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浙江大学学报（医学版）
浙江大学学报（医学版）  2021, Vol. 50 Issue (5): 582-590    DOI: 10.3724/zdxbyxb-2021-0197
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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
宋飞凤,张轶雯,潘宗富,张琪,卢茜璇,黄萍()
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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摘要：

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    
Abstract:

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: huangping@hmc.edu.cn
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引用本文:

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.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0197    或    https://www.zjujournals.com/med/CN/Y2021/V50/I5/582

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.
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