Construction of GSTZ1 recombinant adenovirus and its effect on migration of hepatocarcinoma cells

Yang Yi; Li Jingjing; Liang Li; Gao Qingzhu; Zheng Yaqiu; Pan E; Wang Kai; Tang Ni; Shan Youlan

doi:10.13406/j.cnki.cyxb.001314

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Construction of GSTZ1 recombinant adenovirus and its effect on migration of hepatocarcinoma cells

更新时间：2024-01-17

- Construction of GSTZ1 recombinant adenovirus and its effect on migration of hepatocarcinoma cells
- Journal of Chongqing Medical University Vol. 42, Issue 7, Pages: 821-827(2017)
- 作者机构：
  
  重庆医科大学附属第二医院传染科重庆医科大学感染性疾病分子生物学教育部重点实验室
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.001314
  CLC： R735.7
- Published：2017，
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Yang Yi, Li Jingjing, Liang Li, et al. Construction of GSTZ1 recombinant adenovirus and its effect on migration of hepatocarcinoma cells. [J]. Journal of Chongqing Medical University 42(7):821-827(2017)
DOI：

Yang Yi, Li Jingjing, Liang Li, et al. Construction of GSTZ1 recombinant adenovirus and its effect on migration of hepatocarcinoma cells. [J]. Journal of Chongqing Medical University 42(7):821-827(2017) DOI： 10.13406/j.cnki.cyxb.001314.

摘要

目的:构建人谷胱甘肽-S-转移酶Z1(glutathione S-transferase zeta 1

GSTZ1)重组腺病毒

并初步研究GSTZ1调控粘附连接信号通路(adhesion junction pathway)靶基因SMAD3、IGF1R等影响肝癌细胞的迁移能力。方法:PCR扩增GSTZ1 cDNA序列并克隆到腺病毒载体pAd Track-TO4

构建重组腺病毒质粒p Ad Track-GSTZ1

将重组质粒用Lipofectamine 2000转染至HEK293细胞中

包装、扩增成高滴度重组腺病毒AdGSTZ1。首先

将SMMC-7721细胞设为3组:Mock组、AdGFP组和AdGSTZ1组

Mock组为空白对照组

AdGFP组为感染腺病毒AdGFP的对照组

AdGSTZ1组为感染腺病毒AdGSTZ1的实验组。然后

通过Transwell实验和MTS实验观察其对肝癌细胞迁移增殖能力的影响

进一步经qRT-PCR及Western blot技术研究GSTZ1通过调控粘附连接信号通路关键分子影响肝癌细胞的迁移。结果:成功构建了高滴度重组腺病毒AdGSTZ1

经Western blot鉴定GSTZ1在SMMC-7721细胞中的表达。Transwell实验结果表明

AdGSTZ1组与Mock组及AdGFP组相比

能够明显抑制人肝癌细胞的迁移能力;与Mock组相比

抑制率为(68.10±1.63)%(P=0.000)

与AdGFP组相比

抑制率为(50.70±0.99)%(P=0.000)。MTS实验结果表明

AdGSTZ1组在72 h和96 h的吸光度值分别为(1.13±0.08)和(1.28±0.07)

AdGFP组72 h和96 h的吸光度值分别为(1.28±0.03)和(1.45±0.03)

AdGSTZ1组在96 h的吸光度值与对照组相比明显降低

差异有统计学意义(P<0.01)。另外q RT-PCR及Western blot结果显示

与Mock组和AdGFP组相比

AdGSTZ1感染细胞内粘附连接信号通路中靶基因SMAD3、IGF1R等的表达明显降低。q RT-PCR结果显示

AdGSTZ1组与AdGFP组相比

TGFBR1相对表达量为(0.58±0.13)(t=4.609

P=0.010)

SMAD3相对表达量为(0.51±0.08)(t=8.030

P=0.000)

ERBB2相对表达量为(0.46±0.09)(t=9.384

P=0.000)

IGF1R相对表达量为(0.45±0.02)(t=14.480

P=0.000)

FARP2相对表达量为(0.37±0.13)(t=7.082

P=0.028)。Western blot表明

SMAD3组中

AdGFP组的蛋白相对表达量为(0.96±0.18)

AdGSTZ1实验组的蛋白相对表达量(0.39±0.01)(P=0.001)。IGF1R组中

AdGFP组的蛋白相对表达量为(0.97±0.05)

AdGSTZ1实验组的蛋白相对表达量(0.44±0.02)(P=0.000)。结论:GSTZ1通过调控粘附连接信号通路相关靶基因的表达抑制肝癌细胞的迁移。

Abstract

Objective:To observe the effects of Glutathione S-transferase Zeta 1（GSTZ1）gene on cell migration through regulating target genes of adhesion junction signal pathway such as SMAD3 and IGF1R. Methods:The GSTZ1 cDNA sequence was amplified and cloned into adenoviral plasmid p Ad Track-TO4. Recombinant adenovirus plasmid pAd Track-GSTZ1 was transfected into HEK-293 cells to package and generate high titer recombinant adenoviruses encoding GSTZ1（Ad-GSTZ1）. First

the SMMC-7721 cells were set into three groups

which Mock group as blank control group

AdGFP group as control group infected with adenovirus AdGFP

AdGSTZ1 group as experimental group infected with adenovirus AdGSTZ1. The effect on the migration of hepatocarcinoma cells was observed by Transwell assay

the effect on the proliferation was observed by MTS assay. Further

genes in adhesion junction signal pathway were analyzed by qRT-PCR and Western blot. Results:A high-titer recombinant adenovirus AdGSTZ1 was successfully constructed. Western blot showed GSTZ1 overexpression in AdGSTZ1 infected cells. GSTZ1 inhibited the migration of hepatocarcinoma cells significantly compared with Mock and AdGFP control group. The inhibition rate of AdGSTZ1 group was（68.10±1.63）%（P=0.000）and（50.7±0.99）%（P=0.000）compared with Mock group and AdGFP control respectively. MTS assay showed that the absorbance values at 490 nm were decreased at 96 h compared with AdGFP. In addition

q RT-PCR and Western blot showed that the expressions of genes in adhesion junction signal pathway such as SMAD3

IGF1 R were significantly decreased in AdGSTZ1 infected cells than in Mock and AdGFP infected cells. q RT-PCR showed that compared with those of AdGFP control group

in AdGSTZ1 group the relative expression of TGFBR1 was（0.58±0.13）（t=4.609

P=0.010）

the relative expression of SMAD3 was（0.51 ±0.08）（t =8.030

P =0.000）

the relative expression of ERBB2 was（0.46±0.09）（t=9.384

P=0.000）

the relative expression of IGF1 R was（0.45±0.02）（t=14.480

P=0.000）

the relative expression of FARP2 was（0.37±0.13）（t=7.082

P=0.028）. Western blot showed that in SMAD3 group

the relative quantity of protein expression was（0.96±0.18）and（0.39±0.01）（P=0.001）of AdGFP and AdGSTZ1 respectively. In IGF1 R group

the relative quantity of protein expression was（0.97±0.05）and（0.44±0.02）（P=0.000）of AdGFP and AdGSTZ1 respectively. Conclusion:GSTZ1 inhibited the migration of hepatocarcinoma cells through regulating the expression of target genes in adhesion junction pathway.

关键词

GSTZ1SMMC-7721细胞粘附连接信号通路肝癌细胞迁移

Keywords

GSTZ1SMMC-7721 celladhesion junctionhepatocellular carcinomacell migration

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