Long non-coding RNA GATA6-AS1 is mediated by N6-methyladenosine methylation and inhibits the proliferation and metastasis of gastric cancer

Figure 1 The expression levels of GATA6-AS1 in gastric cancer tissues. The expression levels of GATA6-AS1 in gastric cancer tissues were significantly down-regulated when compared with normal tissues. ^bP < 0.01. STAD: Stomach adenocarcinoma; Num: Number; T: Tumor tissues; N: Normal tissues.

Figure 2 The effects of GATA6-AS1 overexpression on the biological function of gastric cancer cells. A: The overexpression vector was constructed by lentivirus transfection, and the transfection efficiency was verified by reverse transcription-polymerase chain reaction; B: Cell counting kit-8 assays showed that the overexpression of GATA6-AS1 reduced the proliferation ability of HGC-27 and AGS gastric cancer cell lines when compared with the blank and negative controls; C: Trans-well assays showed that when compared with the negative control and blank control groups, the migration and invasion ability of AGS and HGC-27 cells in the GATA6-AS1 overexpression group were decreased. ^aP < 0.05. ^bP < 0.01. Control: Blank control group; NC: Negative control group; OE-GATA6-AS1: GATA6-AS1 overexpression group.

Figure 3 The binding of GATA6-AS1 to methylase. A: RIP experiments confirmed that of the three m6A methylases, fat mass and obesity-associated protein, YTH domain-containing family protein 1 and methyltransferase-like 3, GATA6-AS1 bound most strongly to the m6A demethylase fat mass and obesity-associated protein (FTO) in AGS cells; B: RIP experiment performed in HGC-27 cells also demonstrated that GATA6-AS1 has strong binding ability with m6A demethylase FTO. ^aP < 0.05. ^bP < 0.01. FTO: Fat mass and obesity-associated protein; YTHDF1: YTH domain-containing family protein 1; METTL3: Methyltransferase-like 3.

Figure 4 The expression of fat mass and obesity-associated protein in gastric cancer and its relationship with GATA6-AS1. A: Bioinformatics analysis showed that the expression levels of fat mass and obesity-associated protein (FTO) in gastric cancer tissues were significantly higher than those in normal tissues; B and C: Reverse transcription-polymerase chain reaction and western blotting experiments confirmed that the expression levels of FTO in AGS and HGC-27 gastric cancer cells were higher than those in GES-1 gastric mucosa cells; D: TCGA database analysis revealed that the expression levels of FTO were negatively correlated with GATA6-AS1. ^aP < 0.05. ^bP < 0.01. STAD: Stomach adenocarcinoma; Num: Number; T: Tumor tissues; N: Normal tissues; FTO: Fat mass and obesity-associated protein.

Figure 5 GATA6-AS1 expression was up-regulated after fat mass and obesity-associated protein knockdown. A and B: Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to verify the interference efficiency of small interfering RNA of fat mass and obesity-associated protein had the best interference efficiency; C: The expression of GATA6-AS1 in AGS cells was up-regulated after fat mass and obesity-associated protein expression was down-regulated, as determined by RT-PCR. ^aP < 0.05. ^bP < 0.01. Control: Blank control group; Si-NC: Small interfering RNA transfection/NC group; Si-FTO: Small interfering RNA of fat mass and obesity-associated protein; FTO: Fat mass and obesity-associated protein.

Figure 6 The effect of fat mass and obesity-associated protein up-regulation on the biological function of gastric cancer cells. A: The results of cell counting kit-8 proliferation assays showed that when compared with the negative control group, the proliferation ability of AGS cells in the fat mass and obesity-associated protein (FTO) interference group was decreased; B: Trans-well migration and invasion assays showed that the migration and invasion ability of AGS cells in the FTO interference group were decreased when compared with the negative control group. ^aP < 0.05. ^bP < 0.01. Control: Blank control group; Si-NC: Small interfering RNA transfection/NC group; Si-FTO: Small interfering RNA of fat mass and obesity-associated protein.