白细胞介素-1受体Ⅱ对胃癌细胞的增殖、迁移和侵袭能力的影响

Effects of interleukin-1 receptor Ⅱ on proliferation, migration and invasion of gastric cancer cells

目的探讨人白细胞介素-1受体Ⅱ(IL-1R2)对胃癌细胞生物学行为的影响及其对血管的影响。方法用反转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹法检测胃癌细胞株(AGS、MGC-803、BGC-823、SGC-7901、HGC-27、HSC-39)及人胃黏膜细胞(GES-1)中IL-1R2的mRNA、蛋白水平, 选择MGC-803、BGC-823构建下调及过表达IL-1R2细胞模型。细胞计数试剂盒(CCK-8)细胞增殖、划痕实验、Transwell侵袭实验明确IL-1R2对胃癌细胞增殖、迁移、侵袭能力的影响。细胞周期及凋亡实验探究IL-1R2对细胞周期及凋亡的作用。RT-PCR检测下调及过表达IL-1R2的MGC-803、BGC-823细胞中血管内皮生长因子(vascular endothelial growth factor, VEGF) mRNA水平。下调、过表达IL-1R2细胞组及其对照组与血管内皮细胞(HMEC-1)共培养, 明确IL-1R2对HMEC-1增殖、迁移及VEGF、IL-6 mRNA水平的影响。两组间差异表达采用t检验方法比较;多组间采用方差分析(ANOVA)和Tukey trend检验。结果 RT-PCR、蛋白免疫印迹实验证明胃癌细胞株(AGS、MGC-803、BGC-823、SGC-7901、HGC-27)中IL-1R2的mRNA及蛋白水平较GES-1显著升高, 差异有统计学意义(mRNA:1.944±0.310、4.777±0.320、2.214±0.100、3.235±0.121、4.809±0.190比1.000±0.118, t=2.971、11.54、10.01、16.06、18.81, P<0.05, P<0.01;蛋白:2.010±0.151、4.445±0.362、1.988±0.113、2.821±0.083、4.777±0.280比1.000±0.087, t=5.973、11.54、10.01、16.06、18.81, P<0.01, P<0.01)。细胞增殖、划痕实验、Transwell侵袭实验提示IL-1R2可促进胃癌细胞的增殖(下调组48、72、96 h:0.298±0.028比0.438±0.030、0.418±0.030比0.592±0.026、0.599±0.025比0.779±0.014, t=3.415、4.396、6.193, P<0.05, P<0.01;过表达组72、96 h:0.745±0.051比0.562±0.009、0.886±0.025比0.790±0.015, t=3.577、3.306, P<0.05, P<0.01)、迁移(下调组:0.620±0.070比1.000±0.080, t=3.587, P<0.05;过表达组:1.643±0.116比1.000±0.067, t=4.810, P<0.01)和侵袭(下调组:76.670±4.631比144.300±4.978, t=9.953, P<0.01;过表达组:195.700±4.807比141.700±7.219, t=6.226, P<0.01)。细胞周期实验表明下调IL-1R2的胃癌细胞中S期细胞增多(32.700±0.879比25.500±0.209, t=7.967, P<0.01), 但未进入G2、M期。RT-PCR实验结果表明VEGF mRNA在IL-1R2下调的MGC-803细胞中含量低于空载体对照组(0.573±0.039比1.000±0.011, t=3.721, P<0.05), VEGF mRNA在IL-1R2过表达的BGC-823细胞中含量高于空载体对照组(1.368±0.052比1.000±0.084, t=3.715, P<0.05)。共培养实验提示下调IL-1R2的MGC-803细胞组HMEC-1的侵袭能力显著降低(55.330±3.756比77.670±5.925, t=3.183, P<0.05), 而过表达IL-1R2的BGC-823组HMEC-1的侵袭能力显著增强(103.700±4.978比80.330±3.383, t=3.877, P<0.05);下调IL-1R2的MGC-803细胞与HMEC-1共培养, HMEC-1中VEGF mRNA含量低于空载体对照组(0.446±0.023比1.000±0.029, t=15.03, P<0.01), IL-6 mRNA水平与空载体对照组比较, 差异无统计学意义(P>0.05);过表达IL-1R2的BGC-823细胞与HMEC-1共培养, HMEC-1中VEGF mRNA高于空载体对照组(1.896±0.236比1.000±0.048, t=3.717, P<0.05), IL-6 mRNA水平与空载体对照组比较, 差异无统计学意义(P>0.05)。结论 IL-1R2能促进胃癌细胞的增殖、迁移、侵袭能力, 同时能促进肿瘤血管的生成。

