Circ_0074027 binds to EIF4A3 and promotes gastric cancer progression

Abstract

Circular RNAs (circRNAs) have been reported to play an important role in the progression of numerous types of human cancer. The aim of the present study was to determine the effects of circRNA_0074027 (circ_0074027) in gastric cancer (GC), and to elucidate the underlying mechanisms of action. For this purpose, the expression of circ_0074027 in GC cell lines was detected using reverse transcription-quantitative PCR. The effects of circ_0074027 on the proliferation and migration of GC cells were investigated using Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. The Circular RNA Interactome was used to predict that eukaryotic translation initiation factor 4A3 (EIF4A3) could bind to circ_0074027, which was confirmed using an RNA immunoprecipitation assay. The expression and function of EIF4A3 in GC cells were also determined using western blot analysis, as well as CCK-8, colony formation, wound-healing and Transwell assays. The results revealed that circ_0074027 was highly expressed in GC cell lines in the form of a closed loop. In addition, circ_0074027-knockdown inhibited cellular proliferation and motility. Furthermore, EIF4A3 was predicted to be targeted by circ_0074027 and a positive association was identified between them. The overexpression of EIF4A3 reversed the effects of circ_0074027 on the proliferation and motility of GC cells. In conclusion, the findings of the present study demonstrated that circ_0074027 bound to EIF4A3 and promoted the proliferation and migration capacities of GC cells.

Keywords: circ_0074027; eukaryotic translation initiation factor 4A3; gastric cancer.

Figure 1.

Expression levels of circ_0074027 are increased in gastric cancer. (A) RT-qPCR was performed to quantify circ_0074027 expression in GES-1, AGS, MKN45, HGC-27 and SNU-5 cells. (B) Expression levels of circ_0074027 and PITX1 in MKN45 cells with or without RNase R treatment were detected using RT-qPCR. ***P<0.001 vs. GES-1 or RNase (−). RT-qPCR, reverse transcription-quantitative PCR; circ_0074027, circular RNA_0074927; PITX1, paired like homeodomain 1.

Figure 2.

Knockdown of circ_0074027 impedes gastric cancer cell proliferation and motility. MKN45 cells were transfected with si-NC, si-circ-1 or si-circ-2. (A) Expression of circ_0074027 was detected using RT-qPCR following transfection. (B) Viability of MKN45 cells was assessed using a Cell Counting Kit-8 assay. (C) Colony formation capacity of MKN45 cells following transfection was assessed using a colony formation assay. Migratory ability of MKN45 cells following transfection was analyzed using (D) Transwell and (E) wound-healing assays (magnification, ×100). *P<0.05 ***P<0.001 vs. siNC. RT-qPCR, reverse transcription-quantitative PCR; circ_0074027, circular RNA_0074927; sicirc, small interfering RNA of circ_0074027; si, small interfering RNA; NC, negative control.

Figure 3.

circ_0074027 is positively associated with EIF4A3. (A) Circular RNA Interactome analysis predicted genes that bound to circ_0074027. (B) circ_0074027 with EIF4A3 enrichment, which was measured using an RNA immunoprecipitation assay. (C) Gene expression of EIF4A3 profiled across tumor samples and tissues from normal patients, acquired from the Gene Expression Profiling Interactive Analysis database. (D) Expression of EIF4A3 was detected using RT-qPCR in gastric cancer cells following transfection. (E) Expression of EIF4A3 was detected using RT-qPCR in MKN45 cells following transfection with si-NC, si-circ-1 or si-circ-2. *P<0.05 and ***P<0.001 vs. GES-1 or siNC. RT-qPCR, reverse transcription-quantitative PCR; circ_0074027, circular RNA_0074927; sicirc, small interfering RNA of circ_0074027; si, small interfering RNA; NC, negative control; EIF4A3, eukaryotic translation initiation factor 4A3; STAD, stomach adenocarcinoma.

Figure 4.

Overexpression of EIF4A3 reverses the suppressive effects of circ_0074027 silencing on gastric cancer cell proliferation. MKN45 cells were transfected with pcDNA3.1, pc-EIF4A3 and si-circ-1. (A) Expression of EIF4A3 was detected using western blot analysis in MKN45 cells following transfection. (B) Viability of MKN45 cells was assessed using a Cell Counting Kit-8 assay. (C) Colony numbers of MKN45 cells following transfection were determined using a colony formation assay. ***P<0.001 vs. siNC; ^#P<0.001 and ^###P<0.001 vs. sicirc-1 + pcDNA3.1. EIF4A3, eukaryotic translation initiation factor 4A3; circ_0074027, circular RNA_0074927; sicirc, small interfering RNA of circ_0074027; si, small interfering RNA; NC, negative control.

Figure 5.

Overexpression of EIF4A3 counteracts the inhibitory effects of circ_0074027 silencing on gastric cancer cell motility. (A) Migratory ability of MKN45 cells following transfection was analyzed by Transwell assay (magnification, ×100). (B) Migratory ability of MKN45 cells following transfection was analyzed using a wound-healing assay (magnification, ×100). ***P<0.001 vs. siNC; ^##P<0.01, ^###P<0.001 vs. sicirc-1 + pcDNA3.1. EIF4A3, eukaryotic translation initiation factor 4A3; circ_0074027, circular RNA_0074927; sicirc, small interfering RNA of circ_0074027; si, small interfering RNA.