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浙江大学学报(医学版)  2021, Vol. 50 Issue (5): 582-590    DOI: 10.3724/zdxbyxb-2021-0197
浙江省人民医院 杭州医学院附属人民医院临床药学中心药学部,浙江 杭州 310014
Resveratrol inhibits the migration, invasion and epithelial-mesenchymal transition in liver cancer cells through up-regulating miR-186-5p expression in vitro
SONG Feifeng,ZHANG Yiwen,PAN Zongfu,ZHANG Qi,LU Xixuan,HUANG Ping()
Department of Pharmacy, Clinical Pharmacy Center, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital of Hangzhou Medical College, Hangzhou 310014, China
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目的:探讨白藜芦醇抑制肝癌细胞转移的分子机制。方法:利用CCK-8法检测白藜芦醇对肝癌细胞HepG2和Huh7存活率的影响,筛选后续实验适合的浓度;实时定量RT-PCR检测肝癌组织和肝癌细胞中miR-186-5p的表达;细胞划痕实验、Transwell小室实验和蛋白质印迹法分别检测白藜芦醇或miR-186-5p表达变化对肝癌细胞HepG2和Huh7迁移、侵袭和上皮-间质转化相关蛋白表达的影响。结果:6.25?μmol/L白藜芦醇对肝癌细胞HepG2和Huh7的存活率无显著影响,遂后续实验中白藜芦醇的浓度选择6.25?μmol/L。实验结果显示, 白藜芦醇可以抑制肝癌细胞的迁移和侵袭,并增加上皮钙黏素表达,降低波形蛋白和Twist1表达(均P<0.05)。与癌旁组织和正常肝细胞相比,肝癌组织和肝癌细胞中miR-186-5p表达均下调(均P<0.05)。白藜芦醇能诱导肝癌细胞中miR-186-5p的表达(均P<0.01)。上调miR-186-5p表达可显著抑制肝癌细胞的迁移、侵袭和上皮-间质转化(均P<0.05),而敲低miR-186-5p表达能阻断白藜芦醇对肝癌细胞迁移、侵袭和上皮-间质转化的抑制作用。结论:白藜芦醇能体外抑制肝癌转移,其机制可能与上调miR-186-5p表达有关。

关键词: 肝肿瘤白藜芦醇miR-186-5p迁移侵袭上皮-间质转化体外实验    

Objective: To investigate the molecular mechanism of resveratrol inhibiting the metastasis of liver cancer in vitro. Methods:HepG2 and Huh7 cells were treated with different concentrations of resveratrol, and the cell viability was determined by CCK-8 assay to determine the optimal concentration of resveratrol for subsequent experiments. The expressions of miR-186-5p in liver cancer tissues and liver cancer cells were determined by quantitative real-time RT-PCR. The migration and invasion of HepG2 and Huh7 cells were detected by wound healing assay and Transwell assay, and the expression levels of epithelial-mesenchymal transition (EMT) related proteins were determined by Western blotting. Results:Resveratrol with concentration of 6.25?μmol/L had no effect on the viability of HepG2 and Huh7 cells, so the concentration of resveratrol in subsequent experiments was 6.25?μmol/L. Resveratrol inhibited the wound healing and invasion of liver cancer cells; increased the expression of E-cadherin, and decreased the expression of vimentin and Twist1. The expression of miR-186-5p was significantly down-regulated in liver cancer tissues and cells compared with the adjacent tissues and normal liver cells (both P<0.05). Furthermore, resveratrol induced the expression of miR-186-5p in liver cancer cells (bothP<0.01). Overexpression of miR-186-5p suppressed the migration, invasion and EMT of liver cancer cells. Knockdown of miR-186-5p blocked the inhibition effects of resveratrol on the migration, invasion and EMT of liver cancer cells.Conclusion: Resveratrol could inhibit the metastasis of liver cancer in vitro, which might be associated with up-regulating miR-186-5p.

