Research ArticleExperimental Studies
Open Access

Circular RNA hsa_circ_0004650 Enhances 5-Fluorouracil Resistance in Gastric Cancer via Sponging miR-145-5p

FUSHENG ZHOU, JIALONG ZHUO, XUDONG XU, DUYI PAN, CHENWEN CAI, JIAXIN HUANG, XIANGUANG ZHAO, QIQI MAO, XIAOYUN JIANG, XU SUN, LIANG ZHONG, NAN GAO and JIAJIE CHEN
Anticancer Research January 2025, 45 (1) 173-187; DOI: https://doi.org/10.21873/anticanres.17403

Abstract

Background/Aim: Chemotherapy based on 5-fluorouracil (5-Fu) is the first-line treatment for advanced gastric cancer (GC) patients. Importantly, 5-Fu resistance is recognized as a major obstacle for the successful treatment of GC. Circular RNAs (circRNAs) are non-coding RNAs involved in the pathogenesis of GC. However, their role in the mechanism of 5-Fu resistance in GC remains largely unknown. The purpose of this study was to explore and elucidate the biological function and molecular mechanism of circRNAs underlying 5-Fu resistance in GC. Materials and Methods: High-throughput sequencing results for intersection analysis were used to select a novel differentially expressed circRNA hsa_circ_0004650. The expression levels of the new circRNA between 5-Fu-sensitive and 5-Fu-resistant GC cells were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), and biological behaviors, such as proliferation and apoptosis of GC cells, were observed after silencing the hsa_circ_0004650. The mechanism of hsa_circ_0004650 sponges miR-145-5p to regulate 5-Fu resistance in GC cells was investigated by luciferase reporter assay, qRT-PCR, CCK-8 assay, Calcein AM/PI double fluorescence staining and flow cytometry. Results: hsa_circ_0004650 was identified as a differentially expressed circRNA between 5-Fu-sensitive GC and 5-Fu-resistant GC cells. Hsa_circ_0004650 was up-regulated in 5-Fu-resistant GC cells. Silencing of hsa_circ_0004650 in 5-Fu-resistant GC cells, the survival rates of cells treated with increasing doses of 5-Fu for 24 h and 48 h were decreased (p<0.01); the mortality rates of SGC-7901-5-Fu cells were increased (17.86%±0.6 vs. 44.86%±1.52; p<0.001), and those of BGC-823-5-Fu cells were increased (8.17%±7.80 vs. 26.61%±1.12; p<0.001); and the apoptosis rates of cells treated with the same concentration of 5-Fu were increased (p<0.001). Mechanistically, miR-145-5p was confirmed as a downstream target of hsa_circ_0004650. By the down-regulation of the expression of miR-145-5p in 5-Fu-resistant GC cells, the survival rates of cells treated with increasing doses of 5-Fu for 24 h and 48 h were increased (p<0.05); the mortality rates of SGC-7901-5-Fu cells were decreased (12.86%±1.10 vs. 7.83%±0.53; p<0.01), those of BGC-823-5-Fu cells were decreased as well (16.99%±1.31 vs. 11.40%±0.72; p<0.01); and the apoptosis rates of cells treated with the same concentration of 5-Fu were decreased (p<0.001). Conclusion: The circRNA hsa_circ_0004650 promotes chemotherapy resistance to 5-Fu in GC cells through sponge adsorption of miR-145-5p, which offers a potential approach to overcome 5-Fu resistance in GC.

Key Words:

Gastric cancer (GC) is a common malignancy in the alimentary tract with high morbidity and high mortality (1). Due to many patients with GC lacking characteristic manifestations and having a low endoscopic acceptance rate in early stages of GC, patient diagnosis unusually occurs at advanced stages (2). For advanced GC patients, the 5-year survival rate is relatively low, ranging from 10% to 30%, which imposes a huge burden on society, families, and individuals (3, 4). The standard treatment for GC is surgical resection of the tumor, combined chemotherapy and local radiotherapy (5). Chemotherapy remains a crucial clinical treatment method for numerous advanced GC patients. Currently, 5-fluorouracil (5-Fu) is a first-line chemotherapeutic agent in the treatment of cancer (6). Moreover, 5-Fu has been approved by the US Federal Drug Administration for the GC treatment (7). Also, 5-Fu has demonstrated high efficacy and safety in clinical application (8). However, GC patients often develop resistance to 5-Fu after several 5-Fu-based treatment cycles, which directly affects the efficacy and prognosis (9). Hence, solving the issue of 5-Fu resistance on clinical practice in GC is urgently needed (10).

Circular RNAs (circRNAs) are a special class of non-coding transcripts that are considered essential regulatory factors in various biological processes in eukaryotes (11). Unlike traditional linear RNAs, circRNAs do not have 5 ‘cap and 3′ poly (A) tail structure. CircRNAs are mainly located in the cytoplasm or stored in exosomes and are much more resistant to degradation by exonucleases (12). Besides, circRNAs have been proposed to implicate in the regulation of biological processes, such as the occurrence, development, invasion, and metastasis of GC (13, 14). In the last years, increasing reports indicated that circRNAs are involved in the regulation of chemotherapy resistance in GC. Yao et al. (15) found that circ-PVT1 expression was up-regulated in the serum of cisplatin resistant GC patients and in the extracellular vesicles of cisplatin resistant GC cells. Moreover, they demonstrated that circ-PVT1 regulated the autophagy, invasion, and apoptosis of GC cells by targeting the miR-30a-5p/YAP1 axis. Notably, there is relatively little research on the regulation of 5-Fu resistance by circRNAs in GC. Recently, Xu et al. (16) have conducted preliminary exploration on the regulation of 5-Fu resistance mechanism of circNRIP1 in GC cells under low oxygen conditions. The study revealed that silencing circNRIP1 enhanced the sensitivity of GC cells to 5-Fu, and miR-138-5p was also confirmed as a downstream target of circNRIP1.

In our previous study, we successfully constructed a 5-Fu-resistant GC cell line (SGC-7901-5-Fu cells) by using a low concentration gradient increment method on 5-Fu-sensitive GC cells (SGC-7901 cells) (17). RNA sequencing (RNA-seq) revealed 31 circRNAs that were differentially expressed in SGC-7901-5-Fu cells, of which, 10 circRNAs were up-regulated and 21 circRNAs were down-regulated. Our preliminary research prompted that circRNAs may be involved in the regulation of 5-Fu resistance in GC. Nevertheless, the specific regulatory mechanisms have not been thoroughly studied.

