LPS通过TLR4/MyD88/NF-κB途径促进人肝癌细胞增殖及转移能力
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摘要
目的研究脂多糖(lipopolysaccharide,LPS)对人肝癌细胞HepG2增殖、粘附、侵袭、迁移能力的影响,并阐明相关机制。方法 (1)细胞增殖及粘附能力采用MTT法检测;(2)细胞侵袭和迁移能力采用Transwell小室模型检测;(3) Toll样受体4(toll-like receptor 4,TLR4)、髓样分化因子88(myeloid differentiation factor,MyD88)及核因子-κB(nuclear factor-κB,NF-κB)的蛋白和mRNA表达水平分别采用Western blot和RT-PCR测定。结果 (1) LPS(浓度分别为10、100、1 000 ng/m L)刺激Hep G224、48、72 h,与对照组相比,细胞增殖能力均增高,差异有统计学意义(均P <0. 05),且呈浓度和时间依赖性。(2)经100 ng/m L的LPS干预HepG2 48 h后,干预组细胞粘附能力明显高于对照组(P <0. 01);侵袭和迁移实验中穿膜细胞数比较,干预组均显著多于对照组(P <0. 01)。(3) LPS可明显上调TLR4、MyD88及NF-κB蛋白及mRNA表达水平(P <0. 05)。结论 LPS可能通过TLR4/MyD88/NF-κB途径促进人肝癌细胞增殖及转移能力。
Objective To observe the effect of LPS on the proliferative and metastatic ability of HepG2,and clarified the related mechanism. Methods(1) The proliferation and adhesive ability determined by MTT assay. Transwell assay was used for detecting the invasive and migratory ability.(3) The mRNA and protein expression of TLR4、MyD88 and NF-κB were evaluated by RT-PCR and Western blot. Results(1) LPS(10,100,1000 ng/m L) interfered with HepG2 for 24,48 and 72 hours respectively. The proliferation ability of HepG2 cells was significantly higher than that of control group(all P < 0. 05). The proliferation ability of HepG2 cells increased with the increase of dose and time.(2) The adhesive ability was significantly promoted with treatment of LPS(100 ng/m L)than the control group(P < 0. 01); the number cells of invasive or migratory through Matrigel was significantly increased in response to LPS(100 ng/m L)(P < 0. 01).(3) The mRNA and protein expression of TLR4、MyD88 and NF-κB were significantly up-regulated after treatment of LPS(P < 0. 05). Conclusion LPS may promote the proliferation and metastasis of human hepatocellular carcinoma cells through TLR4/MyD88/NF-κB pathway.
Objective To observe the effect of LPS on the proliferative and metastatic ability of HepG2,and clarified the related mechanism. Methods(1) The proliferation and adhesive ability determined by MTT assay. Transwell assay was used for detecting the invasive and migratory ability.(3) The mRNA and protein expression of TLR4、MyD88 and NF-κB were evaluated by RT-PCR and Western blot. Results(1) LPS(10,100,1000 ng/m L) interfered with HepG2 for 24,48 and 72 hours respectively. The proliferation ability of HepG2 cells was significantly higher than that of control group(all P < 0. 05). The proliferation ability of HepG2 cells increased with the increase of dose and time.(2) The adhesive ability was significantly promoted with treatment of LPS(100 ng/m L)than the control group(P < 0. 01); the number cells of invasive or migratory through Matrigel was significantly increased in response to LPS(100 ng/m L)(P < 0. 01).(3) The mRNA and protein expression of TLR4、MyD88 and NF-κB were significantly up-regulated after treatment of LPS(P < 0. 05). Conclusion LPS may promote the proliferation and metastasis of human hepatocellular carcinoma cells through TLR4/MyD88/NF-κB pathway.
引文
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[16] Mio I, Mms YK, Masafumi N. Lipopolysaccharide(LPS)Increases the Invasive Ability of Pancreatic Cancer Cells Through the TLR4/My D88 Signaling Pathway[J]. J Surg Oncol,2009,100(8):725-731.
[17] Park GB,Kim D. TLR4-mediated galectin-1 production triggers epithelial-mesenchymal transition in colon cancer cells through ADAM10-and ADAM17-associated lactate production[J]. Mol Cell Biochem,2017,425(1-2):191-202.
[18] He A,Shao J,Zhang Y,et al. CD200Fc reduces LPS-induced IL-1βactivation in human cervical cancer cells by modulating TLR4-NF-κB and NLRP3 inflammasome pathway[J]. Oncotarget,2017,8(20):33214-33224.
[19] Wang CH,Wang PJ,Hsieh YC,et al. Resistin facilitates breast cancer progression via TLR4-mediated induction of mesenchymal phenotypes and stemness properties[J]. Oncogene,2018,37(5):589-600.