Key words: Liver neoplasms    Resveratrol    MiR-186-5p    Migration    Invasion    Epithelial-mesenchymal transition    In vitro experiment
收稿日期: 2021-07-19 出版日期: 2021-12-29
:  R735.7  
基金资助: 国家自然科学基金(82003853);浙江省自然科学基金(LQ20H310005,LYY21H310009);浙江省医药卫生科技计划(2021KY046)
通讯作者: 黄萍     E-mail:
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宋飞凤,张轶雯,潘宗富,张琪,卢茜璇,黄萍. 白藜芦醇通过上调miR-186-5p表达抑制肝癌细胞迁移、侵袭和上皮-间质转化[J]. 浙江大学学报(医学版), 2021, 50(5): 582-590.

SONG Feifeng,ZHANG Yiwen,PAN Zongfu,ZHANG Qi,LU Xixuan,HUANG Ping. Resveratrol inhibits the migration, invasion and epithelial-mesenchymal transition in liver cancer cells through up-regulating miR-186-5p expression in vitro. J Zhejiang Univ (Med Sci), 2021, 50(5): 582-590.


图 2  溶剂对照组和白藜芦醇组细胞上皮-间质转化相关蛋白表达比较A:两组上皮-间质转化相关蛋白电泳图(1:溶剂对照组,2:白藜芦醇组);B:HepG2细胞蛋白相对表达量比较; C:Huh7细胞蛋白相对表达量比较.与溶剂对照组比较,<0.05,<0.01.GAPDH:甘油醛-3-磷酸脱氢酶.
图 1  溶剂对照组和白藜芦醇组侵袭细胞观察与溶剂对照组比较,HepG2 和 Huh7细胞白藜芦醇组侵袭细胞数均减少. 标尺=250 μm.
图 3  miR-186-5p模拟物组和模拟物对照组创伤愈合能力及上皮–间质转化相关蛋白表达量比较A:两组创伤愈合率比较; B:HepG2细胞蛋白相对表达量比较; C:Huh7细胞蛋白相对表达量比较.与模拟物对照组比较,<0.05,<0.01.
图 4  miR-186-5p模拟物组和模拟物对照组Transwell小室实验结果与模拟物对照组比较,两种肝癌细胞miR-186-5p模拟物组侵袭细胞数减少.标尺=250 μm.
图 5  miR-186-5p高表达肝癌细胞上皮-间质转化相关蛋白电泳图1:模拟物对照组;2:miR-186-5p模拟物组.GAPDH:甘油醛-3-磷酸脱氢酶.
图 6  miR-186-5p抑制剂和白藜芦醇合用与否各组创伤愈合能力及上皮-间质转化相关蛋白表达量比较A:各组创伤愈合率比较; B:HepG2细胞蛋白相对表达量比较; C:Huh7细胞蛋白相对表达量比较. 与抑制剂对照+溶剂对照组比较,<0.05; 与miR-186-5p抑制剂+溶剂对照组比较,<0.05;与抑制剂对照组+白藜芦醇组比较,<0.05.
图 7  miR-186-5p抑制剂和白藜芦醇合用与否各组细胞侵袭数变化与溶剂对照组比较,白藜芦醇组侵袭细胞数减少.与抑制剂对照+白藜芦醇组比较,miR-186-5p抑制剂+白藜芦醇组侵袭细胞数增加. 标尺=250 μm.
图 8  miR-186-5p抑制剂和白藜芦醇合用与否各组细胞EMT相关蛋白表达比较1.抑制剂对照+溶剂对照组;2.抑制剂对照+白藜芦醇组;3.miR-186-5p抑制剂+溶剂对照组;4.miR-186-5p抑制剂+白藜芦醇组.GAPDH:甘油醛-3-磷酸脱氢酶.
1 ZHENG R S, SUN K X, ZHANG S W, et al. Report of cancer epidemiology in China, 2015[J]. Chin J Oncol, 2019, 41(1): 19-28
2 LVJ, ZHANGS, WUH, et al.Deubiquitinase PSMD14 enhances hepatocellular carcinoma growth and metastasis by stabilizing GRB2[J]Cancer Lett, 2020, 22-34.
doi: 10.1016/j.canlet.2019.10.025
3 CHENT W, YINF F, YUANY M, et al.CHML promotes liver cancer metastasis by facilitating Rab14 recycle[J]Nat Commun, 2019, 10( 1): 2510-2514.