Herein, we investigated the contribution of hsa_circ_0004650 to 5-Fu resistance in GC, and its down-stream regulatory mechanism was also explored. The results manifested that hsa_circ_0004650 functioned as a miR-145-5p sponge to maintain resistance to 5-Fu in GC, which provides a potential therapeutic therapy for overcoming 5-Fu resistance in GC.

Materials and Methods

Cell lines. Human GC cell lines (SGC-7901 and BGC-823 cells) were purchased from iCell Bioscience (Shanghai, PR China). Tool cell line HEK-293T cells was obtained from iCell Bioscience. All the cells had been tested and authenticated by the corporation. 5-Fu-resistant GC cell lines (SGC-7901-5-Fu and BGC-823-5-Fu cells) were constructed from 5-Fu-sensitive GC cell lines by low concentration gradient increment method, starting from addition of 10 nmol/l 5-Fu (Yuanye Bio-Technology, Shanghai, PR China). The drug-resistant GC cell lines were successfully constructed, when the GC cells can grow stably in culture medium containing 10 μmol/l 5-Fu. The cells were incubated in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco), and grown in 5% CO2 incubator at 37°C. In addition, 5-Fu-resistant GC cells required the addition of culture medium containing 10μmol/l 5-Fu to maintain resistance.

RNA-seq. RNA-seq data of SGC-7901 and SGC-7901-5-Fu cells were available at Dryad (https://Datadryad.org/stash/dataset/doi:10.5061/dryad.v15dv41wv). RNA-seq analysis of BGC-823 and BGC-823-5-Fu cells were implemented with the assistance of Tiangen Biochemical Technology Co., LTD. (Beijing, PR China). Firstly, the quality of the sample RNA was carried out, including 1% agarose electrophoresis to detect degradation and impurities, preliminary quantification using Nanodrop to detect RNA concentration and purity, and testing the integrity of the RNA sample using Agilent 2100 RNA Nano 6000 Assay Kit (Agilent Technologies, Santa Clara, CA, USA). Then, the transcriptome library was prepared by using the method of removing ribosomal RNA. After the library check was completed, sequencing was performed on Illumina HiSeqTM4000 sequencer (Illumina, San Diego, CA, USA). Finally, transcriptome analysis was conducted on sequencing data. By comparing the reference genome, we can discover the expression of species transcripts, identify specific expression sequences, perform differential expression analysis, and analyze functional genes.

qRT-PCR validation of different circRNAs and miRNAs levels. Total RNA was extracted from cells using the FastPure® Cell/Tissue Total RNA Isolation Kit (Vazyme, Nanjing, PR China) following the product manual. The cDNA was synthesized according to the procedure of HiScript® III RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme). And the ChamQ Universal SYBR qPCR Master Mix kit (Vazyme) was used for qRT-PCR according to the manufacturer’s guidelines. In order to accurately and efficiently measure miRNA expression in different cells, we used the miRcute miRNA Isolation Kit (Tiangen) for miRNA extraction, miRcute Plus miRNA First-Strand cDNA Kit (Tiangen) for cDNA synthesis and miRcute Plus miRNA qPCR Kit (Tiangen) for qRT-PCR, as directed by the manufacturer’s instructions. The upstream primer sequence of hsa_circ_0004650 was 5′-ACAGTAGGCAGCAACCAAGC-3′, the downstream primer sequence was 5′-CTGGGATCTTAGGGCC TCTC-3′; the upstream primer sequence of β-actin gene was 5′-AGCGAGCATCCCCCAAAGTT-3′, and the downstream primer sequence is 5′-GGGCACGAAGGCTCATCATT-3′. The upstream primer sequence of miR-145-5p was 5′-GTCCAGTTTTCCCAGG AATCCCT-3′, and downstream primer was universal primer provided by the company. The upstream primer sequence of U6 gene was 5′- CTCGCTTCGGCAGCACA-3′, and the downstream primer sequence was 5′-AACGCTTCACGAATTTGCGT-3′. Each sample was subjected to qRT-PCR reactions using the IQ5 Multicolor qRT-PCR assay system (Bio-Rad, Hercules, CA, USA) with three replicates per group. Relative gene expression was computed through the 2−ΔΔCt method using β-actin and U6 as endogenous controls.

The siRNA synthesis, miRNA inhibitor synthesis, and cell transfection. We screened the siRNA with relative advantages to improve the efficiency of cell transfection. The siRNA of hsa_circ_0004650 was designed and synthesized by Shanghai Gima Pharmaceutical Technology Co., LTD. (Shanghai, PR China), and scrambled sequence siRNA was selected as the negative control group. The miRNA inhibitor inhibits the action of mature miRNA molecules by specific binding, and down-regulates the regulatory effects caused by miRNA. We used miR-145-5p inhibitor to down-regulate miR-145-5p, and miRNA inhibitor control was selected as the negative control (miRNA NC) group. Cell transfection was performed when the cells grew to 80-90% fusion degree with three replicates per group. Before transfection, the old culture medium was discarded and the cells were washed with serum-free medium Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) three times. Then, 1ml Opti-MEM was added to 6-well plates per well. Next, the 10 μl transfection agent Lipofectamine 2000 (Thermo Fisher Scientific) was diluted with 250 μl Opti-MEM. Meanwhile, the configured siRNA or miRNA inhibitor was diluted with 250 μl Opti-MEM. The transfection mixture was prepared by mixing the two diluents evenly and incubated at room temperature for 20 min. Next, the transfection mixture was added to each well and placed in an incubator. After incubation for 6 h, supernatant from these wells were replaced with complete culture medium and cultured for 48 h for subsequent analyses.

Detecting cell proliferation using CCK-8 assay. According to the experimental purpose, GC cells in logarithmic growth stage were collected and seeded into 96-well plates (5×104 cells/ml) with three replicates per group. Each group of cells was treated with increasing doses of 5-Fu, when the GC cells were adhered to the well. The GC cells continued to be cultured to the appointed time, and then 10 μl/well CCK-8 solution (Dojindo, Shanghai, PR China) was added. After the GC cells were incubated for 2 h, the absorbance of each group at 450 nm was measured by microplate reader (BioTek, Winooski, VT, USA). Finally, the cell inhibition rate of each group was calculated.

Detection of cell viability using Calcein acetoxymethyl ester (CAM)/propidium iodide (PI) dual-fluorescence staining. CAM and PI double fluorescence staining assay (Beyotime, Shanghai, PR China) was used to detect the cell viability with three replicates per group. CAM-stained live cells, whereas PI-stained dead cells. After GC cells adhesion, cells of each group were intervened with 50 μmol/l 5-Fu. Then the GC cells were cultured for 48 h, washed twice with phosphate buffered saline (PBS), and added 100 μl CAM/PI detection solution per well. Finally, the GC cells were incubated for 30 min and then observed the staining effect of the cells under a fluorescence microscope.

