Down-regulation of miR-4443/TIMP2 signaling pathway enhances sorafenib sensitivity in HepG2 cells

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Down-regulation of miR-4443/TIMP2 signaling pathway enhances sorafenib sensitivity in HepG2 cells

Basic Medicine | 更新时间：2026-01-09

- Down-regulation of miR-4443/TIMP2 signaling pathway enhances sorafenib sensitivity in HepG2 cells
- Journal of Third Military Medical University Vol. 43, Issue 3, Pages: 188-194(2021)
- 作者机构：
  
  400038 重庆，陆军军医大学(第三军医大学)基础医学院生物化学与分子生物学教研室
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81572375);the General Program of Natural Science Foundation of Chongqing(CSTC2018jcyjA2018)
- DOI：10.16016/j.1000-5404.202008219
  CLC： R394.6;R735.7;R979.1
- Received：26 August 2020，
  
  Revised：2020-10-29，
  
  Published：15 February 2021
- 稿件说明：
移动端阅览
Hanxi XIAO, Yueting ZHANG, Liangbo SUN, et al. Down-regulation of miR-4443/TIMP2 signaling pathway enhances sorafenib sensitivity in HepG2 cells[J]. Journal of Third Military Medical University, 2021, 43(3): 188-194.
DOI：

Hanxi XIAO, Yueting ZHANG, Liangbo SUN, et al. Down-regulation of miR-4443/TIMP2 signaling pathway enhances sorafenib sensitivity in HepG2 cells[J]. Journal of Third Military Medical University, 2021, 43(3): 188-194. DOI： 10.16016/j.1000-5404.202008219.

摘要

目的

2

探讨下调miR-4443/TIMP2信号通路后肝癌HepG2细胞对索拉非尼敏感性的影响。

方法

2

将HepG2细胞分为对照组(DMSO)与索拉非尼处理组(10 μmol/L)，索拉非尼处理HepG2细胞24 h后，RT-qPCR检测miR-4443的变化；脂质体法将miR-4443模拟物与miR-4443抑制剂转染HepG2细胞，通过CCK-8检测转染miR-4443模拟物或抑制剂后联用索拉非尼对HepG2细胞活性的影响；生物信息学预测miR-4443的下游靶基因，索拉非尼处理24 h后通过RT-qPCR和Western blot分别检测下游靶基因TIMP2 mRNA及其蛋白水平的变化；转染miR-4443模拟物、抑制剂后，RT-qPCR和Western blot分别检测TIMP2 mRNA以及蛋白含量的变化；在HepG2细胞中通过脂质体法转染TIMP2 siRNA，Western blot检测TIMP2蛋白含量；CCK-8检测敲低TIMP2后联用索拉非尼对HepG2细胞活性的影响。

结果

2

与对照组相比，索拉非尼处理组HepG2细胞中miR-4443的含量显著降低(

P

<

0.05)；转染miR-4443模拟物可显著降低索拉非尼对细胞活性的抑制(

P

<

0.05)，而转染其抑制剂则可显著增强索拉非尼的这一作用(

P

<

0.05)；生物信息学预测结果显示miR-4443结合的下游靶基因为TIMP2；索拉非尼处理后HepG2细胞中TIMP2的mRNA及蛋白水平显著升高(

P

<

0.05)；而在HepG2细胞中转染miR-4443的模拟物或抑制剂则可使TIMP2的mRNA表达量相应下降或上升，转染模拟物后TIMP2蛋白表达显著降低(

P

<

0.05)，表明miR-4443与TIMP2间存在调控关系；用TIMP2 siRNA降低TIMP2表达后，可显著增强索拉非尼对HepG2细胞活性的抑制效果(

P

<

0.05)。

结论

2

下调miR-4443/TIMP2信号通路可以增强HepG2细胞对索拉非尼的敏感性。

Abstract

Objective

2

To investigate the significance of down-regulating the miR-4443/TIMP2 pathway on sorafenib sensitivity in HepG2 cells.

Methods

2

HepG2 cells were divided into control group (DMSO) and sorafenib treatment group (10 μmol/L). After treatment for 24 h

the mRNA level of miR-4443 was measured by RT-qPCR. CCK-8 assay was used to study the effects of transfection with miR-4443 mimics or pretreatment of its inhibitor on the cell viability of HepG2 cells treated with sorafenib for 24 h. Bioinformatics analysis was used to predict the downstream target gene of miR-4443. The expression of TIMP2 at mRNA and protein levels was assessed by RT-qPCR and Western blotting in the HepG2 cells treated with sorafenib for 24 h

transfected with miR-4443 mimics or pretreatment of its inhibitor followed by sorafenib treatment. Then TIMP2 siRNA was transfected into HepG2 cells by liposome transfection

the content of TIMP2 protein was detected by Western blotting

and the effect of TIMP2 knockdown combined with sorafenib treatment on the viability of HepG2 cells was measured with CCK-8 assay.

Results

2

Sorafenib treatment decreased the expression of miR-4443 in HepG2 cells (

P

<

0.05). Overexpression of miR-4443 (transfection of miR-4443 mimics) significantly decreased the inhibitory effect of sorafenib on cell viability (

P

<

0.05)

while pretreatment of miR-4443 inhibitor could enhance the effect of the drug (

P

<

0.05). Bioinformatics analysis indicated that the downstream target gene of miR-4443 was TIMP2. The mRNA and protein levels of TIMP2 were obviously increased in HepG2 cells after sorafenib treatment (

P

<

0.05)

but the transfection of miR-4443 mimics or pretreatment of its inhibitor would down- or up-regulate the mRNA level of TIMP2

and the transfection of miR-4443 mimics decreased the protein level of TIMP2

indicating the regulative relationship between the 2 molecules. The transfection of TIMP2 siRNA decreased the expression of TIMP2 and then enhanced the inhibitory effect of sorafenib on HepG2 viability (

P

<

0.05).

Conclusion

2

Down-regulation of the miR-4443/TIMP2 pathway enhances sorafenib sensitivity in HepG2 cells.

关键词

Keywords

references

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