Long noncoding RNA NR2F1-AS1 plays a carcinogenic role in gastric cancer by recruiting transcriptional factor SPI1 to upregulate ST8SIA1 expression

Abstract

Gastric cancer (GC) is a highly malignant solid tumor of the digestive tract, which is associated with a high mortality rate. Long non-coding RNA (lncRNA) nuclear receptor subfamily 2 group F member 1 antisense RNA 1 (NR2F1-AS1) has been reported to exert a tumor-promoting effect in some types of cancer. The present study aimed to investigate the role of NR2F1-AS1 in GC. The expression levels of NR2F1-AS1 and its potential target gene were measured in GC cell lines. Bioinformatics analysis, an RNA immunoprecipitation assay and a chromatin immunoprecipitation assay were used to determine the binding relationship between NR2F1-AS1 and downstream genes. The effect of NR2F1-AS1 regulatory axis on AGC cell viability, proliferation, migration, invasion and epithelial-mesenchymal transition was evaluated. The results of the present study revealed that the knockdown of NR2F1-AS1 inhibited the proliferation, invasion and migration of GC cells. NR2F1-AS1 also upregulated the expression levels of ST8SIA1 by recruiting transcriptional factor SPI1. Thus, the effects of the knockdown of NR2F1-AS1 on GC cell functions were suggested to occur via regulation of ST8SIA1. In conclusion, the findings of the current study indicated that NR2F1-AS1 may promote the proliferation, invasion and migration of GC cells by recruiting SPI1, to upregulate ST8SIA1 expression. Thus, the regulation of their expression levels may provide a novel direction for the treatment of GC.

Keywords: Gastric cancer; LncRNA NR2F1-AS; SPI1; ST8SIA1.

Figure 1.

Knockdown of long non-coding RNA NR2F1-AS1 suppresses the viability and proliferation of gastric cancer cells. (a) Relative mRNA expression of NR2F1-AS1 in different cell lines was measured using RT-qPCR. ***P < 0.001 vs. GES-1. (b) Relative mRNA expression of NR2F1-AS1 in the control and transfection group was detected using RT-qPCR. ***P < 0.001 vs. shRNA. (c) MTT and (d and e) colony formation assays were performed to determine the viability and proliferation of NR2F1-AS1-silenced AGS cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs. shRNA-NC

Figure 2.

Knockdown of long non-coding RNA NR2F1-AS1 suppresses the invasion and migration of gastric cancer cells. (a) Relative migration rate was detected using a wound healing assay. (b) Relative cell invasion rate was detected using a Transwell assay. (c) Expression levels of epithelial-mesenchymal transition-related proteins were measured using Western blotting. ***P < 0.001 vs. control or shRNA-NC

Figure 3.

NR2F1-AS1 upregulates ST8SIA1 expression by recruiting SPI1. (a) lncRNA Modulator Atlas in Pan-cancer database was used to determine that NR2F1-AS1 bound with ST8SIA1 by recruiting SPI1. Relative (b) protein and (c) mRNA expression levels of ST8SIA1 in GES-1 and AGS cells were detected using Western blotting and RT-qPCR, respectively. Relative (d) protein and (e) mRNA expression levels were detected of ST8SIA1 in the control and transfection group using Western blotting and RT-qPCR, respectively. (f) shRNA-NR2F1-AS1 co-transfected with the pGL3-ST8SIA1-WT/MUT luciferase reporter was used to confirm the binding relationship between NR2F1-AS1 and ST8SIA1. (g) RNA pull-down and (h) RNA immunoprecipitation assays were performed to detect the binding relationship between NR2F1-AS1 and SPI1. (i) Chromatin immunoprecipitation assay was performed to further verify the binding between ST8SIA1 and SPI1. *P < 0.05, **P < 0.01, ***P < 0.001 vs. GES-1, shRNA-NC or IgG

Figure 4.

Knockdown of NR2F1-AS1 suppressed proliferation of gastric cancer cells through ST8SIA1. Relative (a) protein and (b) mRNA expression levels in the control and SPI1 knockdown groups were detected using Western blotting and RT-qPCR, respectively. (c) Relative mRNA expression levels in the control and Oe-NR2F1-AS1 group were detected using RT-qPCR. Relative (d) protein and (e) mRNA expression levels in the control and Oe-ST8SIA1 group were detected using Western blotting and RT-qPCR, respectively. Relative (f) protein and (g) mRNA expression levels in the control and co-transfection group were detected using Western blotting and RT-qPCR, respectively. (h) MTT and (i) colony formation assays were used to analyze the viability and proliferation of NR2F1-AS1-silenced and ST8SIA1-OeAGS cells. **P < 0.01, ***P < 0.001 vs. shRNA-NC; ^###P < 0.001 vs. shRNA-NR2F1-AS1 + Oe-NC; ^$$P < 0.01, ^$$$P < 0.001 vs. shRNA-NC + Oe-ST8SIA1

Figure 5.

Knockdown of NR2F1-AS1 suppresses the invasion and migration of gastric cancer cells by regulating ST8SIA1. (a) Relative migration rate was detected using a wound healing assay. (b) Relative cell invasion rate was detected using a Transwell assay. (c) Expression levels of epithelial-mesenchymal transition-related proteins were measured using Western blotting. ***P < 0.001 vs. shRNA-NC; ^###P < 0.001 vs. shRNA-NR2F1-AS1 + Oe-NC; ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001 vs. shRNA-NC + Oe-ST8SIA1

Figure 6.

NR2F1-AS1 upregulates the expression levels of ST8SIA1 by recruiting SPI1