Detection of cell apoptosis by flow cytometry. Cell apoptosis was measured via an Annexin V-FITC/PI Apoptosis Detection Kit (Dojindo) following the procedures with three replicates per group. After 48 h of cell transfection, GC cells of each group were treated with 50 μmol/l 5-Fu. And GC cells were collected after 48 h and washed twice with PBS. Next, 300 μl binding buffer were re-suspended containing 5 μl Annexin V-FITC and 5 μl PI. The cell complex was incubated in a dark environment at 4°C for 15 min, and then analyzed using flow cytometry (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Luciferase reporter assay. Firstly, the hsa_circ_0004650 wild type (hsa_circ_0004650-WT) double luciferase reporter plasmid was constructed according to the binding site predicted by the bioinformatics website Circinteractome (https://circinteractome.nia.nih.gov). Next, we mutated the predicted binding sites of hsa_circ_000465 and miR-145-5p, and hsa_circ_0004650 mutant (hsa_circ_0004650-MUT) double luciferase reporter plasmid was constructed. After seeding in 96-well plates, HEK-293T cells were co-transfected with miRNA inhibitors and plasmids when the cell growth density was 70%-80%. Each group had three replicates. After culturing for 48 h, we added 100 μl Passive Lysis Buffer into each well, gently shook at room temperature for 15 min, and collected the cell lysate solution. Finally, the cell lysate solution was performed using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Statistical analysis. We used the SPSS 17.0 software (SPSS, Chicago, IL, USA) to process and analyze the data. The data were given as the mean±standard deviation. The Prism 5.0 software (GraphPad, San Diego, CA, USA) was used to draw graphs. The screening conditions for the differentially expressed circRNAs in different groups were as follows: Fold change (FC) ≥2, p<0.05. The R language was used to draw the volcano map and heat map for the differentially expressed circRNAs. The intersection analysis of two high-throughput sequencing data was performed using Venn diagram. The difference of data between control group and experimental group was analyzed using with Student’s t-test. A probability value of p<0.05 was considered to be the limit of statistical significance.

Results

Mining and analysis of RNA-seq data. In our previous study, RNA-seq was performed on SGC-7901 and SGC-7901-5-Fu cells. For further study, we fully mined and analyzed the high-throughput sequencing data. We analyzed the characteristics of differentially expressed circRNAs in Ensembl database (http://www.ensembl.org/) and collected the circRNAs indexed in circBase database (http://www.circbase.org/). The results showed that 23 circRNAs were differentially expressed in SGC-7901-5-Fu cells compared with SGC-7901 cells, among which 5 circRNAs were up-regulated and 18 circRNAs were down-regulated, as shown in Table I.

View this table:
Table I.

Differentially expressed circRNAs indexed by circBase between SGC-7901 and SGC-7901-5-Fu cells.

Next, we used the same method to construct the other 5-Fu-resistant GC cells. BGC-823-5-Fu cells were successfully constructed by using low concentration gradient increment method on the BGC-823 cells. The RNA-seq was performed on two kinds of cells. After data normalization, FC ≥2 and p<0.05 were used as the screening condition. The results showed that compared with BGC-823 cells, a total of 2268 different circRNAs were differentially expressed in BGC-823-5-Fu cells, among which 759 were up-regulated and 1509 were down-regulated, as shown in Figure 1.

Figure 1.
Figure 1.

The volcano plot and heat maps of circRNA differentially expressed in BGC-823-5-Fu cells. (A) The horizontal coordinate represented the log2 fold change in circRNAs expression, and the ordinate represented the −log10 (p-value) of the change in circRNAs expression. The green dots represented down-regulated circRNAs, the red dots represented up-regulated circRNAs, and black dots represented insignificantly differentially expressed circRNAs. (B) circRNAs represented vertically, with red indicating up-regulated circRNAs and blue indicating down-regulated circRNAs.

The intersection analysis of up-regulated and down-regulated circRNAs in 5-Fu-resistant GC cells from two high-throughput sequencing screenings was performed. As shown in Figure 2, it was found that there was one circRNA in the intersection of up-regulated circRNAs, namely hsa_circ_0004650. Besides, there were two circRNAs in the intersection of downregulated circRNAs, namely hsa_circ_0007717 and hsa_circ_0012152. Because the overall expression trend of circRNAs is low, the selection of circRNAs with up-regulated expression is more conducive to clinical detection and application. Ultimately, hsa_circ_0004650 was selected as the research object.

Figure 2.
Figure 2.

Venn diagram of differentially expressed circRNAs in high-throughput sequencing screenings. There were 23 differentially expressed circRNAs in SGC-7901-5-Fu cells, of which 5 were up-regulated and 18 were down-regulated. In BGC-823-5-Fu cells, 2268 distinct circRNAs were differentially expressed, of which759 were up-regulated and 1509 were down-regulated. (A) The intersection analysis of up-regulated circRNAs between SGC-7901-5-Fu and BGC-823-5-Fu cells; (B) The intersection analysis of down-regulated circRNAs between SGC-7901-5-Fu and BGC-823-5-Fu cells. Red circles represented BGC-823-5-Fu cells and yellow circles represented SGC-7901-5-Fu cells.

Hsa_circ_0004650 was up-regulated in 5-Fu-resistant GC cells. To understand the potential role of hsa_circ_0004650 in GC cells, we examined the expression levels of hsa_circ_0004650 in SGC-7901, BGC-823, SGC-7901-5-Fu and BGC-823-5-Fu cells, respectively using qRT-PCR. The expression of hsa_circ_0004650 in SGC-7901-5-Fu and BGC-823-5-Fu cells were significantly up-regulated compared to the SGC-7901 and BGC-823 cells (p<0.01; Figure 3). The results of qRT-PCR validation were consistent with those of RNA-seq, indicating the reliability of RNA-seq technology. Thereby, we speculated that the high expression of hsa_circ_0004650 may be involved in the regulation of 5-Fu resistance in GC cells.

Figure 3.
Figure 3.

The expression of hsa_circ_0004650 was upregulated in 5-Fu resistant gastric cancer cell lines. (A) Relative expression of hsa_circ_0004650 in SGC-7901 and SGC-7901-5-Fu cells; (B) Relative expression of hsa_circ_0004650 in BGC-823 and BGC-823-5-Fu cells. **p<0.01, ***p<0.001.

