沉默PRMT6基因对胃癌细胞增殖和迁移的影响

doi:10.3760/cma.j.cn371439-20200221-00042

摘要/Abstract

摘要：

目的研究沉默蛋白质精氨酸甲基转移酶6(PRMT6)基因对胃癌细胞株MGC-803增殖和迁移的影响,并探讨其相关分子机制。方法实时荧光定量聚合酶链反应(qRT-PCR)与Western blotting检测人正常胃黏膜上皮细胞GSE-1和人胃癌细胞株(AGS、SGC-7901、MGC-803)中PRMT6 mRNA与蛋白的表达水平。胃癌细胞株MGC-803分为沉默对照组(NC)、PRMT6沉默组(si-PRMT6)、过表达核转录因子-κB(NF-κB/p65)组(pcDNA-p65)、si-PRMT6+pcDNA-p65组及PRMT6沉默同时过表达基质金属蛋白酶9(MMP9)组(si-PRMT6+pcDNA-MMP9)。Western blotting检测PRMT6、NF-κB/p65和MMP9的蛋白表达水平;CCK-8实验、Transwell实验分别检测细胞增殖、迁移率。结果 qRT-PCR结果显示,PRMT6 mRNA 在GSE-1、AGS、SGC-7901、MGC-803细胞中的相对表达水平分别为1.041±0.114、2.141±0.132、2.716±0.231、2.825±0.300,4组间差异有统计学意义(F=46.082,P<0.001),与GSE-1细胞相比,PRMT6 mRNA表达水平在AGS、SGC-7901、MGC-803细胞中明显升高(均P<0.001)。Western blotting结果显示,PRMT6 蛋白在GSE-1、AGS、SGC-7901、MGC-803细胞中的相对表达水平分别为1.090±0.101、2.847±0.331、2.925±0.419、3.278±0.463,4组间差异有统计学意义(F=22.683,P<0.001),与GSE-1细胞相比,PRMT6蛋白表达水平在AGS、SGC-7901、MGC-803细胞中明显升高(P=0.008;P=0.002;P=0.003)。MGC-803细胞沉默PRMT6 48 h后,NC组和si-PRMT6组中的PRMT6 mRNA表达水平分别为0.921±0.110、0.303±0.045;PRMT6蛋白表达水平为分别为1.032±0.105、0.289±0.043;细胞增殖活性分别为0.917±0.089、0.660±0.069;细胞迁移率为(89.122±5.109)%、(30.831±4.463)%;p-p65/p65的蛋白相对表达量比值分别为0.947±0.143、0.285±0.023;si-PRMT6组PRMT6 mRNA表达水平、PRMT6蛋白表达水平、细胞增殖活性、细胞迁移率、p-p65/p65的蛋白相对表达量比值均显著低于NC组(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结果显示,NC组、si-PRMT6组、pcDNA-p65组与si-PRMT6+pcDNA-p65组中MMP9的蛋白相对表达量为1.202±0.138、0.318±0.018、2.849±0.217与1.595±0.194,4组间差异有统计学意义(F=127.410,P<0.001),进一步两两比较,si-PRMT6组MMP9的蛋白相对表达量显著低于NC组(P<0.001),而si-PRMT6+pcDNA-p65组MMP9的蛋白相对表达量显著低于pcDNA-p65组(P=0.002)。MGC-803细胞转染si-PRMT6同时转染pcDNA-p65或pcDNA-MMP9 48 h后,NC组、si-PRMT6组、si-PRMT6+pcDNA-p65及si-PRMT6+pcDNA-MMP9组细胞增殖活性分别为0.923±0.054、0.608±0.024、0.818±0.035与0.807±0.029,4组间差异有统计学意义(F=37.343,P<0.001),进一步两两比较,si-PRMT6+pcDNA-p65组与si-PRMT6+pcDNA-MMP9组细胞增殖活性显著高于si-PRMT6组(均P<0.001);4组的细胞迁移率分别为(85.195±3.176)%、(28.419±1.845)%、(60.490±7.231)%与(53.653±6.761)%,4组间差异有统计学意义(F=59.672,P<0.001);进一步两两比较,si-PRMT6+pcDNA-p65组与si-PRMT6+pcDNA-MMP9组细胞迁移率显著高于si-PRMT6组(P=0.002;P=0.003)。结论 PRMT6沉默可抑制NF-κB/MMP9信号通路的活化,进而抑制胃癌细胞MGC-803的增殖和迁移。

关键词: 胃肿瘤, NF-κB, 基质金属蛋白酶9, 蛋白质精氨酸甲基转移酶6

Abstract:

Objective To 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. Methods The 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. Results The 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 (all P<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 (both P<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). Conclusion PRMT6 silencing can inhibit the proliferation and migration of gastric cancer cells MGC-803 via deactivation of NF-κB/MMP9 signaling pathway.

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

刘家铭, 原姗姗, 王雨, 司望利. 沉默PRMT6基因对胃癌细胞增殖和迁移的影响[J]. 国际肿瘤学杂志, 2020, 47(7): 385-390.

Liu Jiaming, Yuan Shanshan, Wang Yu, Si Wangli. Effects of silencing PRMT6 gene on proliferation and migration of gastric cancer cells[J]. Journal of International Oncology, 2020, 47(7): 385-390.

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组别	si-PRMT6转染时间
组别	48 h	72 h	96 h
沉默对照组	0.917±0.089	1.353±0.110	1.572±0.128
si-PRMT6组	0.660±0.069	0.812±0.077	0.898±0.104
t值	3.954	6.979	7.079
P值	0.017	0.002	0.002