微小RNA-107对脂多糖诱导人鼻黏膜上皮细胞增殖、凋亡及炎症因子的影响及机制研究
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摘要
目的检测微小RNA(microRNA,miR)-107对细菌脂多糖(lipopolysaccharides,LPS)诱导的人鼻黏膜上皮细胞(human nasal mucosal epithelial cells,HNMECs)增殖、凋亡及炎症因子的影响及可能的分子机制。方法以1 mg/L LPS诱导HNMECs建立炎症反应模型,采用实时荧光定量PCR检测诱导前后miR-107和高迁移率族蛋白B1(high mobility group box 1,HMGBl)的表达量变化。HNMECs中转染miR-107激动剂(agomir)和激动剂对照(agomir control),并采用噻唑蓝比色法、流式细胞术及酶联免疫吸附试验观察细胞增殖、凋亡及炎症因子的变化。利用双荧光素酶报告基因和蛋白质印迹试验验证HMGBl是否为miR-107的靶基因。结果 miR-107在LPS诱导后HNMECs中的表达量较诱导前显著降低(t=9.35,P<0.05),而HMGBl在LPS诱导后HNMECs中的表达量较诱导前显著升高(t=13.07,P<0.05)。与转染agomir control相比,LPS诱导后HNMECs中转染miR-107agomir可显著抑制细胞增殖(F=17.12,P <0.05),降低炎症因子肿瘤坏死因子α(TNF-α)(t=6.11,P <0.05)、白细胞介素1β(interleukin-1β,IL-1β)(t=6.90,P<0.05)及IL-6(t=8.18,P <0.05)的表达水平,并提高细胞凋亡率(t=7.49,P<0.05)。HMGBl是miR-107的靶基因,LPS诱导后HNMECs中转染miR-107 agomir可显著降低HMGBl蛋白的表达量(t=28.56,P<0.05)。结论 miR-107在LPS诱导后HNMECs中表达下调,而HMGBl表达上调。过表达miR-107可显著抑制LPS诱导后HNMECs增殖和炎症因子的表达,同时促进细胞凋亡。HMGBl是miR-107的靶基因,提示miR-107可能通过调控HMGBl发挥抑制作用。
OBJECTIVE To detect the effects of miR-107 on the proliferation, apoptosis and inflammatory factors of human nasal mucosal epithelial cells(HNMECs) induced by Lipopolysaccharides(LPS), and to elucidate the potential molecular mechanism. METHODS The expression changes of miR-107 and HMGBl(High mobility group box 1) in 1 mg/L LPS induced HNMECs before and after LPS induction were detected by using quantitative realtime PCR. LPS induced-HNMECs were transfected with miR-107 agomir and agomir control, and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide(MTT) assay, flow cytometry and enzyme-linked immunosorbent assay(ELISA) were performed to detect cell proliferation, apoptosis and inflammatory factors. Dual luciferase reporter assay and Western blots were applied to verify whether HMGB1 was a target gene of miR-107. RESULTS The expression of miR-107 in HNMECs after LPS induction was significantly lower than that before induction(t =9.35, P<0.05), while the expression of HMGB1 in HNMECs after LPS induction was significantly higher than that before induction(t =13.07, P<0.05). Compared with transfected agomir control, transfection of miR-107 agomir in HNMECs significantly inhibited cell proliferation(F =17.12, P <0.05) and decreased inflammatory factor TNF-α(t=6.11, P<0.05). IL-1β(t=6.90, P<0.05) and IL-6(t=8.18, P<0.05) expression levels, and increased apoptosis rate(t =7.49, P <0.05). HMGB1 was a target gene of miR-107, and transfection of miR-107 agomir in HNMECs after LPS induction could significantly reduce the expression of HMGB1 protein(t =28.56, P <0.05). CONCLUSION miR-107 is upregulated in HNMECs after LPS induction, while HMGB1 is down-regulated. Overexpression of miR-107 significantly inhibits the proliferation of HNMECs and the expression of inflammatory factors after LPS induction, and promotes apoptosis. HMGB1 is a target gene of miR-107, suggesting that miR-107 may play a regulatory role by regulating HMGB1.