Silencing the hsa_circ_0004650 increased the sensitivity of 5-Fu-resistant GC cells to 5-Fu. We selected 5-Fu-resistant GC cells as the research models, silenced the hsa_circ_0004650 by RNA interference technology, and then intervened the cells with 5-Fu. CCK-8 assay was used to detect the survival rates of GC cells treated with increasing doses of 5-Fu (0, 5, 10, 20, 50 μmol/l) for 24 h and 48 h. CAM/PI double fluorescence staining was used to detect the dead and live cells, and flow cytometry was used to detect cell apoptosis. The results indicated that in 5-Fu-resistant GC cells, the cell survival rates of the si-circ-0004650 group were significantly lower than those of the si-NC group, and the differences were statistically significant (p<0.01; Figure 4). In addition, in SGC-7901-5-Fu cells, the mortality rates of cells transfected with si-circ-0004650 group were 44.86%±1.52, while the mortality rates of cells transfected with si-NC were 17.86%±0.60, indicating that the mortality rates of the si-circ-0004650 group were significantly higher than those of the si-NC group, with a statistically significant difference (p<0.001; Figure 5). Similarly, the mortality rates of BGC-823-5-Fu cells transfected with si-circ-0004650 were 26.61%±1.12, while the mortality rates of cells transfected with si-NC were 8.17%±7.80, indicating that the mortality rates of the si-circ-0004650 group were significantly higher than those of the si-NC group, with a statistically significant difference (p<0.001; Figure 6). The above results showed that down-regulation of hsa_circ_0004650 in 5-Fu-resistant GC cells decreased the proliferation activity. Additionally, after the 5-Fu-resistant GC cells were induced by the same concentration of 5-Fu, the apoptosis rates of the si-circ-0004650 group were significantly higher than those of the si-NC group (p<0.001; Figure 7). The results showed that down-regulation of hsa_circ_0004650 in 5-Fu-resistant GC cells significantly promoted apoptosis. Therefore, silencing the hsa_circ_ 0004650 could improve the sensitivity of 5-Fu-resistant GC cells to 5-Fu.

Figure 4.
Figure 4.

The survival rates of 5-Fu-resistant GC cells after silencing the hsa_circ_0004650. (A) The survival rates of SGC-7901-5-Fu cells treated with increasing doses of 5-Fu for 24 h; (B) The survival rates of SGC-7901-5-Fu cells treated with increasing doses of 5-Fu for 48 h; (C) The survival rates of BGC-823-5-Fu cells treated with increasing doses of 5-Fu for 24 h; (D) The survival rates of BGC-823-5-Fu cells treated with increasing doses of 5-Fu for 48 h. The si-circ-0004650 group represented down-regulatinghsa_circ_0004650 expression and si-NC group represented negative control group. **p<0.01, ***p<0.001.

Figure 5.
Figure 5.

Silencing the hsa_circ_0004650 in SGC-7901-5-Fu cells promoted cell death. The mortality rates of cells transfected with si-circ-0004650 group were 44.86%±1.52, while the mortality rates of cells transfected with si-NC were 17.86%±0.60. Among them, red represented dead cells, while green represented live cells. The si-circ-0004650 group represented down-regulating hsa_circ_0004650 expression and si-NC group represented negative control group.

Figure 6.
Figure 6.

Silencing the hsa_circ_0004650 in BGC-823-5-Fu cells promoted cell death. The mortality rates of cells transfected with si-circ-0004650 were 26.61%±1.12, while the mortality rates of cells transfected with si-NC were 8.17%±7.80. Among them, red represented dead cells, while green represented live cells. The si-circ-0004650 group represented down-regulating hsa_circ_0004650 expression and si-NC group represented negative control group.

Figure 7.
Figure 7.

Effect of silencing the hsa_circ_0004650 on cell apoptosis in 5-Fu-resistant GC cells. (A) Apoptosis of SGC-7901-5-Fu cells induced by 50 μmol/l 5-Fu after silencing the hsa_circ_0004650; (B) Apoptosis of BGC-823-5-Fu cells induced by 50 μmol/l 5-Fu after silencing the hsa_circ_0004650. The si-circ-0004650 group represented down-regulating hsa_circ_0004650 expression and si-NC group represented negative control group. ***p<0.001.

Hsa_circ_0004650 exerted function by sponging miR-145-5p. To investigate the mechanism by which hsa_circ_ 0004650 regulates 5-Fu resistance in GC cells, Circinteractome was used to predict the existence of binding sites between hsa_circ_0004650 and miR-145-5p. To verify the targeted binding, we used HEK-293T cells and further validated them with luciferase reporter assay. The results showed that compared to the miRNA NC group, the luciferase activity of hsa_circ_0004650-WT transfected with miR-145-5p inhibitor was markedly increased (p<0.01). Meanwhile, the luciferase activity of hsa_circ_0004650-MUT transfected with miR-145-5p inhibitor failed to change compared with the miRNA NC group, as shown in Figure 8. The above results indicated that hsa_circ_000465 and miR-145-5p have targeted binding.

Figure 8.
Figure 8.

Luciferase reporter assay to detect the binding of hsa_circ_0004650 to miR-145-5p. **p<0.01.

Next, we detected the expression of miR-145-5p in 5-Fu-sensitive GC cells and 5-Fu-resistant GC cells by qRT-PCR. The expression of miR-145-5p in SGC-7901-5-Fu cells was remarkably lower than that in SGC-7901 cells, and the difference was statistically significant (p<0.01; Figure 9A). Similarly, the expression of miR-145-5p in BGC-823-5-Fu cells was remarkably lower than that in BGC-823 cells, and the difference was statistically significant (p<0.05; Figure 9B). The results suggested that miR-145-5p presented low expression levels in 5-Fu-resistant GC cells.

Figure 9.
Figure 9.

Expression levels of miR-145-5p in 5-Fu-sensitive GC cells and 5-Fu-resistant GC cells. (A) The expression of miR-145-5p in SGC-7901 and SGC-7901-5-Fu cells; (B) The expression of miR-145-5p in BGC-823 and BGC-823-5-Fu cells. *p<0.05, **p<0.01.

Furthermore, siRNA was used to silence the expression of hsa_circ_0004650 in 5-Fu-resistant GC cells, and qRT-PCR was used to detect the changes of miR-145-5p in each group. In the SGC-7901-5-Fu cells, the expression of miR-145-5p in the si-circ-0004650 was significantly higher than that in the si-NC group, and the difference was statistically significant (p<0.001; Figure 10A). Similarly, in the BGC-823-5-Fu cells, the expression of miR-145-5p in the si-circ-0004650 group was significantly higher than that in the si-NC group, and the difference was statistically significant (p<0.001; Figure 10B). The results indicated that silencing the expression of hsa_circ_0004650 gene could up-regulate the expression of miR-145-5p gene. That is, hsa_circ_0004650 could target and negatively regulate the expression of miR-145-5p.

