分子伴侣自噬通过缓解G0/G1期阻滞维持细胞周期蛋白表达促进HER2阳性乳腺癌SK-BR-3细胞卡铂耐药

李智群; 朱元超; 尹伊; 代姣; 陈莎

doi:10.16016/j.2097-0927.202607067

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分子伴侣自噬通过缓解G0/G1期阻滞维持细胞周期蛋白表达促进HER2阳性乳腺癌SK-BR-3细胞卡铂耐药

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- 分子伴侣自噬通过缓解G0/G1期阻滞维持细胞周期蛋白表达促进HER2阳性乳腺癌SK-BR-3细胞卡铂耐药
- Chaperone-mediated autophagy promotes carboplatin resistance in HER2-positive breast cancer SK-BR-3 cells by alleviating G0/G1 arrest and maintaining cell cycle protein expression
- 陆军军医大学学报 2026年48卷第18期页码：2593-2603
- 作者机构：
  
  重庆医科大学药学院重庆高校生物化学与分子药理学重点实验室药理学系，重庆
- 作者简介：
  
  陈莎，E-mail：191037@cqmu.edu.cn
- 基金信息：
  
  重庆市教委科研项目(KJQN202200458);中国博士后科学基金(2022MD713710)
- DOI：10.16016/j.2097-0927.202607067
  中图分类号： R737.9;R915;R979.19
- 收稿：2026-07-16，
  
  修回：2026-08-07，
  
  录用：2026-08-18，
  
  纸质出版：2026-09-30
- 稿件说明：
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李智群，朱元超，尹伊，等.分子伴侣自噬通过缓解G0/G1期阻滞维持细胞周期蛋白表达促进HER2阳性乳腺癌SK-BR-3细胞卡铂耐药［J］.陆军军医大学学报，2026，48（18）：2593-2603.DOI：10.16016/j.2097-0927.202607067.

Li Z Q，Zhu Y C，Yin Y，et al.Chaperone-mediated autophagy promotes carboplatin resistance in HER2-positive breast cancer SK-BR-3 cells by alleviating G0/G1 arrest and maintaining cell cycle protein expression［J］.J Army Med Univ，2026，48（18）：2593-2603.DOI：10.16016/j.2097-0927.202607067.
李智群，朱元超，尹伊，等.分子伴侣自噬通过缓解G0/G1期阻滞维持细胞周期蛋白表达促进HER2阳性乳腺癌SK-BR-3细胞卡铂耐药［J］.陆军军医大学学报，2026，48（18）：2593-2603.DOI：10.16016/j.2097-0927.202607067. DOI：

Li Z Q，Zhu Y C，Yin Y，et al.Chaperone-mediated autophagy promotes carboplatin resistance in HER2-positive breast cancer SK-BR-3 cells by alleviating G0/G1 arrest and maintaining cell cycle protein expression［J］.J Army Med Univ，2026，48（18）：2593-2603.DOI：10.16016/j.2097-0927.202607067. DOI：

摘要

目的

卡铂可通过诱导DNA损伤和细胞周期阻滞来抑制肿瘤细胞增殖，而细胞周期异常调控可能参与卡铂耐药，但具体调控机制尚待进一步阐明。本研究旨在探讨分子伴侣自噬（chaperone-mediated autophagy，CMA）对人表皮生长因子受体2（human epidermal growth factor receptor 2， HER2）阳性乳腺癌SK-BR-3细胞卡铂耐药的影响及其与细胞周期调控的关系。

方法

①慢病毒感染构建CMA活性不同的SK-BR-3细胞株，通过荧光显微镜观察感染荧光，Western blot检测溶酶体相关膜蛋白2A（lysosome-associated membrane protein 2A，LAMP2A）表达，并结合LysoTracker Red免疫荧光评价CMA活性。②采用MTT法检测不同浓度卡铂处理后的细胞活力并确定半数抑制浓度（half-maximal inhibitory concentration

）；根据IC

测定结果，以20 μmol/L卡铂连续处理各组细胞7 d，检测细胞增殖变化，并观察不同浓度卡铂处理后细胞密度及贴壁状态。③对高CMA活性组及其对照组进行RNA-seq测序，将差异表达基因（differentially expressed genes，DEGs）与细胞周期相关基因取交集，并进行GO和KEGG富集分析。④流式细胞术检测卡铂处理后各组细胞周期分布，Western blot检测CDK4、CyclinD1和CyclinE1的表达水平。

结果

①成功构建CMA活性差异的SK-BR-3细胞模型。与shControl组相比，shLAMP2A组LAMP2A蛋白表达降低（

=0.000 3），LysoTracker Red荧光强度降低（

0.000 1）；与Control vector组相比，pCMV6-LAMP2A组LAMP2A蛋白表达升高（

0.000 1），LysoTracker Red荧光强度升高（

=0.000 2）。②MTT结果显示，卡铂对SK-BR-3细胞的IC

约为20 μmol/L；20 μmol/L卡铂连续处理7 d后，shLAMP2A组细胞增殖水平低于shControl组，pCMV6-LAMP2A组细胞增殖水平高于Control vector组（

