MicroRNA-9 inhibits the gastric cancer cell proliferation by targeting TNFAIP8

Abstract

Background and objectives: MicroRNA-9 is frequently dysregulated in many human carcinoma types, including gastric cancer (GC). Previous studies demonstrated that the expression of TNFAIP8 in GC is correlated with tumour occurrence, development, invasion, metastasis and prognosis. However, till now, the relationship between MicroRNA-9 and TNFAIP8 in GC has not been reported.

Materials and methods: Levels of miR-9 and TNFAIP8 expression in GC tissues and in human GC cell lines were studied using qualitative real-time PCR (qRT-PCR) and Western blotting. Cell viability was detected using the CCK-8 and clone formation assays. A dual-luciferase reporter system was used to confirm the target gene of miR-9.

Results: We found that the expression level of MicroRNA-9 in GC tissues and cell lines was significantly lower than that in adjacent non-cancerous tissues and human immortalized gastric epithelial cell (GES) line, respectively. In addition, overexpression of MicroRNA-9 markedly inhibited GC cell proliferation in vitro and tumour growth in vivo. Further experiments revealed that TNFAIP8 was a direct and functional target of MicroRNA-9 in GC and overexpression of MicroRNA-9 obviously down-regulated the expression of TNFAIP8, which was involved in the gastric carcinogenesis and cancer progression.

Conclusion: Our results suggested that MicroRNA-9-TNFAIP8 might represent a promising diagnostic biomarker for GC patients and could be a potential therapeutic target in the prevention and treatment of GC.

Keywords: TNFAIP8; biomarker; gastric cancer; miR-9; proliferation.

Figure 1

Expression of TNFAIP8 is up‐regulated in gastric cancer tissues and cell lines. A, Quantitative PCR results of TNFAIP8 mRNA levels in GC cell lines. B, Western blotting results of TNFAIP8 protein levels in different GC cell lines. TNFAIP8 protein levels are higher in the five GC cell lines than in the human immortalized gastric epithelial cell line (GES). C, Quantitative PCR results of TNFAIP8 mRNA levels in GC tissues (T) and in adjacent non‐cancerous gastric tissues (ANT). TNFAIP8 mRNA levels are higher in the GC tissues (T) than in adjacent non‐cancerous gastric tissues (ANT) (n=16). D, Western blotting results of TNFAIP8 protein levels in GC tissues and adjacent non‐cancerous gastric tissues. TNFAIP8 protein levels are higher in GC tissues (T) than in adjacent non‐cancerous gastric tissues (ANT) from the same patient (n=16). Each bar represents the mean ± SD of three independent experiments. **P<.01

Figure 2

TNFAIP8 promotes GC cell proliferation. A, Western blotting results of TNFAIP8 protein levels after knocking down TNFAIP8 in different GC cell lines. Knocking‐down TNFAIP8 decreased the protein level of TNFAIP8 in MKN45 and MGC803 cells. B, Cell proliferation was examined by CCK8 assay after knocking down TNFAIP8. Knocking‐down TNFAIP8 inhibited cell proliferation of MKN45 and MGC803 cells compared to cells in the control mock‐treated group. C, Knocking‐down TNFAIP8 suppressed clone formation in MKN45 and MGC803 cells. D, Overexpressing TNFAIP8 increased the protein level of TNFAIP8 in MKN45 and MGC803 cells. E, Overexpressing TNFAIP8 promoted cell proliferation of MKN45 and MGC803 cells compared to cells in the control mock‐treated group. F, Overexpressing TNFAIP8 promoted clone formation in MKN45 and MGC803 cells. Each bar represents the mean ± SD of three independent experiments. *P<.05, **P<.01

Figure 3

TNFAIP8 is a direct target of miR‐9. A, miRNAs were computationally predicted using two independent miRNA databases. B, qPCR results of miR‐9 mRNA levels after transfecting GC cells with miR‐9 mimics or anti‐miR‐9. Overexpression of miR‐9 markedly increased the mRNA level of miR‐9. Conversely, knocking‐down miR‐9 significantly decreased the mRNA level of miR‐9. C, Western blotting results showed that the overexpression of miR‐9 significantly reduced TNFAIP8 protein levels in MKN45 and MGC803 cells, whereas knocking‐down miR‐9 has the opposite results. D, qPCR results of TNFAIP8 mRNA levels after transfecting GC cells with miR‐9 mimics or anti‐miR‐9. E, Bioinformatics predicted wild type or mutant TNFAIP8 3′‐UTR miRNA binding sites were inserted into luciferase reporter plasmids. F, In luciferase activity assays, miR‐9 suppressed luciferase activity of the wild type but not mutant TNFAIP8 3′‐UTR constructs in MKN45. Each bar represents the mean ± SD of three independent experiments. *P<.05, **P<.01

Figure 4

miR‐9 inhibits cell proliferation. A, Overexpression of miR‐9 inhibits MKN45 and MGC‐803 cell proliferation in CCK8 assays. However, knocking down of miR‐9 promotes MKN45 and MGC‐803 cell proliferation. B, Overexpressing miR‐9 suppressed clone formation in MKN45 and MGC803 cells. However, knocking‐down miR‐9 promotes clone formation in MKN45 and MGC803 cells. C, Overexpressing TNFAIP8 rescued miR‐9‐mediated inhibition of TNFAIP8 expression levels in MKN45 and MGC803. D, Restoration of TNFAIP8 rescued miR‐9‐mediated inhibition of cell proliferation. E, MKN45 and MGC803 cells were infected with a lentivirus encoding miR‐9 mimics or an empty lentivirus (control) for 96 h. F, After 96 h of infection, the TNFAIP8 protein levels were examined by Western blotting in MKN45 and MGC803. G, After 96 h of infection, the TNFAIP8 mRNA levels were examined by qPCR in MKN45 and MGC803. H, Representative images of tumours from mice in each group. Each bar represents the mean ± SD of three independent experiments. *P<.05, **P<.01. **P<.01 vs control, #**P<.01 vs miR‐9+ov‐TNFAIP8

Figure 5

Clinical relevance of miR‐9‐TNFAIP8 in fresh GC tissues. A, The relative levels of miR‐9 expression in 16 pairs of fresh GC tissues were determined by qPCR. B, Statistical analysis suggested an inverse correlation between miR‐9 expression and TNFAIP8 protein expression. Each bar represents the mean ± SD of three independent experiments