Figure 10.
Figure 10.

Effect of silencing the hsa_circ_000465 on the expression of miR-145-5p. (A) The expression of miR-145-5p after silencing the expression of hsa_circ_000465 in the SGC-7901-5-Fu cells; (B) The expression of miR-145-5p after silencing the expression of hsa_circ_000465 in the BGC-823-5-Fu cells. The si-circ-0004650 group represented down-regulating hsa_circ_0004650 expression and si-NC group represented negative control group. ***p<0.001.

Down-regulation of miR-145-5p enhanced 5-Fu resistance of 5-Fu-resistant GC cells. We first down-regulated the expression of miR-145-5p in 5-Fu-resistant GC cells then intervened with increasing doses of 5-Fu, and finally determined the survival rates for 24 h and 48 h by CCK-8 assay. The results suggested that the survival rates in the miR-145-5p inhibitor group were significantly higher than those in the miRNA NC group, and the differences were statistically significant (p<0.05; Figure 11).

Figure 11.
Figure 11.

Survival rates of 5-Fu-resistant GC cells after down-regulation of miR-145-5p. (A) The survival rates of SGC-7901-5-Fu cells intervened with increasing doses of 5-Fu for 24 h; (B) The survival rates of SGC-7901-5-Fu cells intervened with increasing doses of 5-Fu for 48 h; (C) The survival rates of BGC-823-5-Fu cells intervened with increasing doses of 5-Fu for 24 h; (D) The survival rates of BGC-823-5-Fu cells intervened with increasing doses of 5-Fu for 48 h. The anti-miR-145-5p group represented down-regulating miR-145-5p expression and anti-miR-NC group represented negative control group. *p<0.05, **p<0.01, ***p<0.001.

Furthermore, CAM/PI double fluorescence staining was used to detect the viability of 5-Fu-resistant GC cells after down-regulating miR-145-5p. In SGC-7901-5-Fu cells, the mortality rates of cells transfected with miR-145-5p inhibitor group were 7.83%±0.53, while the mortality rates of cells transfected with miRNA NC were 12.86%±1.10, indicating that the mortality rates of the miR-145-5p inhibitor group were significantly lower than those of the miRNA NC group, with a statistically significant difference (p<0.01; Figure 12). In addition, the mortality rates of BGC-823-5-Fu cells transfected with miR-145-5p inhibitor group were 11.40%±0.72, while the mortality rates of cells transfected with miRNA NC were 16.99%±1.31, indicating that the mortality rates of the miR-145-5p inhibitor group were significantly lower than those of the miRNA NC group, with a statistically significant difference (p<0.01; Figure 13). Therefore, it is speculated that miR-145-5p also played an important role in the 5-Fu resistance mechanism of GC cells.

Figure 12.
Figure 12.

Effect of down-regulating miR-145-5p on cell viability in the SGC-7901-5-Fu cells. The mortality rates of cells transfected with miR-145-5p inhibitor group were 7.83%±0.53, while the mortality rates of cells transfected with miRNA NC were 12.86%±1.10. Among them, red represented dead cells and green represented live cells. The anti-miR-145-5p group represented down-regulating miR-145-5p expression and anti-miR-NC group represented negative control group.

Figure 13.
Figure 13.

Effect of down-regulating miR-145-5p on cell viability in the BGC-823-5-Fu cells. The mortality rates of cells transfected with miR-145-5p inhibitor group were 11.40%±0.72, while the mortality rates of cells transfected with miRNA NC were 16.99%±1.31. Among them, red represented dead cells and green represented live cells. The anti-miR-145-5p group represented down-regulating miR-145-5p expression and anti-miR-NC group represented negative control group.

We down-regulated the expression of miR-145-5p with miR-145-5p inhibitor, and then used flow cytometry analysis to detect the cell apoptosis of SGC-7901-5-Fu and BGC-823-5-Fu cells induced by the same concentration of 5-Fu. The results showed that in 5-Fu-resistant GC cells, the apoptosis rates of miR-145-5p inhibitor group were significantly lower than those of miRNA NC group, and the differences were statistically significant (p<0.001; Figure 14). To summarize, 5-Fu-resistant GC cells were more resistant to 5-Fu after down-regulating the miR-145-5p.

Figure 14.
Figure 14.

Effect of down-regulating the miR-145-5p on cell apoptosis of 5-Fu-resistant GC cells. (A) Apoptosis of SGC-7901-5-Fu cells induced by 5-Fu after down-regulating the miR-145-5p; (B) Apoptosis of BGC-823-5-Fu cells induced by 5-Fu after down-regulation the miR-145-5p. The anti-miR-145-5p group represented down-regulating miR-145-5p expression and anti-miR-NC group represented negative control group. ***p<0.001.

Discussion

Chemotherapy resistance severely limits the therapeutic effect of GC patients. It is quite important to identify the potential mechanisms of chemotherapy resistance and develop treatment strategies. In this study, we found that hsa_circ_0004650 was involved in the regulation of 5-Fu resistance in GC. Hsa_circ_0004650 is a novel circRNA with a splicing sequence length of 4052 bp, originating from the long arm of chromosome 12. The expression of hsa_circ_0004650 was significantly up-regulated in 5-Fu-resistant GC cells, and knockdown of hsa_circ_0004650 could improve the sensitivity of GC cells to 5-Fu. Hence, we believe that hsa_circ_0004650 was involved in regulating the drug resistance of cells to 5-Fu in GC cells.

The circRNAs are closely related to proliferation, invasion, migration, and apoptosis of GC cells (18). For instance, circRBM33 up-regulated the levels of downstream product interleukin-6 through sponge adsorption of miR-149, thus promoting the proliferation process of GC cells (19). In addition, accumulating evidence suggested that circRNAs can affect the resistance of chemotherapy drugs in GC cells (15, 20). Related studies mainly focused on cisplatin resistance in GC. As an example, circCUL2 may function as a tumor suppressor and regulator of cisplatin sensitivity through miR-142-3p/ROCK2-mediated autophagy activation (21). However, the participation of circRNA in 5-Fu resistance is still obscure in GC. Therefore, it is valuable to study the mechanism of circRNA regulating 5-Fu resistance in GC patients.

