PUF60 Promotes Chemoresistance Through Drug Efflux and Reducing Apoptosis in Gastric Cancer

Abstract

Background: Chemotherapy resistance is a great challenge in the treatment of gastric cancer (GC), so it is urgent to explore the prognostic markers of chemoresistance. PUF60 (Poly (U)-binding splicing factor 60) is a nucleic acid-binding protein that has been shown to regulate transcription and link to tumorigenesis in various cancers. However, its biological role and function in chemotherapy resistance of GC is unclear. Methods: The expression and prognostic value of PUF60 in GC chemotherapy-resistant patients were analyzed by databases and K-M Plotter. The functional effect of PUF60 on chemoresistance in GC was studied by by RNA interference, CCK8 test, colony formation test and apoptosis detection. Moreover, further validation and mechanism exploration were conducted in clinical samples. Results: PUF60 was highly expressed in both GC and chemoresistant tissues, and was positively correlated with poor prognosis in GC patients treated with 5-fluorouracil (5-FU). In addition, the knockdown of PUF60 significantly reduced the proliferation of human gastric cancer cells and increased sensitivity to chemotherapy drugs, such as 5-FU and cisplatin (CDDP). Mechanistically, PUF60 enhances chemotherapy resistance in gastric cancer (GC) cells by actively excluding chemotherapy drugs via the recombinant ATP Binding Cassette Transporter A1 (ABCA1) and ATP Binding Cassette Subfamily C Member 1 (ABCC1). This process further affects the cell cycle, reduces cell apoptosis, and ultimately promotes resistance to chemotherapy in GC. Conclusion: PUF60 promotes chemoresistance in GC, resulting in poor prognosis of GC patients treated with 5-FU, and providing a new idea for overcoming the chemoresistance in GC.

Keywords: Chemoresistance; Gastric cancer; PUF60; Prognostic biomarkers.

Figure 1

PUF60 expression was elevated and related to poor prognosis in GC Patients treated with 5-FU-based Chemotherapy. (A) Volcano plots of the differentially expressed genes (DEGs) screened by GEO2R in GSE14210. (B) The top 10 hub genes of above upregulated DEGs by MCC degree. (C) The mRNA expression level of 10 hub genes in the two groups. (D) Prognostic value of PUF60 in GC patients based on 5-FU chemotherapy by Kaplan-Meier plotter analysis. OS: overall survival, FP: first progression, PPS: post progression survival. According to the degree of chemotherapy response of the patients, they were divided into non-sensitive group (n=6) and sensitive group (n=7). The 13 GC patients here had paired tumor and normal tissues. ns, p≥ 0.05, *p < 0.05.

Figure 2

Expression level of PUF60 in GC by databases and experimental validation. (A) The expression level of PUF60 in pan-tumor cell lines analyzed by the CCLE database. (B-C) Expression level of PUF60 in unpaired or paired GC and normal tissues analyzed by TCGA database; (D-F) Expression level of PUF60 in GC and normal tissues analyzed by GEO database; (G) Comparison of the mRNA levels of PUF60 in paired GC samples. (H) Representative IF images of PUF60 from GC samples in gastric tumor (T) and adjacent normal tissues (NAT) by IF. **p < 0.01, ***p < 0.001.

Figure 3

Expression level of PUF60 in different chemotherapy sensitivity groups. (A-B) The protein levels and corresponding bar graphs of PUF60 expression in GC tissues of the three groups. (C) The mRNA levels of PUF60 in GC tissues of the three groups; (D-E) Representative IHC images of PUF60 from GC samples and corresponding bar graphs. The groups are classified according to the degree of chemotherapy sensitivity, namely, PD: disease progression. SD: stable disease. PR: partial response. ns, p≥ 0.05, **p < 0.01, ****p < 0.0001.

Figure 4

Knockdown of PUF60 in human gastric cancer cell line HGC-27 and its drug sensitivity to 5-FU and CDDP. (A) Expression level of PUF60 in different human gastric cancer cell lines. (B) Interference efficiency verification of PUF60 in HGC-27 cells (Ctrl, sh1, sh2). Left: western blot gels. Right: protein quantification, interference group values were compared with the control group. (C) The mRNA quantification in HGC-27; (D) Relative cell viability of HGC-27 cells expressing shNC and shPUF60 treated with different concentrations of CDDP and 5-FU by CCK 8 experiment. (E) Flow cytometry for detection of apoptosis by Annexin/7-AAD double staining in HGC-27 cells expressing shNC and shPUF60. Data are presented as the means±SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 5

Low expression of PUF60 decreased the proliferative capacity and colony formation of HGC-27 cells expressing shNC (Ctrl) and shPUF60 (sh1, sh2). There are representative images of macroscopic and microscopic observation and relevant bar graphs for quantification. (A) Without drug treatment. (B) 5-FU treatment (0.5uM, 1uM, 1.5uM). (C) CDDP treatment (0.25uM, 0.5uM, 1 uM). Data are presented as the means±SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

Mechanisms Underlying PUF60's Role in Enhancing Chemoresistance in GC. (A) Functional Enrichment Analysis: This panel presents the functional enrichment analysis of PUF60 in GC, suggesting its potential roles and interactions within the cellular context. (B) mRNA Expression Levels of ABC Protein Transporters: The expression levels of various ABC protein transporters (ABCA1, ABCC1, ABCC2, ABCG2) are shown. These data indicate how PUF60 may regulate the expression of these transporters, thereby affecting drug resistance. (C) mRNA Expression Levels of Apoptosis-Related Factors: The expression levels of apoptosis-related factors (caspase-3/cas3, caspase-9/cas9, p53) are presented. These factors are crucial for cancer cell death and their regulation by PUF60 may contribute to chemoresistance. (D-E) mRNA Expression Levels of Cell Cycle Factors: The expression levels of cell cycle factors, including all cyclin-dependent kinases and cyclin family members, are displayed. These data illuminate how PUF60 may disturb the normal cell cycle progression, thereby promoting chemoresistance. (F) mRNA Expression Levels of FEN 1 and Associated m6A Methylation Genes: The expression levels of FEN 1 and its associated m6A methylation genes (FTO, METTL14, METTL3, WTAP, YTHPF1, YTHPF2, YTHPF3) are shown. These data hint at a potential mechanism through which PUF60 may regulate DNA repair and replication processes, contributing to chemoresistance. Data are presented as the means±SEM. *p < 0.05, **p < 0.01.