doi: 10.1038/s41467-019-10364-0
4 L’HERMITTEA, PHAMS, CADOUXM, et al.Lect2 controls inflammatory monocytes to constrain the growth and progression of hepatocellular carcinoma[J]Hepatology, 2019, 69( 1): 160-178.
doi: 10.1002/hep.30140
5 HAIDERC, HNATJ, WAGNERR, et al.Transforming growth factor-β and axl induce CXCL5 and neutrophil recruitment in hepatocellular carcinoma[J]Hepatology, 2019, 69( 1): 222-236.
doi: 10.1002/hep.30166
6 YUANT, CHENZ, YANF, et al.Deubiquitinating enzyme USP10 promotes hepatocellular carcinoma metastasis through deubiquitinating and stabilizing smad4 protein[J]Mol Oncol, 2020, 14( 1): 197-210.
doi: 10.1002/1878-0261.12596
7 KIME, KIMD, LEEJ S, et al.Capicua suppresses hepatocellular carcinoma progression by controlling the ETV4-MMP1 axis[J]Hepatology, 2018, 67( 6): 2287-2301.
doi: 10.1002/hep.29738
8 HUANGX, ZHUH L. Resveratrol and its analogues: promising antitumor agents[J]Anticancer Agents Med Chem, 2011, 11( 5): 479-490.
doi: 10.2174/187152011795677427
9 RAUFA, IMRANM, SULERIAH A R, et al.A comprehensive review of the health perspectives of resveratrol[J]Food Funct, 2017, 8( 12): 4284-4305.
doi: 10.1039/C7FO01300K
10 VARGASJ E, PUGAR, LENZG, et al.Cellular mechanisms triggered by the cotreatment of resveratrol and doxorubicin in breast cancer: a translational in vitro–in silico model[J]Oxid Med Cell Longev, 2020, 5432651.
doi: 10.1155/2020/5432651
11 KUOI M, LEEJ J, WANGY S, et al.Potential enhancement of host immunity and anti-tumor efficacy of nanoscale curcumin and resveratrol in colorectal cancers by modulated electro-hyperthermia[J]BMC Cancer, 2020, 20( 1): 603.
doi: 10.1186/s12885-020-07072-0
12 YANGH, ZHENGY, LIT W H, et al.Methionine adenosyltransferase 2B, HuR, and sirtuin 1 protein cross-talk impacts on the effect of resveratrol on apoptosis and growth in liver cancer cells[J]J Biol Chem, 2013, 288( 32): 23161-23170.
doi: 10.1074/jbc.M113.487157
13 SUNY, ZHOUQ M, LUY Y, et al.Resveratrol inhibits the migration and metastasis of MDA-MB-231 human breast cancer by reversing TGF-β1-induced epithelial-mesenchymal transition[J]Molecules, 2019, 24( 6): 1131-1146.
doi: 10.3390/molecules24061131
14 PETRIB J, KLINGEC M. Regulation of breast cancer metastasis signaling by miRNAs[J]Cancer Metastasis Rev, 2020, 39( 3): 837-886.
doi: 10.1007/s10555-020-09905-7
15 KIMT W, LEEY S, YUNN H, et al.MicroRNA-17-5p regulates EMT by targeting vimentin in colorectal cancer[J]Br J Cancer, 2020, 123( 7): 1123-1130.
doi: 10.1038/s41416-020-0940-5
16 CHANS H, WANGL H. Regulation of cancer metastasis by microRNAs[J]J Biomed Sci, 2015, 22( 1): 9-20.
doi: 10.1186/s12929-015-0113-7
17 LOU W, LIU J, GAO Y, et al. MicroRNA regulation of liver cancer stem cells[J]. Am J Cancer Res, 2018, 8(7): 1126-1141
18 LIJ, XIAL, ZHOUZ, et al.MiR-186-5p upregulation inhibits proliferation, metastasis and epithelial-to-mesenchymal transition of colorectal cancer cell by targeting ZEB1[J]Arch Biochem Biophys, 2018, 53-60.
doi: 10.1016/
19 SHANY, LIP. Long intergenic non-protein coding RNA 665 regulates viability, apoptosis, and autophagy via the MiR-186-5p/MAP4K3 axis in hepatocellular carcinoma[J]Yonsei Med J, 2019, 60( 9): 842-853.