Our study concentrated on the role of hsa_circ_0004650 in the regulation of 5-Fu resistance in GC cells. As an inhibitor of thymidylate synthase, 5-Fu can inhibit the synthesis of RNA and DNA in tumor cells, while DNA damage can induce p53-dependent cell cycle arrest and apoptosis, leading to tumor cells death (22, 23). Additionally,5-Fu acts as a common antitumor agent in GC patients and activates apoptotic signaling by inhibiting cell division (24). In our study, down-regulation of the expression of hsa_circ_0004650 gene promoted the apoptosis of drug-resistant cells. These findings confirmed that hsa_circ_0004650 was involved in the regulation of 5-Fu resistance in GC cells. Targeting hsa_circ_0004650 may be a potentially effective way to overcome 5-Fu resistance.

The miRNA is a small molecule RNA with a length of approximately 19-25 nucleotides, which can negatively regulate gene expression by binding to the 3′-untranslated region of targeted mRNA to affect protein translation (25). There have been reports that miR-185, miR-218, miR-145, miR-27b, miR-30a, miR-107, miR-Bart20-5p, and miR-23b-3p are involved in 5-Fu resistance of GC (26). The mechanism of 5-Fu resistance in GC regulated by circRNA is complex, in which the competing endogenous RNA (ceRNA) potentially acts as a principal factor. That is, circRNAs can act as ceRNAs to interact with target microRNAs (miRNAs) molecules, and indirectly affects the expression of downstream target genes. It is currently widely recognized that circRNA can act as a post-transcriptional regulator of gene expression by reducing miRNA activity (27). We speculated that hsa_circ_0004650 and miR-145-5p have complementary binding sites based on bioinformatics. The targeting binding of hsa_circ_0004650 to miR-145-5p was verified by dual luciferase assay. The miR-145-5p, as an important regulatory factor, has been found to participate in the regulation of the development, proliferation, differentiation, migration and invasion of GC (28-30). Besides, miR-145-5p has been shown to promote G1-S transition by regulating the expression of key cell cycle regulators CDK4, CDK6, and cyclin D1 in GC cells (31). Also, miR-145-5p is a potential biological marker of tumor metastasis (32). Additionally, related research shows that overall survival time was worse in patients with low miR-145-5p expression compared to those with high miR-145-5p expression (33). In the present work, the expression of miR-145-5p in 5-Fu-resistant GC cells was remarkably lower than that in 5-Fu-sensitive GC cells. More importantly, inhibition of miR-145-5p improved the 5-Fu resistance of GC cells. Based on these results, we speculated that hsa_circ_0004650 inhibited apoptosis by sponging miR-145-5p, thereby promoting the proliferation and 5-Fu resistance of GC cells.

In conclusion, we have shown that hsa_circ_0004650 is highly expressed in 5-Fu-resistant GC cells and that it can availably sponge miR-145-5p, thereby inhibiting apoptosis and ultimately facilitating 5-Fu resistance. However, there are some limitations in this study. Firstly, the expression of hsa_circ_0004650 in tissues and serum of 5-Fu-resistant GC patients has not been verified. Secondly, downstream regulatory mechanism of miR-145-5p was not discussed. Finally, in vivo experiments, we did not use animal models of GC to further verify. These drawbacks should be further explored in subsequent studies.

Conclusion

Hsa_circ_0004650, as a ceRNA, induced resistance to 5-Fu in GC cells. We speculated that hsa_circ_0004650 promoted the 5-Fu resistance of GC cells by sponging miR-145-5p. Thus, hsa_circ_0004650 may be a promising therapeutic target for overcoming 5-Fu resistance in GC.

Footnotes

  • Authors’ Contributions

    Fusheng Zhou, Jialong Zhuo, Nan Gao collected the data, prepared the table and wrote the article. Xudong Xu, Duyi Pan, Chenwen Cai, Jiaxin Huang, Xianguang Zhao, Qiqi Mao, Xiaoyun Jiang and Xu Sun conceived the study, aided in the research work and checked the article. Liang Zhong and Jiajie Chen reviewed the article. All Authors have seen and approved the final draft before submission.

  • Funding

    This work was supported by the Intramural Funds for Huashan Hospital affiliated with Fudan University, Shanghai, China (Nos. HSBY2020010 and HSBY2020004).

  • Conflicts of Interest

    The Authors have no competing interests to declare that are relevant to the content of this article.

