Abstract:

ObjectiveTo study the effects of silencing protein arginine methyltransferase 6 (PRMT6) gene on cell proliferation and migration of gastric cancer cell line MGC-803, and explore its related molecular mechanism.MethodsThe expression levels of PRMT6 mRNA and protein in human normal gastric mucosa epithelial cell line GES-1 and human gastric cancer cell lines (AGS, SGC-7901, MGC-803) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The gastric cancer cell line MGC-803 was divided into silencing control group (NC group), PRMT6 silencing group (si-PRMT6 group), nuclear factor-κB (NF-κB/p65) overexpression group (pcDNA-p65 group),si-PRMT6+pcDNA-p65 group and PRMT6 silencing and matrix metalloproteinase 9 (MMP9) overexpression group (si-PRMT6+pcDNA-MMP9 group). Western blotting was used to detect the protein expression levels of PRMT6, NF-κB/p65 and MMP9. CCK-8 kit and Transwell assay were used to measure cell proliferation and migration rates.ResultsThe results of qRT-PCR showed that the relative expression levels of PRMT6 mRNA in GSE-1, AGS, SGC-7901 and MGC-803 cells were 1.041±0.114, 2.141±0.132, 2.716±0.231, 2.825±0.300, and the difference among the four groups was statistically significant (F=46.082,P<0.001). Compared with GSE-1 cells, PRMT6 mRNA expression levels were significantly increased in AGS, SGC-7901 and MGC-803 cells (allP<0.001). Western blotting results showed that the relative expression levels of PRMT6 protein in GSE-1, AGS, SGC-7901 and MGC-803 cells were 1.090±0.101, 2.847±0.331, 2.925±0.419 and 3.278±0.463, with a statistically significant difference (F=22.683,P<0.001). Compared with GSE-1 cells, PRMT6 protein expression levels were significantly increased in AGS, SGC-7901 and MGC-803 cells, with statistically significant differences (P=0.008;P=0.002;P=0.003). After 48 hours of silencing PRMT6 in MGC-803 cells, in NC group and si-PRMT6 group, the PRMT6 mRNA expression levels were 0.921±0.110 and 0.303±0.045, the PRMT6 protein expression levels were 1.032±0.105 and 0.289±0.043, the cell proliferation activities were 0.917±0.089 and 0.660±0.069, the cell migration rates were (89.122±5.109)% and (30.831±4.463)%, and the p-p65/p65 protein relative expression ratios were 0.947±0.143 and 0.285±0.023. The relative expression levels of PRMT6 mRNA and protein, cell proliferation activity, cell migration rate, protein relative expression ratio of p-p65/p65 in si-PRMT6 group were significantly lower than those in NC group, with statistically significant differences (t=9.006,P<0.001;t=11.338,P<0.001;t=3.954,P=0.017;t=14.881,P<0.001;t=7.919,P<0.001). Western blotting results showed that the MMP9 protein relative expression levels in NC group, si-PRMT6 group, pcDNA-p65 group and si-PRMT6+pcDNA-p65 group were 1.202±0.138, 0.318±0.018, 2.849±0.217 and 1.595±0.194, with a statistically significant difference (F=127.410,P<0.001). Further pairwise comparison showed that the protein relative expression level of MMP9 in si-PRMT6 group was significantly lower than that in NC group (P<0.001), while that in si-PRMT6+pcDNA-p65 group was significantly lower than that in pcDNA-p65 group (P=0.002). Then, MGC-803 cells were co-transfected with si-PRMT6 and pcDNA-p65 or pcDNA-MMP9 for 48 h. The cell proliferation activities in NC group, si-PRMT6 group, si-PRMT6+pcDNA-p65 group and si-PRMT6+pcDNA-MMP9 group were 0.923±0.054, 0.608±0.024, 0.818±0.035 and 0.807±0.029, with a statistically significant difference (F=37.343,P<0.001). Further pairwise comparison showed that the cell proliferation activity of si-PRMT6+pcDNA-p65 group or si-PRMT6+pcDNA-MMP9 group was significantly higher than that of si-PRMT6 group (bothP<0.001). The cell migration rates of above four groups were (85.195±3.176)%, (28.419±1.845)%, (60.490±7.231)% and (53.653±6.761)%, with a statistically significant difference (F=59.672,P<0.001). Further pairwise comparison showed that the cell migration rate of si-PRMT6+pcDNA-p65 group or si-PRMT6+pcDNA-MMP9 group was significantly higher than that of si-PRMT6 group (P=0.002;P=0.003).ConclusionPRMT6 silencing can inhibit the proliferation and migration of gastric cancer cells MGC-803viadeactivation of NF-κB/MMP9 signaling pathway.

Key words:Stomach neoplasms,NF-kappa B,Matrix metallopeptidase 9,Protein arginine methyltransferase 6