Immune-related long noncoding RNA zinc finger protein 710-AS1-201 promotes the metastasis and invasion of gastric cancer cells

Abstract

Background: Gastric cancer (GC) is a prevalent malignant tumor of the gastrointestinal system. ZNF710 is a transcription factor (TF), and zinc finger protein 710 (ZNF710)-AS1-201 is an immune-related long noncoding RNA (lncRNA) that is upregulated in GC cells.

Aim: To assess the correlation between ZNF710-AS1-201 and immune microenvironment features and to investigate the roles of ZNF710-AS1-201 in the invasion and metastasis processes of GC cells.

Methods: We obtained data from The Cancer Genome Atlas and Wujin Hospital. We assessed cell growth, migration, invasion, and programmed cell death using cell counting kit-8, EdU, scratch, Transwell, and flow cytometry assays. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to identify the potential downstream targets of ZNF710-AS1-201.

Results: In GC tissues with low ZNF710-AS1-201 expression, immunoassays detected significant infiltration of various antitumor immune cells, such as memory CD8 T cells and activated CD4 T cells. In the low-expression group, the half-maximal inhibitory concentrations (IC₅₀s) of 5-fluorouracil, cisplatin, gemcitabine, and trametinib were lower, whereas the IC₅₀s of dasatinib and vorinostat were higher. The malignant degree of GC was higher and the stage was later in the high-expression group. Additionally, patients with high expression of ZNF710-AS1-201 had lower overall survival and disease-free survival rates. In vitro, the overexpression of ZNF710-AS1-201 greatly enhanced growth, metastasis, and infiltration while suppressing cell death in HGC-27 cells. In contrast, the reduced expression of ZNF710-AS1-201 greatly hindered cell growth, enhanced apoptosis, and suppressed the metastasis and invasion of MKN-45 cells. The expression changes in ZNF710 were significant, but the corresponding changes in isocitrate dehydrogenase-2, Semaphorin 4B, ARHGAP10, RGMB, hsa-miR-93-5p, and ZNF710-AS1-202 were not consistent or statistically significant after overexpression or knockdown of ZNF710-AS1-201, as determined by qRT-PCR.

Conclusion: Immune-related lncRNA ZNF710-AS1-201 facilitates the metastasis and invasion of GC cells. It appears that ZNF710-AS1-201 and ZNF710 have potential as effective targets for therapeutic intervention in GC. Nevertheless, it is still necessary to determine the specific targets of the ZNF710 TF.

Keywords: Apoptosis; Gastric cancer; Invasion; Metastasis; Proliferation; ZNF710-AS1-201.

Figure 1

Immune correlation analysis of zinc finger protein 710-AS1-201. A: The infiltration levels of 28 immune cells in the high and low expression (LExp) groups; B: The stromal score, immune score, and ESTIMATE score in the high and LExp groups; C: The expression levels of 19 immune checkpoints in the high and LExp groups. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ns: Not significant.

Figure 2

Sensitivity to different antitumor drugs in the high and low expression groups. A: Half-maximal inhibitory concentrations (IC₅₀s) of 5-fluorouracil; B: IC₅₀s of axitinib; C: IC₅₀s of cisplatin; D: IC₅₀s of gemcitabine; E: IC₅₀s of trametinib; F: IC₅₀s of vorinostat. IC₅₀s: Half-maximal inhibitory concentrations.

Figure 3

Gene ontology and kyoto encyclopedia of genes and genomes analysis of zinc finger protein 710-AS1-201. A: Gene ontology assessment of differentially expressed genes (DEGs) between the high and low expression (LExp) groups; B: Kyoto encyclopedia of genes and genomes evaluation of DEGs between the high and LExp groups.

Figure 4

Zinc finger protein 710-AS1-201 is upregulated in gastric cancer tissues and cell lines. A: The expression level of zinc finger protein 710 (ZNF710)-AS1-201 in 64 paired gastric cancer and nontumoral tissues was determined by quantitative real-time polymerase chain reaction (qRT-PCR) assay; B and C: Patients with high expression of ZNF710-AS1-201 had lower overall survival and disease-free survival rates; D: The expression level of ZNF710-AS1-201 in a normal human gastric mucosa cell line and gastric cancer cell lines (HGC-27, AGS, NCI-N87 and MKN-45) was determined by qRT-PCR; E: The expression level of ZNF710-AS1-201 in HGC-27 cells after transfection with the overexpression plasmid; F: The expression level of ZNF710-AS1-201 in MKN-45 cells after transfection with the shRNA plasmid. Data are shown as the mean ± SD. ZNF710: Zinc finger protein 710. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ns: Not significant.

Figure 5

Zinc finger protein 710-AS1-201 promotes gastric cancer cell viability and proliferation. A: Cell viability was examined by cell counting kit-8 assay after cells were transfected for 24 h, 48 h, and 72 h; B: The cell proliferation rate was detected by EdU assay. ^aP < 0.05; ^bP < 0.01.

Figure 6

Zinc finger protein 710-AS1-201 inhibits gastric cancer cell apoptosis. A: Quantitative real-time polymerase chain reaction was used to detect the expression levels of apoptosis-related genes (Bax and Bcl-2); B: Western blot analysis was used to detect the expression levels of apoptosis-related proteins (Bax and Bcl-2); C: Cell apoptosis in HGC-27 cells was examined by flow cytometry after transfection with the zinc finger protein 710 (ZNF710)-AS1-201 overexpression plasmid for 24 h, 48 h, and 72 h; D: Cell apoptosis in MKN-45 cells was examined by flow cytometry after transfection with the ZNF710-AS1-201 shRNA plasmid for 24 h, 48 h, and 72 h. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ns: Not significant.

Figure 7

Zinc finger protein 710-AS1-201 promotes gastric cancer cell metastasis and invasion. A: Cell metastasis in HGC-27 cells was examined by scratch assays after transfection of zinc finger protein 710 (ZNF710)-AS1-201-overexpressing plasmids for 24 h, 48 h, and 72 h; B: Cell metastasis in MKN-45 cells was examined by scratch assay after transfection with ZNF710-AS1-201 shRNA plasmid for 24 h, 48 h, and 72 h; C: Cell invasion was examined by Transwell assay after transfection for 72 h. ZNF710: Zinc finger protein 710. ^aP < 0.05; ^bP < 0.01; ns: Not significant.

Figure 8

Prediction of downstream targets of zinc finger protein 710-AS1-201. A: The expression levels of genes in HGC-27 cells transfected with the zinc finger protein 710 (ZNF710)-AS1-201 overexpression plasmid were determined by quantitative real-time polymerase chain reaction (qRT-PCR); B: The expression levels of genes in MKN-45 cells transfected with the ZNF710-AS1-201 shRNA plasmid were determined by qRT-PCR. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ns: Not significant.