LPS对乳腺癌MDA-MB-231细胞上皮间质转化的诱导作用及其对β-catenin表达的影响

doi:10.13481/j.1671-587x.20200217

摘要/Abstract

摘要： 目的：探讨脂多糖（LPS）对人乳腺癌MDA-MB-231细胞中上皮间质转化（EMT）标志物及β-连环素（β-catenin）表达的影响，阐明其可能机制。方法：人乳腺癌MDA-MB-231细胞分为对照组和不同浓度（5、10、20和40 mg·L^-1）LPS组，倒置显微镜观察各组MDA-MB-231细胞的形态表现，免疫荧光实验检测各组MDA-MB-231细胞中β-catenin表达及定位，实时荧光定量PCR（RT-qPCR）法和Western blotting法检测各组MDA-MB-231细胞中EMT标志物E-钙黏蛋白（E-cadherin）、波形蛋白（Vimentin）和β-catenin mRNA及蛋白表达水平。结果：对照组MDA-MB-231细胞形态表现为上皮细胞表型，不同浓度LPS组MDA-MB-231细胞形态表现为间质细胞表型。免疫荧光实验，β-catenin表达定位主要在细胞核。与对照组比较，不同浓度LPS组细胞中Vimentin和β-catenin mRNA及蛋白表达水平明显升高（P<0.05或P<0.01），以20 mg·L^-1LPS组升高最为明显；与对照组比较，不同浓度LPS组细胞中E-cadherin mRNA和蛋白表达水平明显降低（P<0.05或P<0.01），以20 mg·L^-1 LPS组降低最为明显。结论：LPS通过下调E-cadherin表达、上调Vimentin表达促进乳腺癌MDA-MB-231细胞发生EMT、侵袭和转移，其作用机制可能与Wnt/β-catenin信号通路有关。

关键词: 乳腺肿瘤, 脂多糖, 上皮间质转化, Wnt/β-catenin信号通路

Abstract: Objective: To investigate the effect of lipopolysaccharide (LPS) on the expressions of epithelial-mesenchymal transition (EMT) markers and β-catenin in the breast cancer MDA-MB-231 cells, and to clarify its possible mechanism. Methods: The breast cancer MDA-MB-231 cells were divided into control group and different concentrations (5, 10, 20, and 40 mg·L^-1) of LPS groups. Inverted microscope was used to observe the morphology of MDA-MB-231 cells in various groups. Immunofluorescence test was used to detect the β-catenin expression and location in the MDA-MB-231 cells in various groups. Real-time quantitative PCR(RT-qPCR) and Western blotting methods were used to detect the expression levels of the EMT markers E-cadherin, Vimentin and β-catenin mRNA and proteins in the MDA-MB-231 cells in various groups. Results: The morphology of MDA-MB-231 cells in control group was epithelial phenotype, and the morphology of MDA-MB-231 cells in different concentrations of LPS groups were the phenotype of mesenchymal cells. The results of immunofluorescence staining showed that the expression of β-catenin was mainly located in the nucleus. Compared with control group, the expression levels of Vimentin and β-catenin mRNA and proteins in the MDA-MB-231 cells in different concentrations of LPS groups were increased (P<0.05 or P<0.01), especially in 20 mg·L^-1 LPS group. Compared with control group, the expression levels of E-cadherin mRNA and proteins in the MDA-MB-231 cells in different concentrations of LPS groups were decreased (P<0.05 or P<0.01), especially in 20 mg·L^-1 LPS group. Conclusion: LPS could promote the EMT, invasion and metastasis of the breast cancer MDA-MB-231 cells by down-regulating the E-cadherin expression and up-regulating the Vimentin expression, and its mechanism may be related to Wnt/β-catenin signaling pathway.

Key words: breast neoplasms, lipopolysaccharide, epithelial mesenchymal transition, Wnt/β-catenin signaling pathway

中图分类号:

R737.9

陈州华, 龚辉, 冯磊, 肖玉洁, 黄立中. LPS对乳腺癌MDA-MB-231细胞上皮间质转化的诱导作用及其对β-catenin表达的影响[J]. 吉林大学学报(医学版), 2020, 46(02): 309-315.

CHEN Zhouhua, GONG Hui, FENG Lei, XIAO Yujie, HUANG Lizhong. Induction of LPS on epithelial mesenchymal transition in breast cancer MDA-MB-231 cells and its effect on β-catenin expression[J]. Journal of Jilin University(Medicine Edition), 2020, 46(02): 309-315.

