Silent information regulator 3 modulates lysosome function to inhibit apoptosis of U87 cell by mTOR pathway

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Silent information regulator 3 modulates lysosome function to inhibit apoptosis of U87 cell by mTOR pathway

Basic Medicine | 更新时间：2026-01-09

- Silent information regulator 3 modulates lysosome function to inhibit apoptosis of U87 cell by mTOR pathway
- Journal of Army Medical University Vol. 45, Issue 18, Pages: 1916-1926(2023)
- 作者机构：
  
  1.710021 西安，西安医学院临床医学院：中尼友好拉吉姆医学实验室
  2.710021 西安，西安市肿瘤早诊创新转化重点实验室
  3.710021 西安，陕西省呼吸病预防与诊治工程研究中心
  4.710077 西安，西安医学院第一附属医院神经内科
- 作者简介：
- 基金信息：
  
  the Young Talents Support Project of Shaanxi Provincial Colleges and Universities and Commission of Science and Technology(20200314);the General Program of Shaanxi Provincial Department of Science and Technology(2021JM-495);the Special Research Project of Shaanxi Provincial Department of Education(21JK0890);the Innovation and Entrepreneurship Training Program for College Students(S202111840075)
- DOI：10.16016/j.2097-0927.202306039
  CLC： R394.3;R730.23;R739.41
- Received：13 June 2023，
  
  Revised：2023-07-12，
  
  Published：30 September 2023
- 稿件说明：
移动端阅览
Xudong ZHANG, Litianyi SHAN, Rong WANG, et al. Silent information regulator 3 modulates lysosome function to inhibit apoptosis of U87 cell by mTOR pathway[J]. Journal of Army Medical University, 2023, 45(18): 1916-1926.
DOI：

Xudong ZHANG, Litianyi SHAN, Rong WANG, et al. Silent information regulator 3 modulates lysosome function to inhibit apoptosis of U87 cell by mTOR pathway[J]. Journal of Army Medical University, 2023, 45(18): 1916-1926. DOI： 10.16016/j.2097-0927.202306039.

摘要

目的

2

探讨沉默调节蛋白3(sirtuin 3，Sirt3)在胶质瘤细胞U87中的作用，为胶质瘤的早期诊治提供新靶点。

方法

2

基于中国脑胶质瘤数据库(CGGA)、GEPIA2数据库，通过生物信息学分析Sirt3在胶质瘤不同分级的表达情况，通过Western blot检测6位胶质瘤患者癌旁与肿瘤组织中Sirt3的蛋白水平。利用shRNA慢病毒、CRISPR/Cas9系统干预U87细胞Sirt3的表达，采用TUNEL染色、PI-Annexin V双荧光标记检测细胞凋亡情况，Western blot检测凋亡相关蛋白Bax、Cleaved-caspase3及LAMP1、LAMP2、LAMP2A、TFEB、mTOR等蛋白的表达。利用mTOR小分子抑制剂Rapamycin处理Sirt3敲减U87细胞，检测相关蛋白及细胞凋亡的变化。对Sirt3敲除前后的U87细胞进行核质分离实验，并检测p-TFEB蛋白表达的变化。在敲减及敲除Sirt3的U87细胞中分别过表达TFEB及Sirt3后，检测溶酶体膜蛋白表达的变化情况。

结果

2

Sirt3在高级别胶质瘤中高表达，且与患者的低生存率密切相关。敲减Sirt3表达后细胞凋亡增加(

P

<

0.01)，溶酶体膜蛋白LAMP1、LAMP2、LAMP2A显著下调(

P

<

0.01)，p-mTOR显著增加(

P

<

0.01)，通过核质分离实验发现敲除Sirt3后p-TFEB在胞质中增加(

P

<

0.01)。在Sirt3敲除组过表达Sirt3后，LAMP1、LAMP2蛋白水平恢复，且溶酶体数量显著增加(

P

<

0.01)；在Sirt3敲减组过表达TFEB后，LAMPs蛋白表达明显上调(

P

<

0.01)。

结论

2

Sirt3可能通过调控mTOR信号通路影响TFEB的核质分布，上调溶酶体膜蛋白LAMPs表达增强胶质瘤细胞溶酶体功能，进而促进胶质瘤细胞存活和恶性增殖，提示Sirt3可能是胶质瘤临床诊断和进展的分子标志物。

Abstract

Objective

2

To investigate the role of silent information regulator 3 (Sirt3) in glioma U87 cells

and provide new targets for early diagnosis and treatment of glioma.

Methods

2

Based on the China Brain Glioma Database (CGGA) and GEPIA2 database

the expression of Sirt3 in different grades of glioma was analyzed by bioinformatics

and the protein level of Sirt3 in the adjacent and tumor samples of 6 glioma patients was further detected by Western blotting. Then

shRNA lentivirus and CRISPR/Cas9 system were used to interfere with the expression of Sirt3 in U87 cells

and TUNEL staining

PI-Annexin V double fluorescent labeling and Western blotting were used to detect the expression of apoptosis-related proteins(Bax

Cleaved-caspase3) and lysosome membrane proteins (LAMP1

LAMP2

LAMP2A)

TFEB

mTOR

and other proteins. Then mTOR small molecule inhibitor

rapamycin was employed to treat Sirt3 knockdown cells

the changes in related proteins and cell apoptosis were detected. In addition

nuclear and cytoplasmic separation experiments were carried out on U87 cells before and after Sirt3 knockout

and protein change of p-TFEB was detected. Finally

the change of lysosome membrane protein after overexpression of TFEB and Sirt3 was detected in U87 cells with knocking down and out of Sirt3.

Results

2

Sirt3 was significantly highly expressed in glioma tissue

and the level was closely associated with poor survival of the glioma patients. Sirt3 knockdown significantly enhanced cell apoptosis (

P

<

0.01)

down-regulated the expression of LAMP1

LAMP2 and LAMP2A (

P

<

0.01)

and improved the expression of p-mTOR (

P

<

0.01). Additionally

Sirt3 knockout also

up-regulated p-TFEB protein level in the cytoplasm fraction (

P

<

0.01) while

in the Sirt3 knockout U87 cells

its overexpression reserved the protein levels of LAMP1 and LAMP2 and increased the amount of lysosome. In the Sirt3 knockdown cells

overexpression of TFEB resulted in up-regulation of LAMPs proteins(

P

<

0.01).

Conclusion

2

Sirt3 may affect the nuclear-cytoplasmic distribution of TFEB by regulating the mTOR signaling pathway

up-regulate the expression of LAMPs

enhance the function of lysosome

and then promote the survival and malignant proliferation of glioma cells. Our study suggests that Sirt3 may be a molecular marker for clinical diagnosis and progression of glioma.

关键词

Keywords

references

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