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

Cell culture and drug resistant cell construction

Human GC tumor cell lines SGC-7901 and MKN-45 were purchased from iCell Bioscience (Shanghai, China). The 5-Fu-sensitive gastric cancer line, SGC-7901, was cultured in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA) and 1% penicillin-streptomycin (Gibco, Gaithersburg, MD, USA) at 37 °C in a 5% CO₂ incubator. The cells were digested with 0.25% trypsin and routinely passaged when the cells grew to 70–80% confluency. The stepwise addition method was used, starting from addition of 10 nmol/L 5-Fu (Yuanye Bio-Technology, Shanghai, China) until the cells can grew stably in culture medium containing 10 μM 5-Fu; the constructed drug-resistant cell line was named SGC-7901-5-Fu. The drugs were withdrawn 2 weeks before the experiment and routinely cultivated for future use.

Detecting cell proliferation using CCK-8 assay

SGC-7901 and SGC-7901-5-Fu cells in the logarithmic growth phase were seeded in 96-well plates (5 × 10⁴ cells/mL) with three replicate wells in each group. After the cells adhered on the second day, the two kinds of cells were treated with different concentrations of 5-Fu (0.1, 1, 5, 10, 20, and 50 μmol/L), and 10 μL/well CCK-8 assay reagent was added after 48 h. After culturing the cells for 2 h, a microplate reader was used to measure the absorbance of each well at 450 nm. The half maximal inhibitory concentration (IC₅₀) value of 5-Fu and the drug resistance indices of the two cell lines were then calculated.

RNA library construction and RNA-seq sequencing

The Illumina RNA-Seq procedure (Illumina, San Diego, CA, USA) for RNA library construction was conducted by Nanjing Pisenuo Gene Technology (Nanjing, China). The standard for library construction was RNA integrity number (RIN) ≥ 7, 28 S/18 S ratio > 0.7. The HiSeqTM 2,500 sequencer (Illumina, San Diego, CA, USA) was then used for sequencing with a sample load of 1 μg. The sequencing results were compared and annotated with the database as analysis background data, and the screening conditions for differential expression of circRNA in the two cells line were defined as fold changes (FC) ≥ 2 and P < 0.05.

Analysis of GO enrichment and KEGG pathway analysis of genes derived from the differential circRNAUse GO (Gene Ontology) database (http://www.geneontology.org) was used to perform gene function enrichment analysis on genes derived from differential circRNA, then count the numbers of differential genes included in each GO entry were counted, and the hypergeometric distribution test method was used to calculate the significance of the enrichment of differential transcripts in each GO entry. KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.genome.jp/kegg/pathway.html) was used to analyze the signal pathways (Pathway) of the differential genes, and also used of hypergeometric distribution test to calculatethe significances of the enrichment of differential genes in each pathway entry. The above calculations resulted in a value of P that reflected the significance of enrichment. The smaller the P value, the more significant the tendency of differential genes to be enriched in the GO or Pathway entry.

Real-time quantitative PCR to verify differential circRNA levels

Total RNA was extracted from SGC7901 and SGC7901-5-Fu cells using TRIzol reagent (TaKaRa, Dalian, China). The RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), then RNA was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, Dalian, China), and the cDNA was used as a template for real-time fluorescence quantitative PCR. The upstream primer sequence of hsacirc_004413 was 5′-AGCTGCTCAGAGAGACACAG-3′, the downstream primer sequence was 5′-TCCTCCCATCAAGCCCATTT-3′; the upstream primer sequence of β-actin gene was 5-GATCCACATCTGCTGGAAGG-3′, and the downstream primer sequence is 5-AAGTGTGACGTT GACATCCG-3′. The PCR reaction conditions: were the following: predenaturation at 95 °C for 30 s; denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s, followed b35 cycles.

Transfection of antisense RNA

The cells were overgrown to 80–90% confluency and transfected. Before transfection, the old culture medium was removed and the cells were washed three times with serum-free culture medium, then 450 µL of serum-free culture medium was added to each well in a 24-well plate. Two μLof Lipofectamine 2,000 was then diluted with 50 μL of OpTI-MEM (Thermo Fisher Scientific, Waltham, MA, USA). Then the antisense RNA (Table S1, synthesized by Gene-Pharma, Suzhou, China) was diluted with 50 μL of opti-MEM and filled with a 1 μg mixture containing 50 μL of the two diluents and incubated at room temperature for 10 min. The 50 μL mixture was then added to each well and placed in an incubator,and was cultured for 24 h for other experiments.

Apoptosis and necrosis staining

After cell transfection, 5 μL of 4′, 6-diamidino-2-phenylindole (DAPI) staining solution was added to each well and incubated at room temperature for 10 min, followed by addition of 5 μL of propidium iodide (PI) staining solution for 10 min at room temperature. After washing with phosphate-buffered saline three times, blue and red fluorescence were observed using fluorescence microscope.

Statistical analysis

We used SPSS 17.0 statistical software for Windows for statistical analyses (SPSS, Chicago, IL, USA). Prism 5.0 software (GraphPad, San Diego, CA, USA) were used for the design of hSACIRC_004413 primers. analyses. The difference of data between control group and experimental group and different experimental groups was analyzed using with Student’s t-test or one-way analysis of variance by SPSS 17.0 statistical software. Data are expressed as the mean+−standard deviation (SD). The results were considered statistically significant at a value of P < 0.05.