  • Received October 18, 2024.
  • Revision received December 2, 2024.
  • Accepted December 9, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. Bray F,
    2. Laversanne M,
    3. Sung H,
    4. Ferlay J,
    5. Siegel RL,
    6. Soerjomataram I,
    7. Jemal A
    : Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74(3): 229-263, 2024. DOI: 10.3322/caac.21834
    OpenUrlCrossRef
  2. ↵
    1. Gao K,
    2. Wu J
    : National trend of gastric cancer mortality in China (2003-2015): a population-based study. Cancer Commun (Lond) 39(1): 24, 2019. DOI: 10.1186/s40880-019-0372-x
    OpenUrlCrossRefPubMed
  3. ↵
    1. Ahmad SA,
    2. Xia BT,
    3. Bailey CE,
    4. Abbott DE,
    5. Helmink BA,
    6. Daly MC,
    7. Thota R,
    8. Schlegal C,
    9. Winer LK,
    10. Ahmad SA,
    11. Al Humaidi AH,
    12. Parikh AA
    : An update on gastric cancer. Curr Probl Surg 53(10): 449-490, 2016. DOI: 10.1067/j.cpsurg.2016.08.001
    OpenUrlCrossRefPubMed
  4. ↵
    1. Sitarz R,
    2. Skierucha M,
    3. Mielko J,
    4. Offerhaus GJA,
    5. Maciejewski R,
    6. Polkowski WP
    : Gastric cancer: epidemiology, prevention, classification, and treatment. Cancer Manag Res 10: 239-248, 2018. DOI: 10.2147/CMAR.S149619
    OpenUrlCrossRefPubMed
  5. ↵
    1. Joshi SS,
    2. Badgwell BD
    : Current treatment and recent progress in gastric cancer. CA Cancer J Clin 71(3): 264-279, 2021. DOI: 10.3322/caac.21657
    OpenUrlCrossRefPubMed
  6. ↵
    1. Nakamura A,
    2. Nakajima G,
    3. Okuyama R,
    4. Kuramochi H,
    5. Kondoh Y,
    6. Kanemura T,
    7. Takechi T,
    8. Yamamoto M,
    9. Hayashi K
    : Enhancement of 5-fluorouracil-induced cytotoxicity by leucovorin in 5-fluorouracil-resistant gastric cancer cells with upregulated expression of thymidylate synthase. Gastric Cancer 17(1): 188-195, 2014. DOI: 10.1007/s10120-013-0249-7
    OpenUrlCrossRefPubMed
  7. ↵
    1. Sakuramoto S,
    2. Sasako M,
    3. Yamaguchi T,
    4. Kinoshita T,
    5. Fujii M,
    6. Nashimoto A,
    7. Furukawa H,
    8. Nakajima T,
    9. Ohashi Y,
    10. Imamura H,
    11. Higashino M,
    12. Yamamura Y,
    13. Kurita A,
    14. Arai K, ACTS-GC Group
    : Adjuvant chemotherapy for gastric cancer with S-1, an oral fluoropyrimidine. N Engl J Med 357(18): 1810-1820, 2007. DOI: 10.1056/NEJMoa072252
    OpenUrlCrossRefPubMed
  8. ↵
    1. Ahmadzadeh A,
    2. Seyedian SS,
    3. Valizadeh A,
    4. Soleimani M,
    5. Nazari P,
    6. Hamidi H
    : The efficacy and safety of modified docetaxel, cisplatin, and 5-fluorouracil vs. epirubicin, oxaliplatin, and capecitabine regimen in the advanced gastric cancer: a randomized controlled clinical trial. Asian Pac J Cancer Prev 21(3): 727-732, 2020. DOI: 10.31557/APJCP.2020.21.3.727
    OpenUrlCrossRefPubMed
  9. ↵
    1. Kim DS,
    2. Min K,
    3. Lee SK
    : Cell cycle dysregulation is associated with 5-fluorouracil resistance in gastric cancer cells. Anticancer Res 40(6): 3247-3254, 2020. DOI: 10.21873/anticanres.14306
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Lu Y,
    2. Jin Z,
    3. Hou J,
    4. Wu X,
    5. Yu Z,
    6. Yao L,
    7. Pan T,
    8. Chang X,
    9. Yu B,
    10. Li J,
    11. Li C,
    12. Yan M,
    13. Yan C,
    14. Zhu Z,
    15. Liu B,
    16. Su L
    : Calponin 1 increases cancer-associated fibroblasts-mediated matrix stiffness to promote chemoresistance in gastric cancer. Matrix Biol 115: 1-15, 2023. DOI: 10.1016/j.matbio.2022.11.005
    OpenUrlCrossRefPubMed
  11. ↵
    1. Holdt LM,
    2. Kohlmaier A,
    3. Teupser D
    : Molecular roles and function of circular RNAs in eukaryotic cells. Cell Mol Life Sci 75(6): 1071-1098, 2018. DOI: 10.1007/s00018-017-2688-5
    OpenUrlCrossRefPubMed
  12. ↵
    1. Saleem A,
    2. Khan MU,
    3. Zahid T,
    4. Khurram I,
    5. Ghani MU,
    6. Ullah I,
    7. Munir R,
    8. Calina D,
    9. Sharifi-Rad J
    : Biological role and regulation of circular RNA as an emerging biomarker and potential therapeutic target for cancer. Mol Biol Rep 51(1): 296, 2024. DOI: 10.1007/s11033-024-09211-3
    OpenUrlCrossRefPubMed
  13. ↵
    1. Zhang W,
    2. Yang Q,
    3. Qian D,
    4. Zhao K,
    5. Tang C,
    6. Ju S
    : Deregulation of circRNA hsa_circ_0009109 promotes tumor growth and initiates autophagy by sponging miR-544a-3p in gastric cancer. Gastroenterol Rep (Oxf) 12: goae008, 2024. DOI: 10.1093/gastro/goae008
    OpenUrlCrossRefPubMed
  14. ↵
    1. Lan ZZ,
    2. Sun FH,
    3. Chen C,
    4. Niu L,
    5. Shi JD,
    6. Zhang WY
    : CircPRDM5 inhibits the proliferation, migration, invasion, and glucose metabolism of gastric cancer cells by reducing GCNT4 expression in a miR-485-3p-dependent manner. Kaohsiung J Med Sci 40(3): 231-243, 2024. DOI: 10.1002/kjm2.12799
    OpenUrlCrossRefPubMed
  15. ↵
    1. Yao W,
    2. Guo P,
    3. Mu Q,
    4. Wang Y
    : Exosome-derived Circ-PVT1 contributes to cisplatin resistance by regulating autophagy, invasion, and apoptosis via miR-30a-5p/YAP1 axis in gastric cancer cells. Cancer Biother Radiopharm 36(4): 347-359, 2020. DOI: 10.1089/cbr.2020.3578
    OpenUrlCrossRefPubMed
  16. ↵
    1. Xu G,
    2. Li M,
    3. Wu J,
    4. Qin C,
    5. Tao Y,
    6. He H
    : Circular RNA circNRIP1 sponges microRNA-138-5p to maintain hypoxia-induced resistance to 5-fluorouracil through HIF-1α-dependent glucose metabolism in gastric carcinoma. Cancer Manag Res 12: 2789-2802, 2020. DOI: 10.2147/CMAR.S246272
    OpenUrlCrossRefPubMed
  17. ↵
    1. Zhou F,
    2. Ding W,
    3. Mao Q,
    4. Jiang X,
    5. Chen J,
    6. Zhao X,
    7. Xu W,
    8. Huang J,
    9. Zhong L,
    10. Sun X
