MiSeq analysis of gene expression profiles in human retinal capillary endothelial cells induced by fulvic acid_Chinese Journal of Ocular Fundus Diseases

Authors：

Xu Manhong , Dong Lijie , Wang Linni , Huang Xinyuan ,  Li Xiaorong

Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin International Joint Research and Development Centre of Ophthalmology and Vision Science, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China;

Corresponding?author：

Li Xiaorong, Email: xiaorli@163.com

Keywords：

Anoxia; Retinal vessels/cytology; Endothelial cells/physiology; Cells, cultured; Intercellular adhesion molecule-1; Fulvic acid

DOI：

10.3760/cma.j.cn511434-20190403-00128

Video：

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Objective To observe the MiSeq sequencing analysis results of fulvic acid (FA) intervention in hypoxia-induced human retinal microvascular endothelial cell (hRMEC) gene expression profile.Methods hRMEC were cultured in vitro and divided into the hypoxia group (hypoxia treatment) and the FA intervention group (FA intervention after hypoxia). The MTT colorimetric method was used to detect the influence of different concentrations and different modes of FA on hRMEC activity. The optimal concentration of FA was chosen. RT-PCR was used to investigated the effect of FA on hypoxia-induced intercellular adhesion molecule-1 (ICAM-1), IL-1β, IL-4, IL-6, IL-6, IL-8, IL-10, MMP-2, TNF-α, TNF-β, other inflammatory factors in hRMEC, and inflammation-related factors mRNA expression. Cells in the hypoxia group and FA intervention group in the logarithmic growth phase were collected. MiSeq sequencing technology was applyed to complete the whole transcriptome sequencing of the two groups of cells, biological data were obtained, and the differentially expressed miRNA were analyzed on this basis. Gene annotation (GO) functionally significant enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway significant enrichment analysis were used to analyze the functions and signal pathways of differential miRNAs. The expression of inflammatory factors and inflammation-related factors were compared between groups. The expression level of the corresponding miRNA in the cell was regulated by miRNA mimic, and its effect on cell function was observed, so as to judge the effect of the miRNA.Results Different concentrations and different modes of action of FA had no effect on the cell viability of hRMEC. The mRNA expression of ICAM-1, IL-1β, IL-6 and TNF-β in the hypoxia group hRMEC were significantly up-regulated compared with the normal group, and the difference was statistically significant (t=3.426, 6.011, 5.282, 6.500; P=0.027, 0.004, 0.006, 0.003); the mRNA expression of ICAM-1, IL-6, TNF-α and TNF-β in the FA intervention group hRMEC was significantly lower than that of the hypoxia group, and the difference was statistically significant (t=9.961, 3.676, 3.613, 3.387; P=0.001, 0.021, 0.023, 0.028). There were 14 differentially expressed miRNAs between the hypoxia group and the FA intervention group, of which 9 were up-regulated genes and 5 were down-regulated genes. The predicted target genes of 4 differential miRNAs (hsa-miR-1285-3p, hsa-miR-30d-3p, hsa-miR-3170, hsa-miR-7976) were all ICAM-1. The results of significant enrichment analysis of GO function showed that the functions of differential genes were mainly enriched in the process of cell development, cell differentiation and single organism development. Significant enrichment analysis of the KEGG pathway showed that the differential miRNA expression was highly enriched in the proteoglycan pathway and the cytokine-cytokine receptor interaction pathway in cancer, and the arachidonic acid metabolism pathway and the amphetamine pathway were the more obvious differential expressions.Conclusion FA may affect the expression level of downstream ICAM-1 mRNA by regulating the expression of multiple miRNAs, thereby affecting the inflammatory state of cells after hypoxia-stimulated hRMEC.

Citation： Xu Manhong, Dong Lijie, Wang Linni, Huang Xinyuan, Li Xiaorong. MiSeq analysis of gene expression profiles in human retinal capillary endothelial cells induced by fulvic acid. Chinese Journal of Ocular Fundus Diseases, 2020, 36(10): 795-803. doi: 10.3760/cma.j.cn511434-20190403-00128 Copy