[20]伍振辉,孟娴,胡佳伟,等. TLR4-MyD88-NF-kB信号通路与肝炎-肝纤维化-肝癌轴相关性研究进展[J].国际药学研究杂志,2017,44(5):396-401.
[2] Chaturvedi VK, Singh A, Dubey SK, et al. Molecular mechanistic insight of hepatitis B virus mediated hepatocellular carcinoma[J]. Microb Pathog,2019,128(3):184-194.
[3] Yu LX,Schwabe RF. The gut microbiome and liver cancer:mechanisms and clinical translation[J]. Nat Rev Gastroenterol Hepatol,2017,14(9):527-539.
[4] Shi H,Dong L,Dang X,et al. Effect of chlorogenic acid on LPSinduced proinflammatory signaling in hepatic stellate cell[J].Inflamm Res,2013,62(6):581-587.
[5] Munn LL. Cancer and Inflammation[J]. Wiley Interdiscip Rev Syst Biol Med,2017,9(2):1-13.
[6]韩殿冰,曾本华,葛成果.内毒素在小鼠肝癌发生过程中的作用[J].河南外科学杂志,2015,21(6):1-3.
[7] Liu J,Xu D,Wang Q,et al. LPS induced miR-181a promotes pancreatic cancer cell migration via targeting PTEN and MAP2K4[J]. Dig Dis Sci,2014,59(7):1452-1460.
[8] Zhu G,Huang Q,Zheng W,et al. LPS Upregulated VEGFR-3Expression Promote Migration and Invasion in Colorectal Cancer via a Mechanism of Increased NF-κB Binding to the Promoter of VEGFR-3[J]. Cell Physiol Biochem,2016,39(5):1665-1678.
[9] Lucien F,Leong HS. The role of extracellular vesicles in cancer microenvironment and metastasis:myths and challenges[J].Biochem Soc Trans,2019,47(1):273-280.
[10] Mishra V,Pathak C. Human Toll-Like Receptor 4(hTLR4):Structural and functional dynamics in cancer[J]. Int J Biol Macromol. 2019,122:425-451.
[11] Rogero MM, Calder PC. Obesity, Inflammation, Toll-Like Receptor 4 and Fatty Acids[J]. Nutrients,2018,10(4):pii:E432.
[12] Wu GJ,Lin YW,Chuang CY,et al. Liver nitrosation and inflammation in septic rats were suppressed by propofol via downregulating TLR4/NF-κB-mediated i NOS and IL-6 gene expressions[J]. Life Sci,2018,15(195):25-32.
[13] Weber SN, Bohner A, Dapito DH,et al. TLR4 Deficiency Protects against Hepatic Fibrosis and Diethylnitrosamine-Induced Pre-Carcinogenic Liver Injury in Fibrotic Liver[J]. PLo S One,2016,11(7):1-12.
[14] Shi H,Dong L,Jiang J,et al. Chlorogenic acid reduces liver inflammation and fibrosis through inhibition of toll-like receptor 4signaling pathway[J]. Toxicology,2013,303(7):107-114.
[15] Liu M,Xu Y,Han X,et al. Dioscin alleviates alcoholic liver fibrosis by attenuating hepatic stellate cell activation via the TLR4/My D88/NF-κB signalling pathway[J]. Sci Rep,2015,5:1-13.
[16] Mio I, Mms YK, Masafumi N. Lipopolysaccharide(LPS)Increases the Invasive Ability of Pancreatic Cancer Cells Through the TLR4/My D88 Signaling Pathway[J]. J Surg Oncol,2009,100(8):725-731.
[17] Park GB,Kim D. TLR4-mediated galectin-1 production triggers epithelial-mesenchymal transition in colon cancer cells through ADAM10-and ADAM17-associated lactate production[J]. Mol Cell Biochem,2017,425(1-2):191-202.
[18] He A,Shao J,Zhang Y,et al. CD200Fc reduces LPS-induced IL-1βactivation in human cervical cancer cells by modulating TLR4-NF-κB and NLRP3 inflammasome pathway[J]. Oncotarget,2017,8(20):33214-33224.
[19] Wang CH,Wang PJ,Hsieh YC,et al. Resistin facilitates breast cancer progression via TLR4-mediated induction of mesenchymal phenotypes and stemness properties[J]. Oncogene,2018,37(5):589-600.
[20]伍振辉,孟娴,胡佳伟,等. TLR4-MyD88-NF-kB信号通路与肝炎-肝纤维化-肝癌轴相关性研究进展[J].国际药学研究杂志,2017,44(5):396-401.