Ethyl Acetate Extract of Qigesan Intervenes in Migration and Invasion of Esophageal Cancer Cells via Inhibiting TGF-<italic style="font-style: italic">β</italic><sub>1</sub> Signaling Pathway

SHANG Yiwan; ZHU Rui; WU Yingshuo; CHEN Xing; ZHOU Zhexu; REN Shanshan; LIU Yan; CHEN Yulong; YANG Lianhe

doi:10.13422/j.cnki.syfjx.202202322

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Ethyl Acetate Extract of Qigesan Intervenes in Migration and Invasion of Esophageal Cancer Cells via Inhibiting TGF-β₁ Signaling Pathway

更新时间：2023-05-17

- Ethyl Acetate Extract of Qigesan Intervenes in Migration and Invasion of Esophageal Cancer Cells via Inhibiting TGF-β₁ Signaling Pathway
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 7, Pages: 66-75(2023)
- 作者机构：
  
  1.河南中医药大学河南省方证信号传导重点实验室，郑州 450046
  2.首都医科大学北京胸科医院，北京 100700
  3.河南中医药大学医学院，郑州 450046
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.202202322
  CLC： R22;R242;R2-031;R735.1
- Published：05 April 2023，
  
  Published Online：08 October 2022，
  
  Received：09 June 2022，
- 稿件说明：
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尚艺婉,朱芮,武颖烁等.启膈散乙酸乙酯部位提取物通过抑制TGF-β₁信号通路干预食管癌细胞的迁移和侵袭[J].中国实验方剂学杂志,2023,29(07):66-75.

SHANG Yiwan,ZHU Rui,WU Yingshuo,et al.Ethyl Acetate Extract of Qigesan Intervenes in Migration and Invasion of Esophageal Cancer Cells via Inhibiting TGF-β₁ Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(07):66-75.
尚艺婉,朱芮,武颖烁等.启膈散乙酸乙酯部位提取物通过抑制TGF-β₁信号通路干预食管癌细胞的迁移和侵袭[J].中国实验方剂学杂志,2023,29(07):66-75. DOI： 10.13422/j.cnki.syfjx.202202322.

SHANG Yiwan,ZHU Rui,WU Yingshuo,et al.Ethyl Acetate Extract of Qigesan Intervenes in Migration and Invasion of Esophageal Cancer Cells via Inhibiting TGF-β₁ Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(07):66-75. DOI： 10.13422/j.cnki.syfjx.202202322.

摘要

目的

探讨启膈散（QGS）对食管癌细胞TE-1迁移和侵袭能力的影响机制。

方法

利用基因芯片技术筛选正常组和启膈散组差异表达基因，分析差异基因的本体功能和信号通路。噻唑蓝（MTT）比色法检测QGS对TE-1细胞活性的影响。后续验证实验分为空白组、转化生长因子-

（TGF-

）组、TGF-

+QGS组、TGF-

+SB431542组。显微镜观察各实验组细胞形态，细胞划痕实验检测各组细胞的迁移和侵袭能力，实时荧光定量聚合酶链式反应（Real-time PCR）检测各组细胞E-钙黏蛋白（E-Cadherin）、波形纤维蛋白（Vimentin）、果蝇母本抗生存因子2（Smad2）和果蝇母本抗生存因子7（Smad7） mRNA的表达水平。蛋白免疫印迹法（Western blot）检测各组细胞E-Cadherin、Vimentin、p-Smad2/3、Smad2/3和Smad7蛋白的表达。

结果

QGS组与空白组之间有1 487个差异基因，其中1 080个下调，407个上调，下调基因占差异基因的72.63%。下调基因涉及的主要生物学过程有细胞骨架蛋白结合、ATP结合、腺苷酸核苷酸结合、腺苷酸核糖核苷酸结合等，涉及的京都基因与基因组百科全书（KEGG）通路主要包括TGF-

信号通路、细胞周期、细胞外基质-受体相互作用蛋白、肿瘤中的通路、卵母细胞减数分裂等；上调基因主要参与RNA结合、DNA结合、转录调节子活性、转录激活子活性、核苷酸结合等生物学过程，涉及的KEGG通路主要包括丝裂原活化蛋白激酶（MAPK）信号通路、膀胱癌、肾细胞癌、癌中通路、p53信号通路等。与空白组比较，QGS（20、30、40、60、80 mg·L