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1. 謝若天, 顏華. 表觀遺傳學在糖尿病視網膜病變中的研究進展[J]. 中華眼底病雜志, 2020, 36(8): 657-660. DOI: 10.3760/cma.j.cn511434-20190213-00045.Xie RT, Yan H. Epigenetic research progression in diabetic retinopathy[J]. Chin J Ocul Fundus Dis, 2020, 36(8): 657-660. DOI: 10.3760/cma.j.cn511434-20190213-00045.
2. 劉玉華, 高玲. 糖尿病視網膜病變治療研究現狀、問題與展望[J]. 中華眼底病雜志, 2016, 32(2): 206-210. DOI: 10.3760/cma.j.issn.1005-1015.2016.02.024.Liu YH, Gao L. The status, problem and progress of diabetic retinopathy treatment[J]. Chin J Ocul Fundus Dis, 2016, 32(2): 206-210. DOI: 10.3760/cma.j.issn.1005-1015.2016.02.024.
3. 李筱榮. 糖尿病視網膜病變基礎研究的熱點和難點[J]. 中華眼底病雜志, 2007, 23(4): 234-237.Li XR. Hotspots and problems of basic research for diabetic retinopathyt[J]. Chin J Ocul Fundus Dis, 2007, 23(4): 234-237.
4. MacCarthy P, Peterson MJ, Malcolm RL, et al. Separation of humic substances by pH gradient desorption from a hydrophobic resin[J]. Analytical Chemistry, 1979, 51(12): 2041-2043. DOI: 10.1021/ac50048a036.
5. Naudé PJ, Cromarty AD, van Rensburg CE. Potassium humate inhibits carrageenan induced paw oedema and a graft-vs-host reaction in rats[J]. Inflammopharmacology, 2010, 18(1): 33-39. DOI: 10.1007/s10787-009-0026-8.
6. Motojima H, Villareal MO, Han J, et al. Microarray analysis of immediate-type allergy in KU812 cells in response to fulvic acid[J]. Cytotechnology, 2011, 63(2): 181-190. DOI: 10.1007/s10616-010-9333-6.
7. Chien SJ, Chen TC, Kuo HC, et al. Fulvic acid attenuates homocysteine-induced cyclooxygenase-2 expression in human monocytes[J/OL]. BMC Complement Altern Med, 2015, 15: 61[2015-03-13]. https://pubmed.ncbi.nlm.nih.gov/25888188/. DOI: 10.1186/s12906-015-0583-x.
8. Sherry L, Millhouse E, Lappin DF, et al. Investigating the biological properties of carbohydrate derived fulvic acid (CHD-FA) as a potential novel therapy for the management of oral biofilm infections[J/OL]. BMC Oral Health, 2013, 13: 47[2013-09-24]. https://pubmed.ncbi.nlm.nih.gov/24063298/. DOI: 10.1186/1472-6831-13-47.
9. Wershaw RL, Pinckney DJ, Booker SE. Chemical structure of humic acids-part 1, a generalized structural model[J]. Journal of Research of the US Geological Survey, 1977, 5(5): 565-569.
10. Lehmann J, Kleber M. The contentious nature of soil organic matter[J]. Nature, 2015, 528(7580): 60-68. DOI: 10.1038/nature16069.
11. Yin J, Xia W, Li Y, et al. COX-2 mediates PM2.5-induced apoptosis and inflammation in vascular endothelial cells[J]. Am J Transl Res, 2017, 9(9): 3967-3976.
12. 張哲, 劉巨平, 東莉潔, 等. 高糖狀態下視網膜血管內皮細胞基因表達譜的RNA-Seq分析[J]. 中華眼底病雜志, 2018, 34(4): 377-381. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.014.Zhang Z, Liu JP, Dong LJ, et al. RNA-Seq analysis of gene expression profiling in retinal vascular endothelial cells under high glucose condition[J]. Chin J Ocul Fundus Dis, 2018, 34(4): 377-381. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.014.
13. Joy SS, Siddiqui K. Molecular and pathophysiological mechanisms of diabetic retinopathy in relation to adhesion molecules[J]. Curr Diabetes Rev, 2019, 15(5): 363-371. DOI: 10.2174/1573399814666181017103844.
14. Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay[J]. Nat Rev Genet, 2010, 11(9): 597-610. DOI: 10.1038/nrg2843.
15. Fabian MR, Sonenberg N, Filipowicz W. Regulation of mRNA translation and stability by microRNAs[J]. Annu Rev Biochem, 2010, 79: 351-379. DOI: 10.1146/annurev-biochem-060308-103103.
16. Gour N, Wills-Karp M. IL-4 and IL-13 signaling in allergic airway disease[J]. Cytokine, 2015, 75(1): 68-78. DOI: 10.1016/j.cyto.2015.05.014.
17. Kuperman DA, Schleimer RP. Interleukin-4, interleukin-13, signal transducer and activator of transcription factor 6, and allergic asthma[J]. Curr Mol Med, 2008, 8(5): 384-392. DOI: 10.2174/156652408785161032.
18. Cerwenka A, Lanier LL. Natural killer cell memory in infection, inflammation and cancer[J]. Nat Rev Immunol, 2016, 16(2): 112-123. DOI: 10.1038/nri.2015.9.
19. Bigger JE, Thomas CA 3rd, Atherton SS. NK cell modulation of murine cytomegalovirus retinitis[J]. J Immunol, 1998, 160(12): 5826-5831.
20. Granger DN, Kubes P. The microcirculation and inflammation: modulation of leukocyte-endothelial cell adhesion[J]. J Leukoc Biol, 1994, 55(5): 662-675. DOI: 10.1002/jlb.55.5.662.
21. Huang WS, Yang JT, Lu CC, et al. Fulvic acid attenuates resistin-induced adhesion of HCT-116 colorectal cancer cells to endothelial cells[J]. Int J Mol Sci, 2015, 16(12): 29370-29382. DOI: 10.3390/ijms161226174.
22. Behl T, Kaur I, Kotwani A. Implication of oxidative stress in progression of diabetic retinopathy[J]. Surv Ophthalmol, 2016, 61(2): 187-196. DOI: 10.1016/j.survophthal.2015.06.001.
23. 王樹林, 金學民. 糖尿病鼠視網膜細胞間黏附分子-1的表達與血視網膜屏障破壞的關系及曲安奈德的治療作用[J]. 中華眼底病雜志, 2006, 22(1): 24-27. DOI: 10.3760/j.issn:1005-1015.2006.01.007.Wang SL, Jin XM. Relationship between expression of retinal intercellular adhesion molecule-1 and blood-retinal barrier rupture and therapeutic effect of triamcinolone acetonide in diabetic rats[J]. Chin J Ocul Fundus Dis, 2006, 22(1): 24-27. DOI: 10.3760/j.issn:1005-1015.2006.01.007.

Chinese Journal of Ocular Fundus Diseases

MiSeq analysis of gene expression profiles in human retinal capillary endothelial cells induced by fulvic acid

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