沉默干扰HMGA2对胃癌细胞Wnt/β-Catenin信号转导通路的影响

Effect of HMGA2 gene silencing on Wnt/β-Catenin signaling pathway in gastric cancer cells

目的:观察小分子RNA沉默HMGA2在胃癌细胞株MKN-45的表达,研究HMGA2对Wnt/β-Catenin通路中主要分子β-Catenin以及下游靶分子c-myc、CyclinD1的表达量及转录活性的影响,探讨HMGA2对Wnt/β-Catenin信号转导通路的影响.方法:构建针对人HMGA2基因的shRNA真核表达载体,瞬时转染人胃癌细胞株MKN-45,用RT-PCR、Western blot分别检测转染后shHMGA2组、scrambled组和空白对照组细胞中HMGA2 mRNA和蛋白的表达情况,评估抑制效应;分别检测转染后3组细胞中的β-Catenin、c-myc、Cyclin D1 mRNA和蛋白的表达水平.结果:转染48h后shHMGA2-1组的HMGA2 mRNA相对表达量(0.58±0.07),与shHMGA2-2组(0.92±0.13)、shHMGA2-3组(0.90±0.16)、scrambled组(1.07±0.14)及空白对照组(1.19±0.09)相比有统计学意义(P<0.05),其他4组相比无统计学意义(P>0.05);转染48h后shHMGA2-1组的HMGA2沉默效果最好,HMGA2 mRNA表达明显下降,抑制率51.3%;故选用质粒shHMGA2-1继续后续实验;Westernblot结果显示转染72h后shHMGA2-1组HMGA2蛋白相对表达量为(0.11±0.03),与scrambled组(0.48±0.12)及空白对照组(0.55±0.08)相比有统计学意义(P<0.05),HMGA2蛋白表达明显下降,抑制率80%;沉默干扰HMGA248h和72h后,β-Caten in mRNA和蛋白表达水平在空白对照组(1.07±0.02,0.69±0.04)与scrambled组(0.91±0.02,0.67±0.10)中无明显差异(P>0.05),而shHMGA2-1组(0.53±0.04,0.44±0.05)较之两组有明显的下降(P<0.05);shHMGA2-1组中c-myc mRNA和蛋白相对表达量为(0.39±0.04,0.25±0.07)较之空白对照组(0.88±0.05,0.75±0.09)、scrambled组(0.84±0.03,0.66±0.10)有明显下降(P<0.05),后两组相比较无统计学意义(P>0.05);shHMGA2-1组中Cyclin D1 mRNA和蛋白相对表达量为(0.31±0.02,0.12±0.01)较之空白对照组(0.52±0.03,0.73±0.12)、scrambled组(0.51±0.01,0.63±0.07)有明显下降(P<0.05),后两组相比较无统计学意义(P>0.05).结论:RNAi载体能有效地抑制HMGA2在胃癌细胞株MKN-45中的表达,沉默干扰HMGA2抑制了Wnt/β-Catenin通路中的主要分子β-Catenin以及下游靶分子c-myc、Cyclin D1的表达,HMGA2基因可能是通过调控Wnt/β-Catenin通路对胃癌细胞生长和凋亡发挥作用的.

AIM：To induce HMGA2 gene silencing with shRNAs in gastric cancer cell line MKN-45 and to study the interaction between HMGA2 and the Wnt/β-Catenin signaling pathway. METHODS： A shRNA eukaryotic expression vector that expresses shRNAs of HMGA2 was constructed and transfected into gastric cancer cell line MKN-45. The mRNA and protein expression of HMGA2 was measured by RT-PCR and Western blot 48 h and 72 h after transfection to evaluate the effect of RNA interference. The mRNA and protein expression of β-Catenin,c-myc and cyclin D1 were also measured by RT-PCR and Western blot.RESULTS： The expression of HMGA2 mRNA 48 h after transfection was significantly lower in the shHMG-A2-1 group than in the shHMGA2-2 group,shHMGA2-3 group,scrambled group and blank control group （0.58±0.07 vs 0.92±0.13,0.90±0.16,1.07±0.14,1.19±0.09,all P〈0.05）,but showed no significant difference among the latter four groups （all P〈0.05）. Since HMGA2 expression was most significantly silenced in the shHMGA2-1 group （51.3% at 48 h）,the plasmid pLLU2G-shHMGA2-1 was chosen for use in subsequent experiments. The expression of HMGA2 protein 72 h after transfection in the shHMGA2-1 group was significantly lower than that in the scrambled group and blank group （0.11±0.03 vs 0.48±0.12,0.55±0.08,both P〈0.05）. The silencing efficiency of transfection of shHMGA2-1 was 80% at 72 h. After silencing the HMGA2 gene,the expression of β-Catenin,c-myc and cyclin D1 mRNAs and proteins was significantly inhibited in the shHMGA2-1 group compared to the blank control group and the scrambled group （β-Catenin mRNA： 0.53±0.04 vs 1.07±0.02,0.91±0.02;β-Catenin protein： 0.44±0.05 vs 0.69±0.04,0.67±0.10; c-myc mRNA： 0.39±0.04 vs 0.88±0.05,0.84±0.03; c-myc protein： 0.25±0.07 vs 0.75±0.09,0.66±0.10; cyclin D1 mRNA： 0.31±0.02 vs 0.52±0.03,0.51±0.01; cyclin D1 protein： 0.12±0.01 vs 0.73±0.12,0.61±0.07; all P〈0.05）.CONCLUSION： The recombinant plasmid PLLU2G-shHMGA2 could effectively inhibit the expression of HMGA2 gene in gastric cancer cell line MKN-45. Silencing of the HMGA2 gene restrained the expression of β-Catenin and its downstream target genes c-myc and cyclin D1. HMGA2 controls the growth and apoptosis of gastric cancer cells possibly via the Wnt/β-Catenin signal pathway.