Objective To investigate the effect of human interleukin-1 receptorⅡ(IL-1R2)on biological behavior of gastric cancer cells and its effect on blood vessels.Methods mRNA and protein levels of IL-1R2 in various gastric cancer cell lines(AGS,MGC-803,BGC-823,SGC-7901,HGC-27,HSC-39)and human gastric mucosa cells(GES-1)were detected by reverse transcriptase-polymerase chain reaction(RT-PCR)and Western blotting.MGC-803 and BGC-823 were selected to construct down-regulated and overexpressed IL-1R2 cell models.The effects of IL-1R2 on the proliferation,migration and invasion of gastric cancer cells were determined by cell counting kit-8(CCK-8)assay,scratch test and Transwell invasion test.The effects of IL-1R2 on cell cycle and apoptosis were investigated by cell cycle and apoptosis experiment.Vascular endothelial growth factor(VEGF)mRNA levels in MGC-803 and BGC-823 cells with down-regulated or overexpressed IL-1R2 were detected by RT-PCR.The cells in the down-regulated and overexpressed IL-1R2 groups and the control group were co-cultured with vascular endothelial cells(HMEC-1)to determine the effects of IL-1R2 on the proliferation and migration of HMEC-1 and the mRNA levels of VEGF and IL-6.The difference expression between the two groups was compared by t test.Analysis of variance(ANOVA)and Tukey trend test were used among the groups.Results RT-PCR and Western blotting showed that the mRNA and protein levels of IL-1R2 in gastric cancer cell lines(AGS,MGC-803,BGC-823,SGC-7901,HGC-27)were significantly higher than GES-1(mRNA:1.944±0.310,4.777±0.320,2.214±0.100,3.235±0.121,4.809±0.190 vs.1.000±0.118,t=2.971,11.54,10.01,16.06,18.81,P<0.05,P<0.01;Protein:2.010±0.151,4.445±0.362,1.988±0.113,2.821±0.083,4.777±0.280 vs.1.000±0.087,t=5.973,11.54,10.01,16.06,18.81,P<0.01,P<0.01).CCK-8 assay,scratch test and Transwell invasion test suggested that IL-1R2 could promote the proliferation of gastric cancer cells(48,72,96 h in down-regulated group:0.298±0.028 vs.0.438±0.030,0.418±0.030 vs.0.592±0.026,0.599±0.025 vs.0.779±0.014,t=3.415,4.396,6.193,P<0.05,P<0.01;in the overexpression group at 72 and 96 h:0.745±0.051 vs.0.562±0.009,0.886±0.025 vs.0.790±0.015,t=3.577,3.306,P<0.05,P<0.01),migration(down regulation group:0.620±0.070 vs.1.000±0.080,t=3.587,P<0.05;overexpression group:1.643±0.116 vs.1.000±0.067,t=4.810,P<0.01)and invasion(down-regulation group:76.670±4.631 vs.144.300±4.978,t=9.953,P<0.01;in the overexpression group,195.700±4.807 vs.141.700±7.219,t=6.226,P<0.01).The cell cycle experiment showed that the number of S phase cells increased in gastric cancer cells down-regulated by IL-1R2(32.700±0.879 vs.25.500±0.209,t=7.967,P<0.01),but did not enter G2 and M phase.The results of RT-PCR showed that the content of VEGF mRNA in MGC-803 cells with down-regulated IL-1R2 was significantly lower than that in empty carrier control group(0.573±0.039 vs.1.000±0.011,t=3.721,P<0.05).VEGF mRNA content in BGC-823 cells with overexpressed IL-1R2 was significantly higher than that in empty carrier control group(1.368±0.052 vs.1.000±0.084,t=3.715,P<0.05).The co-culture experiment indicated that the invasion ability of HMEC-1 in MGC-803 cell group with down-regulated IL-1R2 was significantly decreased(55.330±3.756 vs.77.670±5.925,t=3.183,P<0.05).The invasion ability of HMEC-1 in BGC-823 group overexpressing IL-1R2 was significantly enhanced(103.700±4.978 vs.80.330±3.383,t=3.877,P<0.05).MGC-803 cells with down-regulated IL-1R2 were co-cultured with HMEC-1.VEGF mRNA content in HMEC-1 was lower than that in empty carrier control group(0.446±0.023 vs.1.000±0.029,t=15.03,P<0.01),and IL-6 mRNA level was higher than that in empty carrier control group.There was no significant difference(P>0.05).When BGC-823 cells overexpressing IL-1R2 were co-cultured with HMEC-1,VEGF mRNA in HMEC-1 was higher than that in empty carrier control group(1.896±0.236 vs.1.000±0.048,t=3.717,P<0.05),and IL-6 mRNA level was higher than that in empty carrier control group.There was no significant difference(P>0.05).Conclusion IL-1R2 can promote the proliferation,migration and invasion of gastric cancer cells,and promote the formation of tumor blood vessels.