Establishment of 5-Fu resistant cell lines in GC

The 5-Fu resistant gastric cancer cell line, SGC-7901-5-Fu, was established by low concentration gradient increments, combined with high concentration intermittent shock in vitro, and was named SGC-7901-5-Fu. The proliferation inhibition rates of SGC-7901 and drug resistant cells were detected using the CCK-8 assay. The results showed that the proliferation inhibition of SGC7901 and drug-resistant cells increased gradually with an increase of 5-Fu concentration. Prism5 software (GraphPad, San Diego, CA, USA) was used to calculate the IC₅₀ of both types of cells. The results show the IC₅₀ of drug resistant cells (Fig. 1B) and SGC-7901 cells (Fig. 1A), which was significantly higher than that of the parent cells (>5×).

Differentiation of circRNA expression profiles between SGC-7901 and SGC-7901-5-Fu cells. Next, the circRNA expression profiles of SGC-7901 and SGC-7901-5-Fu cells were analyzed using a high-throughput RNA-seq technology. After normalization, a total of 4,496 circRNA targets were found in SGC-7901 cells (Table S2). As a result, 31 distinct circRNAs were differentially expressed between SGC-7901 and SGC-7901-5-Fu cell lines with a fold change >2.0, and a value of P < 0.05, of which 10 were upregulated and 21 were downregulated (Table S3). These up-and downregulated circRNAs are shown in the heat map in Fig. 2B.

The hsacirc_004413 was highly expressed in 5-Fu resistant SGC-7901-5-Fu cells

First, we determined the expression levels of hsacirc_004413 in GC cell lines (MKN-45,SGC-7901, and SGC7901-5-Fu), and compared with MKN-45, the poorly-differentiated human gastric cancer cells, SGC-7901, and the moderately-differentiated human gastric cancer cells, and SGC-7901-5-Fu cell expressions were increased. Figure 3 shows that the expression levels of hsacirc_004413 in MKN-45 and SGC-7901 cells were significantly lower than that of the drug-resistant SGC-7901-5Fu cells. These results indicated that the increased expression of hsacirc_004413 in SGC-7901-5-Fu cells may be involved in the resistance of gastric cancer cells to 5-Fu.

The hsacirc_004413 knockdown enhanced 5-Fu sensitivity in SGC-7901-5-Fu cells

To confirm the role of hsacirc_004413 in resistance to 5-Fu in SGC-7901-5-Fu cells, antisense RNA with knockdown activity was synthesized. After 24 h of transfection, we investigated the expression levels of hsacirc_004413 in SGC-7901 and SGC-7901-5-fu cells (Figs. 4A, 4B). After knockdown, the expression level was only about 60% that of the non-knocked down group, which indicated that knockdown was successful. There was no significant difference in SGC-7901 cells. As-NC as a negative control group did not affect the expression of hsacirc_004413 in both these cell groups. Then, 10 µM 5-Fu was added to the medium to determine the survival percentage of cells transfected with antisense RNA. The results are shown in Figs. 4C, 4D. After transfection, the cell survival percentage was significantly lower than that of the non-knockdown group and the negative control group. However, no such process was found in SGC-7901 cells. The results suggested that hsacirc_004413 played an important role in the drug resistance mechanism of SGC-7901-5-Fu cells.

The 5-Fu mainly inhibits cell mitosis, which leads to cell apoptosis and necrosis. After hsacirc_004413 treatment, cell apoptosis and necrosis were detected. Figure 5 shows that at the same concentration of 5-Fu, apoptosis (solid line arrow) and necrosis (dotted line arrow) were observed in the sensitive SGC-7901 strain, 48 h later, in which apoptotic cells accounted for the majority of changes and necrotic cells accounted for only a small percentage. The percentages of apoptotic and necrotic cells in drug-resistant strains of SGC-7901-5-Fu were lower than that in sensitive strains. After transfection of drug-resistant strains with siRNA, the percentages of cell apoptosis and necrosis increased significantly. Figure 5 shows that the cells of drug-resistant strains became more sensitive to drugs after interference, consistent with the possibility that hsacirc_004413 played an important role in the resistance mechanism of 5-Fu.

Inhibition of miR-145-5p improved the resistance of sensitive GC cells to 5-Fu

We used bioinformatics to predict microRNAs that may have interact with hsacirc_004413 (Table S5). The miR-145-5p has been shown to inhibit the proliferation of tumor cells in GC by promoting cell apoptosis and increasing sensitivity of tumor cells to chemotherapeutic agents in chronic myeloid leukemia patients (Zeng et al., 2017). To confirm our prediction, we obtained an inhibitor of miR-145-5p (synthesized by Gene-Pharma) and transfected it into the cells. After 24 h, the CCK8 assay was used to detect the survival percentage of cells. The results are shown in Fig. 6. After addition of 5-Fu and NC inhibitors, the cell survival percentage decreased, and there was no restriction difference compared with the addition of 5-Fu only. The cells that were added to 5-Fu and inhibitors showed a significant increase in survival. The survival rate was close to that of the SGC-7901-5-Fu drug resistant cells. After addition of the miR-145-5P inhibitor, the expressions of related genes were inhibited because circRNA adsorbed microRNA and inhibited its effect. We therefore hypothesized that hsacirc_004413 was rich in miR-145-5p binding sites to act as miRNA sponges, preventing miR-145-5p from interacting with mRNAs in the 3′ untranslated region, which may have been involved in inhibiting cell apoptosis. These data suggested that hsacirc_004413 inhibited apoptosis by sponging miR-145-5p, thereby promoting SGC-7901-5-Fu cell proliferation, and chemoresistance.