0.000 1）；随着卡铂浓度升高，各组贴壁细胞数量总体减少，shLAMP2A组较shControl组减少更明显，而pCMV

6-LAMP2A组较Control vector组则保留较多贴壁细胞。③RNA-seq及富集分析显示，高CMA活性相关差异基因主要富集于有丝分裂细胞周期转变、CDK复合物、DNA解旋酶活性及细胞周期相关通路。④流式细胞术和Western blot结果进一步显示，与shControl组相比，shLAMP2A组G0/G1期细胞比例增加（

=0.000 4），S期细胞比例降低（

=0.024 3）；与Control vector组相比，pCMV6-LAMP2A组G0/G1期细胞比例降低（

0.000 1），S期细胞比例升高（

=0.000 3）。shLAMP2A组CDK4、CyclinD1和CyclinE1表达较shControl组降低，而pCMV6-LAMP2A组较Control vector组升高（

0.000 1 ）。

结论

CMA活性增强可减弱卡铂对SK-BR-3细胞的增殖抑制作用，提高细胞在卡铂压力下的存活和增殖能力；其机制可能与缓解G0/G1期阻滞、维持CDK4、CyclinD1和CyclinE1表达及促进G1/S期转换有关。CMA介导的细胞周期调控可能参与SK-BR-3细胞卡铂耐药。

Abstract

Objective

Carboplatin inhibits tumor cell proliferation by inducing DNA damage and cell cycle arrest. Whereas abnormal regulation of the cell cycle may be involved in carboplatin resistance

the underlying regulatory mechanisms remain to be further elucidated. This study aims to investigate the effect of chaperone-mediated autophagy (CMA) on carboplatin resistance in HER2-positive breast cancer SK-BR-3 cells and its relationship with cell cycle regulation.

Methods

① SK-BR-3 cell lines with different levels of CMA activity were established by lentiviral transduction. Transduction-associated fluorescence was observed by fluorescence microscopy

LAMP2A protein expression was detected by Western blotting

and CMA activity was evaluated in combination with LysoTracker immunofluorescence. ② Cell viability after treatment with different concentrations of carboplatin was assessed using MTT assay to determine the half-maximal inhibitory concentration (IC

). Based on the IC

value

the cells in each group were continuously treated with 20 μmol/L carboplatin for 7 d to evaluate changes in cell proliferation

and cell density and attachment status were observed after treatment with different concentrations of carboplatin. ③ RNA sequencing (RNA-seq) was perfo

rmed on the high-CMA-activity group and its corresponding control group. Differentially expressed genes (DEGs) were intersected with cell cycle-related genes

followed by GO and KEGG enrichment analyses. ④ Cell cycle distribution after carboplatin treatment was analyzed by flow cytometry

and the expression levels of CDK4

CyclinD1

and CyclinE1 were detected by Western blotting.

Results

① SK-BR-3 cell models with different levels of CMA activity were successfully established. Compared with the shControl group

the shLAMP2A group showed significantly decreased LAMP2A protein expression (

=0.000 3) and decreased LysoTracker Red fluorescence intensity (

0.000 1). Compared with the Control vector group

the pCMV6-LAMP2A group showed increased LAMP2A protein expression (

0.000 1) and enhanced LysoTracker Red fluorescence intensity (

=0.000 2). ② MTT assay indicated that the IC

value of carboplatin against SK-BR-3 cells was approximately 20 μmol/L. After continuous treatment with 20 μmol/L carboplatin for 7 d

cell proliferation was lower in the shLAMP2A group than the shControl group

whereas it was higher in the pCMV6-LAMP2A group than in the Control vector group (

0.000 1). With the increasing of the carboplatin concentration

the number of adherent cells was generally decreased in all groups; this decrease was more pronounced in the shLAMP2A group than in the shControl group

whereas more adherent cells were retained in the pCMV6-LAMP2A group. ③ RNA-seq and enrichment analyses showed that DEGs associated with high CMA activity were mainly enriched in mitotic cell cycle transition

CDK complexes

DNA helicase activity

and cell cycle-related pathways. ④ Flow cytometry and Western blotting further revealed that

compared with the shControl group

the proportion of cells in G0/G1 phase was increased (

=0.000 4) and that in S phase was decreased (

=0.024 3) in the shLAMP2A group. Compared with the Control vector group

the pCMV6-LAMP2A group showed a decreased proportion of cells in G0/G1 phase (

0.000 1) and an increased proportion of cells in S phase (

=0.000 3). The expression levels of CDK4

CyclinD1

and CyclinE1 were notably lower in the shLAMP2A group than the shControl group

whereas that in the pCMV6-LAMP2A group was higher than that in the Control vector group (

0.000 1)

Conclusion

Enhanced CMA activity attenuates the inhibitory effect of carboplatin on SK-BR-3 cell proliferation and improves cell survival and proliferative capacity under carboplatin stress. The mechanism may be related to enhanced CMA activity alleviating G0/G1 phase arrest

maintaining CDK4

CyclinD1

and CyclinE1 expression

and promoting G1/S phase transition. CMA-mediated regulation of cell cycle may be involved in carboplatin resistance in SK-BR-3 cells.

关键词

Keywords

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