-1

）干预TE-1细胞12、24、36、48、60 h后，细胞活性抑制率明显增加，差异有统计学意义（

0.05），抑制率呈时间和浓度依赖性。与空白组比较，TGF-

组细胞变长，呈成纤维细胞表型。与TGF-

组细胞比较，TGF-

+QGS组细胞变短，形态恢复正常，呈上皮细胞表型；TGF-

+SB431542组细胞形态与TGF-

+QGS组相似。与空白组比较，TGF-

组细胞的迁移和侵袭能力增强，差异有统计学意义（

0.05）。与TGF-

组比较，TGF-

+QGS组和TGF-

+SB431542组细胞迁移和侵袭能力受到抑制而减弱，差异有统计学意义（

0.05）。但TGF-

+QGS组和TGF-

+SB431542组之间的迁移和侵袭能力差异无统计学意义。与空白组比较，TGF-

组Vimentin和Smad2 mRNA的表达水平升高（

0.05），E-Cadherin和Smad7 mRNA表达水平降低，差异有统计学意义（

0.05）。与TGF-

组比较，TGF-

+QGS组和TGF-

+SB431542组的Vimentin和Smad2 mRNA表达水平降低，差异有统计学意义（

0.05），E-Cadherin和Smad7 mRNA的表达水平升高（

0.05）。与空白组比较，TGF-

组Vimentin、p-Smad2/3和Smad2/3的蛋白表达水平升高，差异有统计学意义（

0.05），E-Cadherin和Smad7蛋白表达水平降低，差异有统计学意义（

0.05）。与TGF-

组比较，TGF-

+QGS组和TGF-

+SB431542组的Vimentin、p-Smad2/3和Smad2/3蛋白表达水平降低，差异有统计学意义（

0.05），E-Cadherin和Smad7蛋白表达水平升高，差异有统计学意义（

0.05）。

结论

QGS乙酸乙酯提取物通过TGF-

途径抑制TE-1细胞的上皮-间质转化过程，减少TE-1细胞的迁移和侵袭。

Abstract

Objective

To explore the mechanism of Qigesan （QGS） in intervening in the migration and invasion of esophageal carcinoma TE-1 cells.

Method

Microarray technology was used to screen differentially expressed genes （DEGs） in the normal group and the QGS group， and the ontological functions and signaling pathways of DEGs were analyzed. The thiazolyl tetrazolium （MTT） assay was used to detect the effect of QGS on the viability of TE-1 cells. In the subsequent experiments for verification， a blank group， a transforming growth factor-

（TGF-

） group， a TGF-

+ QGS group， and a TGF-

+ SB431542 group were set up. The cell morphology in each experimental group was observed by microscopy. The migration and invasion abilities of cells were detected by wound healing assay， and the mRNA expression levels of E-Cadherin， vimentin， Smad2， and Smad7 were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. The protein expression of E-Cadherin， vimentin， p-Smad2/3， Smad2/3， and Smad7 was detected by Western blot.

Result

There were 1 487 DEGs between the QGS group and the blank group， including 1 080 down-regulated ones （accounting for 72.63%） and 407 up-regulated ones. The down-regulated genes were mainly involved in biological processes such as cytoskeletal protein binding， ATP binding， adenylate nucleotide binding， and adenylate ribonucleotide binding， and the involved Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways included TGF-

signaling pathway， cell cycle， extracellular matrix-receptor interaction protein， tumor pathways， and oocyte meiosis. The up-regulated genes were mainly involved in RNA binding， DNA binding， transcriptional regulator activity， transcriptional activator activity， and nucleotide binding， and the KEGG pathways involved mainly included mitogen-activated protein kinase （MAPK） signaling pathway， bladder cancer， renal cell carcinoma， cancer pathways， and p53 signaling pathway. Compared with the blank group， the inhibition rate of cell viability of TE-1 cells increased after QGS （20， 30， 40， 60， 80 mg·L

-1

） intervention for 12， 24， 36， 48， 60 h （

0.05）， and the inhibition rate was time- and dose-dependent. Compared with the blank group， the TGF-

group showed lengthened cells with fibroblast phenotype. Compared with the TGF-

group， the TGF-

+ QGS group showed shortened cells with normal morphology and epithelial phenotype. The cell morphology in the TGF-

+ SB431542 group was similar to that of the TGF-

+ QGS group. Compared with the blank group， the TGF-

group showed potentiated ability of cell migration and invasion （

0.05）. Compared with the TGF-

group， the TGF-

+ QGS group and the TGF-

+ SB431542 group showed inhibited and weakened migration and invasion abilities of cells （

0.05）. However， there was no significant difference in migration and invasion abilities between the TGF-

+ QGS group and the TGF-

+ SB431542 group. The mRNA expression levels of vimentin and Smad2 in the TGF-

group were higher （

0.05）， and the mRNA expression levels of E-Cadherin and Smad7 were lower （

0.05） than those in the blank group. Compared with the TGF-

group， the TGF-

+ QGS group and the TGF-

+ SB431542 group exhibited decreased expression levels of vimentin and Smad2 mRNA （

0.05）， and elevated expression levels of E-Cadherin and Smad7 mRNA （

0.05）. Compared with the blank group， the TGF-

group showed up-regulated protein expression levels of vimentin， p-Smad2/3， and Smad2/3 （

0.05）， and reduced protein expression levels of E-Cadherin and Smad7 （

0.05）. Compared with the TGF-

group， the TGF-

+ QGS group and the TGF-

+ SB431542 group displayed decreased protein expression levels of vimentin， p-Smad2/3， and Smad2/3 （

0.05）， and increased protein expression levels of E-Cadherin and Smad7 （

0.05）.

Conclusion

The ethyl acetate extract of QGS inhibits the epithelial-mesenchymal transition （EMT） of TE-1 cells through the TGF-

pathway to reduce the migration and invasion of TE-1 cells.

关键词

食管癌上皮间充质转化迁移侵袭启膈散转化生长因子-β1（TGF-β1）信号通路

Keywords

esophageal cancerepithelial-mesenchymal transitionmigration， invasionQigesantransforming growth factor-β1 （TGF-β1） signaling pathway

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Ethyl Acetate Extract of Qigesan Intervenes in Migration and Invasion of Esophageal Cancer Cells via Inhibiting TGF-β1 Signaling Pathway

DOI：10.13422/j.cnki.syfjx.202202322

摘要

Abstract

关键词

Keywords

references

Ethyl Acetate Extract of Qigesan Intervenes in Migration and Invasion of Esophageal Cancer Cells via Inhibiting TGF-β₁ Signaling Pathway