doi: 10.3349/ymj.2019.60.9.842
20 LANT, YANX, LIZ, et al.Long non-coding RNA PVT1 serves as a competing endogenous RNA for miR-186-5p to promote the tumorigenesis and metastasis of hepatocellular carcinoma[J]Tumour Biol, 2017, 39( 6): 101042831770533.
doi: 10.1177/1010428317705338
21 SMOLIGAJ M, BAURJ A, HAUSENBLASH A. Resveratrol and health——a comprehensive review of human clinical trials[J]Mol Nutr Food Res, 2011, 55( 8): 1129-1141.
doi: 10.1002/mnfr.201100143
22 BROWNV A, PATELK R, VISKADURAKIM, et al.Repeat dose study of the cancer chemopreventive agent resveratrol in healthy volunteers: safety, pharmacokinetics, and effect on the insulin-like growth factor axis[J]Cancer Res, 2010, 70( 22): 9003-9011.
doi: 10.1158/0008-5472.CAN-10-2364
23 KOJ H, SETHIG, UMJ Y, et al.The role of resveratrol in cancer therapy[J]Int J Mol Sci, 2017, 18( 12): 2589.
doi: 10.3390/ijms18122589
24 YUANK, XIEK, LANT, et al.TXNDC12 promotes EMT and metastasis of hepatocellular carcinoma cells via activation of β-catenin[J]Cell Death Differ, 2020, 27( 4): 1355-1368.
doi: 10.1038/s41418-019-0421-7
25 ZHAOY R, WANGJ L, XUC, et al.HEG1 indicates poor prognosis and promotes hepatocellular carcinoma invasion, metastasis, and EMT by activating Wnt/β-catenin signaling[J]Clin Sci, 2019, 133( 14): 1645-1662.
doi: 10.1042/CS20190225
26 MENGJ, AIX, LEIY, et al.USP5 promotes epithelial-mesenchymal transition by stabilizing SLUG in hepatocellular carcinoma[J]Theranostics, 2019, 9( 2): 573-587.
doi: 10.7150/thno.27654
27 XUQ, ZONGL, CHENX, et al.Resveratrol in the treatment of pancreatic cancer[J]Ann N Y Acad Sci, 2015, 1348( 1): 10-19.
doi: 10.1111/nyas.12837
28 WANGH, ZHANGH, TANGL, et al.Resveratrol inhibits TGF-β1-induced epithelial-to-mesenchymal transition and suppresses lung cancer invasion and metastasis[J]Toxicology, 2013, 139-146.
doi: 10.1016/j.tox.2012.09.017
29 JIQ, LIUX, HANZ, et al.Resveratrol suppresses epithelial-to-mesenchymal transition in colorectal cancer through TGF-β1/Smads signaling pathway mediated Snail/E-cadherin expression[J]BMC Cancer, 2015, 15( 1): 97.
doi: 10.1186/s12885-015-1119-y
30 LIW, MAJ, MAQ, et al.Resveratrol inhibits the epithelial-mesenchymal transition of pancreatic cancer cells via suppression of the PI-3K/Akt/NF-κB pathway[J]Curr Med Chem, 2013, 20( 33): 4185-4194.
doi: 10.2174/09298673113209990251
31 WANGX, GAOJ, ZHOUB, et al.Identification of prognostic markers for hepatocellular carcinoma based on miRNA expression profiles[J]Life Sci, 2019, 116596.
doi: 10.1016/j.lfs.2019.116596
32 ZHANGQ, HAOL, SHENZ, et al.MiR‐186‐5p suppresses cell migration, invasion, and epithelial mesenchymal transition in bladder cancer by targeting RAB27A/B[J]Environ Toxicol, 2021, 36( 11): 2174-2185.
doi: 10.1002/tox.23331
33 BRABLETZT, KALLURIR, NIETOM A, et al.EMT in cancer[J]Nat Rev Cancer, 2018, 18( 2): 128-134.
doi: 10.1038/nrc.2017.118
34 GREGORYP A, BERTA G, PATERSONE L, et al.The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1[J]Nat Cell Biol, 2008, 10( 5): 593-601.
doi: 10.1038/ncb1722
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