    : The regulation of hsacirc_004413 promotes proliferation and drug resistance of gastric cancer cells by acting as a competing endogenous RNA for miR-145-5p. PeerJ 10: e12629, 2022. DOI: 10.7717/peerj.12629
    OpenUrlCrossRefPubMed
  18. ↵
    1. Li R,
    2. Jiang J,
    3. Shi H,
    4. Qian H,
    5. Zhang X,
    6. Xu W
    : CircRNA: a rising star in gastric cancer. Cell Mol Life Sci 77(9): 1661-1680, 2020. DOI: 10.1007/s00018-019-03345-5
    OpenUrlCrossRefPubMed
  19. ↵
    1. Wang N,
    2. Lu K,
    3. Qu H,
    4. Wang H,
    5. Chen Y,
    6. Shan T,
    7. Ge X,
    8. Wei Y,
    9. Zhou P,
    10. Xia J
    : CircRBM33 regulates IL-6 to promote gastric cancer progression through targeting miR-149. Biomed Pharmacother 125: 109876, 2020. DOI: 10.1016/j.biopha.2020.109876
    OpenUrlCrossRefPubMed
  20. ↵
    1. Deng P,
    2. Sun M,
    3. Zhao WY,
    4. Hou B,
    5. Li K,
    6. Zhang T,
    7. Gu F
    : Circular RNA circVAPA promotes chemotherapy drug resistance in gastric cancer progression by regulating miR-125b-5p/STAT3 axis. World J Gastroenterol 27(6): 487-500, 2021. DOI: 10.3748/wjg.v27.i6.487
    OpenUrlCrossRefPubMed
  21. ↵
    1. Peng L,
    2. Sang H,
    3. Wei S,
    4. Li Y,
    5. Jin D,
    6. Zhu X,
    7. Li X,
    8. Dang Y,
    9. Zhang G
    : circCUL2 regulates gastric cancer malignant transformation and cisplatin resistance by modulating autophagy activation via miR-142-3p/ROCK2. Mol Cancer 19(1): 156, 2020. DOI: 10.1186/s12943-020-01270-x
    OpenUrlCrossRefPubMed
  22. ↵
    1. Santi DV,
    2. McHenry CS,
    3. Sommer H
    : Mechanism of interaction of thymidylate synthetase with 5-fluorodeoxyuridylate. Biochemistry 13(3): 471-481, 1974. DOI: 10.1021/bi00700a012
    OpenUrlCrossRefPubMed
  23. ↵
    1. Namwat N,
    2. Amimanan P,
    3. Loilome W,
    4. Jearanaikoon P,
    5. Sripa B,
    6. Bhudhisawasdi V,
    7. Tassaneeyakul W
    : Characterization of 5-fluorouracil-resistant cholangiocarcinoma cell lines. Chemotherapy 54(5): 343-351, 2008. DOI: 10.1159/000151541
    OpenUrlCrossRefPubMed
  24. ↵
    1. Christensen S,
    2. Van der Roest B,
    3. Besselink N,
    4. Janssen R,
    5. Boymans S,
    6. Martens JWM,
    7. Yaspo ML,
    8. Priestley P,
    9. Kuijk E,
    10. Cuppen E,
    11. Van Hoeck A
    : 5-Fluorouracil treatment induces characteristic T>G mutations in human cancer. Nat Commun 10(1): 4571, 2019. DOI: 10.1038/s41467-019-12594-8
    OpenUrlCrossRefPubMed
  25. ↵
    1. Bartel DP
    : MicroRNAs: target recognition and regulatory functions. Cell 136(2): 215-233, 2009. DOI: 10.1016/j.cell.2009.01.002
    OpenUrlCrossRefPubMed
  26. ↵
    1. Meng X,
    2. Bai X,
    3. Ke A,
    4. Li K,
    5. Lei Y,
    6. Ding S,
    7. Dai D
    : Long non-coding RNAs in drug resistance of gastric cancer: complex mechanisms and potential clinical applications. Biomolecules 14(6): 608, 2024. DOI: 10.3390/biom14060608
    OpenUrlCrossRefPubMed
  27. ↵
    1. Fontemaggi G
    : Non-coding RNA regulatory networks in post-transcriptional regulation of VEGFA in cancer. IUBMB Life 75(1): 30-39, 2023. DOI: 10.1002/iub.2620
    OpenUrlCrossRefPubMed
  28. ↵
    1. Zhou T,
    2. Chen S,
    3. Mao X
    : Mir-145-5p affects the differentiation of gastric cancer by targeting klf5 directly. J Cell Physiol 234(5): 7634-7644, 2019. DOI: 10.1002/jcp.27525
    OpenUrlCrossRefPubMed
    1. Zhang Z,
    2. Wang C,
    3. Zhang Y,
    4. Yu S,
    5. Zhao G,
    6. Xu J
    : CircDUSP16 promotes the tumorigenesis and invasion of gastric cancer by sponging miR-145-5p. Gastric Cancer 23(3): 437-448, 2020. DOI: 10.1007/s10120-019-01018-7
    OpenUrlCrossRefPubMed
  30. ↵
    1. Zhou K,
    2. Song B,
    3. Wei M,
    4. Fang J,
    5. Xu Y
    : MiR-145-5p suppresses the proliferation, migration and invasion of gastric cancer epithelial cells via the ANGPT2/NOD_LIKE_RECEPTOR axis. Cancer Cell Int 20: 416, 2020. DOI: 10.1186/s12935-020-01483-6
    OpenUrlCrossRefPubMed
  31. ↵
    1. Sun Y,
    2. Tian Y,
    3. He J,
    4. Tian Y,
    5. Zhang G,
    6. Zhao R,
    7. Zhu WJ,
    8. Gao P
    : Linc01133 contributes to gastric cancer growth by enhancing YES1-dependent YAP1 nuclear translocation via sponging miR-145-5p. Cell Death Dis 13(1): 51, 2022. DOI: 10.1038/s41419-022-04500-w
    OpenUrlCrossRefPubMed
  32. ↵
    1. Valentini V,
    2. Zelli V,
    3. Gaggiano E,
    4. Silvestri V,
    5. Rizzolo P,
    6. Bucalo A,
    7. Calvieri S,
    8. Grassi S,
    9. Frascione P,
    10. Donati P,
    11. Soda G,
    12. Ottini L,
    13. Richetta AG
    : MiRNAs as potential prognostic biomarkers for metastasis in thin and thick primary cutaneous melanomas. Anticancer Res 39(8): 4085-4093, 2019. DOI: 10.21873/anticanres.13566
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Li CY,
    2. Liang GY,
    3. Yao WZ,
    4. Sui J,
    5. Shen X,
    6. Zhang YQ,
    7. Peng H,
    8. Hong WW,
    9. Ye YC,
    10. Zhang ZY,
    11. Zhang WH,
    12. Yin LH,
    13. Pu YP
    : Identification and functional characterization of microRNAs reveal a potential role in gastric cancer progression. Clin Transl Oncol 19(2): 162-172, 2017. DOI: 10.1007/s12094-016-1516-y
    OpenUrlCrossRefPubMed
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Circular RNA hsa_circ_0004650 Enhances 5-Fluorouracil Resistance in Gastric Cancer via Sponging miR-145-5p
FUSHENG ZHOU, JIALONG ZHUO, XUDONG XU, DUYI PAN, CHENWEN CAI, JIAXIN HUANG, XIANGUANG ZHAO, QIQI MAO, XIAOYUN JIANG, XU SUN, LIANG ZHONG, NAN GAO, JIAJIE CHEN
Anticancer Research Jan 2025, 45 (1) 173-187; DOI: 10.21873/anticanres.17403
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