OBJECTIVE To detect the effects of miR-107 on the proliferation, apoptosis and inflammatory factors of human nasal mucosal epithelial cells(HNMECs) induced by Lipopolysaccharides(LPS), and to elucidate the potential molecular mechanism. METHODS The expression changes of miR-107 and HMGBl(High mobility group box 1) in 1 mg/L LPS induced HNMECs before and after LPS induction were detected by using quantitative realtime PCR. LPS induced-HNMECs were transfected with miR-107 agomir and agomir control, and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide(MTT) assay, flow cytometry and enzyme-linked immunosorbent assay(ELISA) were performed to detect cell proliferation, apoptosis and inflammatory factors. Dual luciferase reporter assay and Western blots were applied to verify whether HMGB1 was a target gene of miR-107. RESULTS The expression of miR-107 in HNMECs after LPS induction was significantly lower than that before induction(t =9.35, P<0.05), while the expression of HMGB1 in HNMECs after LPS induction was significantly higher than that before induction(t =13.07, P<0.05). Compared with transfected agomir control, transfection of miR-107 agomir in HNMECs significantly inhibited cell proliferation(F =17.12, P <0.05) and decreased inflammatory factor TNF-α(t=6.11, P<0.05). IL-1β(t=6.90, P<0.05) and IL-6(t=8.18, P<0.05) expression levels, and increased apoptosis rate(t =7.49, P <0.05). HMGB1 was a target gene of miR-107, and transfection of miR-107 agomir in HNMECs after LPS induction could significantly reduce the expression of HMGB1 protein(t =28.56, P <0.05). CONCLUSION miR-107 is upregulated in HNMECs after LPS induction, while HMGB1 is down-regulated. Overexpression of miR-107 significantly inhibits the proliferation of HNMECs and the expression of inflammatory factors after LPS induction, and promotes apoptosis. HMGB1 is a target gene of miR-107, suggesting that miR-107 may play a regulatory role by regulating HMGB1.
引文
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[3]Bartel DP.MicroRNAs:target recognition and regulatory functions.Cell,2009,136(2):215-233.
[4]Mendell JT,Olson EN.MicroRNAs in stress signaling and human disease.Cell,2012,148(6):1172-1187.
[5]Yang F,Liu W,Yan X,et al.Effects of mir-21 on Cardiac Microvascular Endothelial Cells After Acute Myocardial Infarction in Rats:Role of Phosphatase and Tensin Homolog(PTEN)/Vascular Endothelial Growth Factor(VEGF)Signal Pathway.Med Sci Monit,2016,22:3562-3575.
[6]Shi F,Dong Z,Li H,et al.MicroRNA-137 protects neurons against ischemia/reperfusion injury through regulation of the Notch signaling pathway.Exp Cell Res,2017,352(1):1-8.
[7]Liu CC,Xia M,Zhang YJ,et al.Micro124-mediated AHRexpression regulates the inf lammatory response of chronic rhinosinusitis(CRS)with nasal polyps.Biochem Biophys Res Commun,2018,500(2):145-151.
[8]Xue X,Cao AT,Cao X,et al.Downregulation of microRNA-107in intestinal CD11c(+)myeloid cells in response to microbiota and proinflammatory cytokines increases IL-23p19 expression.Eur JImmunol,2014,44(3):673-682.
[9]Sarma NJ,Tiriveedhi V,Crippin JS,et al.Hepatitis C virusinduced changes in microRNA 107(miRNA-107)and miRNA-449a modulate CCL2 by targeting the interleukin-6 receptor complex in hepatitis.J Virol,2014,88(7):3733-3743.
[10]Schmittgen TD,Livak KJ.Analyzing real-time PCR data by the comparative C(T)method.Nat Protoc,2008,3(6):1101-1108.
[11]Xia G,Bao L,Gao W,et al.Differentially Expressed miRNAin Inflammatory Mucosa of Chronic Rhinosinusitis.J Nanosci Nanotechnol,2015,15(3):2132-2139.
[12]Zhang X H,Z hang Y N,L i H B,e t a l.O verexpression o f m iR-125b,a novel regulator of innate immunity,in eosinophilic chronic rhinosinusitis with nasal polyps.Am J Respir Crit Care Med,2012,185(2):140-151.
[13]Teng Y,Zhang R,Liu C,et al.miR-143 inhibits interleukin-13-induced inf lammatory cytokine and mucus production in nasal epithelial cells from allergic rhinitis patients by targeting IL13Rα1.Biochem Biophys Res Commun,2015,457(1):58-64.
[14]Deng Y,Yan Y,Tan KS,et al.MicroRNA-146a induction during influenza H3N2 virus infection targets and regulates TRAF6levels in human nasal epithelial cells(hNECs).Exp Cell Res,2017,352(2):184-192.
[15]Cheng F,Yang Z,Huang F,et al.microRNA-107 inhibits gastric cancer cell proliferation and metastasis by targeting PI3K/AKTpathway.Microb Pathog,2018,121:110-114.
[16]Sharma P,Saini N,Sharma R.miR-107 functions as a tumor suppressor in human esophageal squamous cell carcinoma and targets Cdc42.Oncol Rep,2017,37(5):3116-3127.
[17]Imbalzano E,Quartuccio S,Di SE,et al.Association between HMGB1 and asthma:a literature review.Clin Mol Allergy,2017,1512.
[18]Lu B,Wang H,Andersson U,et al.Regulation of HMGB1 release by inflammasomes.Protein Cell,2013,4(3):163-167.
[19]Chen D,Bellussi LM,Passali D,et al.LPS may enhance expression and release of HMGB1 in human nasal epithelial cells in vitro.Acta Otorhinolaryngol Ital,2013,33(6):398-404.
[20]Shimizu S,Kouzaki H,Kato T,et al.HMGB1-TLR4 signaling contributes to the secretion of interleukin 6 and interleukin 8 by nasal epithelial cells.Am J Rhinol Allergy,2016,30(3):167-172.