参考文献

[1] 李贺, 郑荣寿, 张思维, 等.2014年中国女性乳腺癌发病与死亡分析[J].中华肿瘤杂志,2018,40(3):166-171.
[2] 郭鱼波, 王春晖, 裴晓华. 乳腺癌细胞上皮间质转化的分子机制及其防治的研究进展[J].中国肿瘤,2018,27(12):933-943.
[3] ZHANG Z H, XIE D D, XU S, et al. Total glucosides of paeony inhibits lipopolysaccharide-induced proliferation, migration and invasion in androgen insensitive prostate cancer cells[J]. PLoS One, 2017,12(8):e0182584.
[4] SEOL M A, PARK J H, JEONG J H, et al. Role of TOPK in lipopolysaccharide-induced breast cancer cell migration and invasion[J]. Oncotarget, 2017,8(25):40190-40203.
[5] CHENG G, YANG S, ZHANG G, et al. Lipopolysaccharide-induced α-catenin downregulation enhances the motility of human colorectal cancer cells in an NF-κB signaling-dependent manner[J].Oncotargets Ther,2016,9:7563-7571.
[6] WANG L L, ZHU R, HUANG Z Q, et al. Lipopolysaccharide-induced toll-like receptor 4 signaling in cancer cells promotes cell survival and proliferation in hepatocellular carcinoma[J]. Dig Dis Sci, 2013,58(8):2223-2236.
[7] PARK G S, KIM J H. LPS Up-regulates ICAM-1 expression in breast cancer cells by stimulating a MyD88-BLT2-ERK-linked cascade, which promotes adhesion to monocytes[J]. Mol Cells, 2015,38(9):821-828.
[8] HUANG T, CHEN Z J, FANG L P. Curcumin inhibits LPS-induced EMT through downregulation of NF-κB-Snail signaling in breast cancer cells[J]. Oncol Rep, 2013, 29(1):117-124.
[9] WONG J H, HO K H, NAM S, et al. Store-operated Ca²⁺ entry facilitates the lipopolysaccharide-induced cyclooxygenase-2 expression in gastric cancer cells[J]. Sci Rep, 2017,7(1):12813.
[10] HE M Q, LIU Y H, SHEN J B, et al. Upregulation of PLZF is associated with neuronal injury in lipopolysaccharide-induced Neuroinflammation[J]. Neurochem Res, 2016,41(11):3063-3073.
[11] 雷莉妍, 王瑞成, 周瑞, 等. 基于NF-κB通路的芒果苷干预脂多糖诱导的细胞炎症作用机制研究[J].中国中西医结合杂志,2019,39(5):597-602.
[12] LIU W M, FAN Y Y, SHI Y, et al. Knockdown of TIPE2 increases the proliferation in lipopolysaccharide-stimulated gastric cancer cells[J]. BMC Cancer, 2018,18(1):857.
[13] 迟强, 艾晓林, 王志勇, 等. 脂多糖经TLR4介导Cx43的表达对膀胱癌细胞增殖和凋亡的影响[J].实用医学杂志,2017,33(5):701-705.
[14] CHE F Y, XIE X L, WANG L, et al. B7-H6 expression is induced by lipopolysaccharide and facilitates cancer invasion and metastasis in human gliomas[J]. Int Immunopharmacol, 2018,59:318-327.
[15] 金一, 马特, 蒋书玲. JNK/MAPK在LPS诱导乳腺癌细胞MCF-7上皮间质转化过程中的作用[J]. 现代肿瘤医学, 2014, 22(6):1272-1275.
[16] KIM Y, LEE E J, JANG H K, et al. Statin pretreatment inhibits the lipopolysaccharide-induced epithelial-mesenchymal transition via the downregulation of toll-like receptor 4 and nuclear factor-κB in human biliary epithelial cells[J].J Gastroenterol Hepatol,2016,31(6):1220-1228.
[17] ZHANG Y Q, LIU Y J, MAO Y F, et al. Resveratrol ameliorates lipopolysaccharide-induced epithelial mesenchymal transition and pulmonary fibrosis through suppression of oxidative stress and transforming growth factor-β1 signaling[J].Clin Nutr,2015,34(4):752-760.
[18] HE Z F, DENG R Z, HUANG X F, et al. Lipopolysaccharide enhances OSCC migration by promoting epithelial-mesenchymal transition[J]. J Oral Pathol Med, 2015,44(9):685-692.
[19] HONG D R, JANG S Y, JANG E H, et al. Shikonin as an inhibitor of the LPS-induced epithelial-to-mesenchymal transition in human breast cancer cells[J]. Int J Mol Med,2015,36(6):1601-1606.
[20] CHO I H, JANG E H, HONG D R, et al. Suppression of LPS-induced epithelial-mesenchymal transition by aqueous extracts of Prunella vulgaris through inhibition of the NF-κB/Snail signaling pathway and regulation of EMT-related protein expression[J]. Oncol Rep, 2015, 34(5):2445-2450.
[21] WU C N, ZHUANG Y W, JIANG S, et al. Interaction between Wnt/β-catenin pathway and microRNAs regulates epithelial-mesenchymal transition in gastric cancer (Review)[J]. Int J Oncol, 2016,48(6):2236-2246.
[22] HE Y J, LIU Z X, QIAO C, et al. Expression and significance of Wnt signaling components and their target genes in breast carcinoma[J]. Mol Med Rep, 2014,9(1):137-143.
[23] BAYERLOVÁ M, KLEMM F, KRAMER F, et al. Newly constructed network models of different WNT signaling cascades applied to breast cancer expression data[J]. PLoS One, 2015,10(12):e0144014.
[24] ZHANG L N, HUANG Y H, ZHAO L. Fusion of macrophages promotes breast cancer cell proliferation, migration and invasion through activating epithelial-mesenchymal transition and Wnt/β-catenin signaling pathway[J].Arch Biochem Biophys,2019,676:108137.
[25] CAI K, JIANG L W, WANG J, et al. Downregulation of β-catenin decreases the tumorigenicity, but promotes epithelial-mesenchymal transition in breast cancer cells[J]. J Cancer Res Ther, 2014, 10(4):1063.
[26] 许光伟, 曹旭晨. β-catenin下调对乳腺癌MDA-MB-231细胞上皮间质转化及侵袭迁移能力的影响[J]. 天津医药, 2015, 43(9):981-984,1093.
[27] DANA N, JAVANMARD S H, VASEGHI G. Effect of lipopolysaccharide on toll-like receptor-4 signals in mouse cancer cells[J]. Bratisl Lek Listy, 2017,118(10):598-601.