【摘要】 目的探讨微RNA(miR)-139-5p对胃癌细胞增殖和侵袭的影响及可能的作用机制。方法采用实时荧光定量聚合酶链反应(PCR)检测永生化胃黏膜上皮细胞GES1和胃癌细胞SGC-7901、AGS和BGC-823中miR-139-5p的表达,Western blot法检测GES1、SGC-7901、AGS和BGC-823细胞中Wnt3a、β-catenin和细胞周期蛋白D1(cyclin D1)蛋白表达。收集对数生长期胃癌细胞SGC-7901,待细胞贴壁生长融合度达50%～60%时将细胞分为miR-139-5p mimics组、miR-NC组和空白对照组,利用脂质体Lipofectamine 2000分别将miR-139-5p mimics、阴性对照质粒转染至miR-139-5pmimics组和miR-NC组SGC-7901细胞,空白对照组细胞不转染任何质粒。采用实时荧光定量PCR法检测3组SGC-7901细胞中miR-139-5p的表达,甲基噻唑基四唑法检测3组SGC-7901细胞活力,Transwell实验检测3组SGC-7901细胞侵袭能力,Western blot法检测3组SGC-7901细胞中Wnt3a、β-catenin和cyclin D1蛋白表达。结果 SGC-7901、AGS、BGC-823和GES1细胞中miR-139-5p相对表达量分别为0.15±0.02、0.62±0.06、0.50±0.05、1.24±0.14; SGC-7901、AGS和BGC-823细胞中miR-139-5p相对表达量显著低于GES1细胞(P <0.05),SGC-7901细胞中miR-139-5p相对表达量显著低于AGS、BGC-823细胞(P <0.05),AGS与BGC-823细胞中miR-139-5p相对表达量比较差异无统计学意义(P> 0.05)。空白对照组、miR-NC组和miR-139-5p mimics组SGC-7901细胞中miR-139-5p相对表达量分别为1.05±0.10、0.96±0.07、2.01±0.23; miR-139-5p mimics组SGC-7901细胞中miR-139-5p相对表达量显著高于空白对照组和miR-NC组(P <0.05),空白对照组与miR-NC组细胞中miR-139-5p相对表达量比较差异无统计学意义(P> 0.05)。转染后72、96 h,miR-139-5p mimics组SGC-7901细胞活力显著低于空白对照组和miR-NC组(P <0.05),空白对照组与miR-NC组SGC-7901细胞活力比较差异无统计学意义(P> 0.05)。空白对照组、miR-NC组和miR-139-5p mimics组穿膜细胞数分别为98.67±10.26、101.45±8.24、49.33±5.12; miR-139-5p mimics组穿膜细胞数显著少于空白对照组和miR-NC组(P <0.05),空白对照组与miR-NC组穿膜细胞数比较差异无统计学意义(P> 0.05)。miR-139-5p mimics组SGC-7901细胞中Wnt3a、β-catenin和cyclin D1蛋白相对表达量显著低于空白对照组和miR-NC组(P <0.05),空白对照组与miR-NC组SGC-7901细胞中Wnt3a、β-catenin和cyclin D1蛋白相对表达量比较差异无统计学意义(P> 0.05)。结论胃癌细胞中miR-139-5p表达下调,miR-139-5p可能通过阻断Wnt/β-catenin信号通路传导而抑制胃癌细胞SGC-7901的增殖和侵袭,miR-139-5p有望成为治疗胃癌的新靶点。更多还原

【Abstract】 Objective To investigate the effect of microRNA( miR)-139-5 p on the proliferation and invasion of gastric cancer cells and its possible mechanism. Methods The expression of miR-139-5 p in immortalized gastric epithelial cells GES1 and gastric cancer cells SGC-7901,AGS and BGC-823 was detected by real time fluorescent quantitative polymerase chain reaction( PCR). The expression of Wnt3 a,β-catenin and cyclin D1 protein in GES1,SGC-7901,AGS and BGC-823 cells was detected by Western blot method. The SGC-7901 cells in logarithmic growth phase were collected and divided into miR-139-5 p mimics group,miR-NC group and blank control group when the confluence of cell adhesion growth reached50%-60%. The miR-139-5 p mimics and negative control plasmid were transfected into SGC-7901 cells in the miR-139-5 p mimics group and miR-NC group by Lipofectamine 2000,respectively. No plasmid was transfected into SGC-7901 cells in the blank control group. The expression of miR-139-5 p in SGC-7901 cells was detected by real time fluorescent quantitative PCR,the viability of SGC-7901 cells was detected by methyl thiazolyl tetrazolium assay,the invasion ability of SGC-7901 cells was detected by Transwell experiment,and the expressions of Wnt3 a,β-catenin and cyclin D1 protein in SGC-7901 cells of the three groups were detected by Western blot method. Results The relative expressions of miR-139-5 p in SGC-7901,AGS,BGC-823 and GES1 cells was 0. 15 ± 0. 02,0. 62 ± 0. 06,0. 50 ± 0. 05 and 1. 24 ± 0. 14,respectively. The relative expression of miR-139-5 p in SGC-7901,AGS and BGC-823 cells was significantly lower than that in GES1 cells( P < 0. 05). The relative expression of miR-139-5 p in SGC-7901 cells was significantly lower than that in AGS and BGC-823 cells( P < 0. 05). There was no significant difference in the relative expression of miR-139-5 p between AGS and BGC-823 cells( P > 0. 05). The relative expression of miR-139-5 p in SGC-7901 cells in the blank control group,miR-NC group and miR-139-5 p mimics group was1. 05 ± 0. 10,0. 96 ± 0. 07 and 2. 01 ± 0. 23,respectively. The relative expression of miR-139-5 p in SGC-7901 cells in the miR-139-5 p mimics group was significantly higher than that in the blank control group and miR-NC group( P < 0. 05). There was no significant difference in the relative expression of miR-139-5 p between the blank control group and miR-NC group( P >0.05). At 72 and 96 hours after transfection,the viability of SGC-7901 cells in the miR-139-5 p mimics group was significantly lower than that in the blank control group and miR-NC group( P < 0. 05),but there was no significant difference in the viability of SGC-7901 cells between the blank control group and miR-NC group( P < 0. 05). The number of transmembrane cells in the blank control group,miR-NC group and miR-139-5 p mimics group was 98. 67 ± 10. 26,101. 45 ± 8. 24 and49. 33 ± 5. 12,respectively. The number of transmembrane cells in the miR-139-5 p mimics group was significantly less than that in the blank control group and miR-NC group( P < 0. 05),but there was no significant difference in the number of transmembrane cells between the blank control group and miR-NC group( P > 0. 05). The relative expressions of Wnt3 a,β-catenin and cyclin D1 protein in SGC-7901 cells in the miR-139-5 p mimics group were significantly lower than those in the blank control group and miR-NC group( P < 0. 05). There was no significant difference in the relative expressions of Wnt3 a,β-catenin and cyclin D1 protein between the blank control group and miR-NC group( P > 0. 05). Conclusion The expression of miR-139-5 p in gastric cancer cells is down-regulated,miR-139-5 p may inhibit the proliferation and invasion of SGC-7901 cells by blocking Wnt/β-catenin signaling pathway,and miR-139-5 p is expected to become a new target for the treatment of gastric cancer.更多还原