Label-free quantitative proteomic study of RUNX3 regulating Herceptin resistance in gastric cancer cells
10.16438/j.0513-4870.2021-0376
- VernacularTitle:RUNX3调控胃癌细胞对赫赛汀耐药的非标记定量蛋白质组学研究
- Author:
Jin-xia CHANG
1
;
Shi-bao WANG
2
;
Jiang-bei YUAN
3
;
Wen-hu LIU
1
Author Information
1. School of Basic Medical Sciences, Department of Pharmacy, North Sichuan Medical College, Nanchong 637100, China
2. Department of Pharmacy, Hanzhong Vocation and Technology College, Hanzhong 723000, China
3. Peking University Shenzhen Hospital, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center, Shenzhen 518036, China
- Publication Type:Research Article
- Keywords:
Runt related transcription factor 3;
Herceptin;
resistance;
label-free quantitative proteomics;
autophagy;
apoptosis
- From:
Acta Pharmaceutica Sinica
2021;56(7):1953-1964
- CountryChina
- Language:Chinese
-
Abstract:
Resistance of tumor cells is a complex biological process involving multiple mechanisms and factors, in which anti-apoptosis is the most important cause of drug resistance. Previous studies have shown that the DNA binding activity of Runt related transcription factor 3 (RUNX3) increased prominently in Herceptin resistant gastric cancer cells (NCI N87R) while the relevance of which to drug resistance has not yet been confirmed. In this study, we employed CRISPR/Cas9 to establish RUNX3 knock-out cell line (△RUNX3/NCI N87R) to investigate the functions of RUNX3 in Herceptin resistance of NCI N87R cells and its potential mechanisms. We investigated proteomics profiling of △RUNX3/NCI N87R cells based on label free quantitative proteomics. Differentially expressed proteins were screened out according to fold change and significance level between △RUNX3/NCI N87R and NCI N87R cells. Pathway enrichment analysis was done using GeneAnalytics database, and gene ontology analysis was conducted by DAVID Bioinformatics Resources database. Protein-protein interaction networks were constructed based on STRING database. The results showed that △RUNX3/NCI N87R cells increased the sensitivity to Herceptin. Proteomic data demonstrated that the expression of 577 genes changed significantly in △RUNX3/NCI N87R cells, among which 191 genes were up-regulated while 386 ones down-regulated comparing with NCI N87R cells. Pathway analysis showed that autophagy, cell cycle, apoptosis, mitochondrial fatty acid β oxidation, neurogenic locus notch homolog protein 1 (NOTCH1), mammalian target of rapamycin (mTOR), Hedgehog and DNA damage response pathways exhibited notable changes based on pathway enrichment ratio and significance level (P < 0.05). These results indicated that RUNX3 knock-out altered multiple signaling pathways of NCI N87R cells. Western blotting manifested that the expression of autophagy regulatory molecules autophagy-related protein (ATG) 13, 7 and BECN1 increased remarkably while cell cycle molecules serine/threonine-protein kinase Chk2 (CHEK2) and apoptosis regulator Bcl-2 (BCL2) decreased prominently in △RUNX3/NCI N87R cells. The p-AKT expression decreased significantly in △RUNX3/NCI N87R cells compared with NCI N87R cells (P < 0.01) and was suppressed by Herceptin. These results indicated that RUNX3 knock-out altered cell cycle, increased inhibition to p-AKT by Herceptin, promoted autophagy and induced cell apoptosis of NCI N87R cells. These results suggested that RUNX3 may be a potential therapeutic target for reversing or reducing Herceptin resistance in gastric cancer cells.
