12): e0190191[2017-12-28]. https://pubmed.ncbi.nlm.nih.gov/29284006/. DOI: 10.1371/journal.pone.0190191.
15. Reimann M, Schrezenmeier J, Richter-Pechanska P, et al. Adaptive T-cell immunity controls senescence-prone MyD88- or CARD11-mutant B-cell lymphomas[J]. Blood, 2021, 137(20): 2785-2799. DOI: 10.1182/blood.2020005244.
16. Wang H, Zhao J, Zhang H, et al. CARD11 blockade suppresses murine collagen-induced arthritis via inhibiting CARD11/Bcl10 assembly and T helper type 17 response[J]. Clin Exp Immunol, 2014, 176(2): 238-245. DOI: 10.1111/cei.12275.
17. Hawiger D, Tran E, Du W, et al. ICOS mediates the development of insulin-dependent diabetes mellitus in nonobese diabetic mice[J]. J Immunol, 2008, 180(5): 3140-3147. DOI: 10.4049/jimmunol.180.5.3140.
18. Kornete M, Sgouroudis E, Piccirillo CA. ICOS-dependent homeostasis and function of Foxp3+ regulatory T cells in islets of nonobese diabetic mice[J]. J Immunol, 2012, 188(3): 1064-1074. DOI: 10.4049/jimmunol.1101303.
19. Maeda S, Fujimoto M, Matsushita T, et al. Inducible costimulator (ICOS) and ICOS ligand signaling has pivotal roles in skin wound healing via cytokine production[J]. Am J Pathol, 2011, 179(5): 2360-2369. DOI: 10.1016/j.ajpath.2011.07.048.
20. Pires S, Jacquet R, Parker D. Inducible costimulator contributes to methicillin-resistant staphylococcus aureus pneumonia[J]. J Infect Dis, 2018, 218(4): 659-668. DOI: 10.1093/infdis/jix664.
21. Kai Y, Tomoda K, Yoneyama H, et al. RNA interference targeting carbohydrate sulfotransferase 3 diminishes macrophage accumulation, inhibits MMP-9 expression and promotes lung recovery in murine pulmonary emphysema[J]. Respir Res, 2015, 16(1): 146. DOI: 10.1186/s12931-015-0310-7.
22. Harden JL, Lewis SM, Lish SR, et al. The tryptophan metabolism enzyme L-kynureninase is a novel inflammatory factor in psoriasis and other inflammatory diseases[J]. J Allergy Clin Immunol, 2016, 137(6): 1830-1840. DOI: 10.1016/j.jaci.2015.09.055.
23. O'farrell K, Fagan E, Connor TJ, et al. Inhibition of the kynurenine pathway protects against reactive microglial-associated reductions in the complexity of primary cortical neurons[J]. Eur J Pharmacol, 2017, 810(1): 163-173. DOI: 10.1016/j.ejphar.2017.07.008.

Chinese Journal of Ocular Fundus Diseases

Transcriptome sequencing of transgelin-2 inhibiting high glucose induced microglia inflammation

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