摘要
目的观察DNA甲基化转移酶抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-CdR)对人结肠癌裸鼠移植瘤生长的抑制作用,探讨其与移植瘤细胞RUNX3基因表达和甲基化的相关性。方法皮下接种SW480细胞建立人结肠癌裸鼠移植瘤模型。应用随机数字表法分为3组,A组为对照组,腹腔注射磷酸盐缓冲液(2μg/g);B组为低剂量实验组,腹腔注射5-Aza-CdR(1μg/g);C组为高剂量实验组,腹腔注射5-Aza-CdR(2μg/g)。各组每周给药3次,连续给药4周,末次给药后处死裸鼠。观察各组移植瘤生长情况及病理形态学改变,并采用TUNEL法检测移植瘤细胞凋亡情况,MSP法检测RUNX3基因的甲基化状态,Real time RT-PCR及免疫组化检测RUNX3的表达水平,分析组间统计学差异。结果给药4周后,A组移植瘤体积为(3.63±1.00)cm3,B组(3.02±1.43)cm3,C组(1.80±0.80)cm3,C组与A、B组比较,差异有统计学意义,F=7.061,P=0.003。TUNEL法检测结果显示,C组移植瘤凋亡指数较高,为(5.66±0.70)%,与A组(1.14±0.45)%、B组(2.83±0.69)%比较,差异有统计学意义,F=53.631,P<0.001。基因甲基化检测发现,A组有较高的RUNX3基因甲基化水平,而B组及C组的RUNX3基因的非甲基化条带明显扩增;而且在RUNX3基因表达上,A组mRNA相对表达量为14.84±0.53,明显低于B组的21.66±0.45、C组的46.72±2.64,差异有统计学意义,F=910.128,P<0.001;A组RUNX3蛋白阳性表达率为(6.84±2.21)%,亦明显低于B组(14.54±3.98)%及C组(16.25±2.71)%,差异有统计学意义,F=10.745,P=0.004。结论 5-Aza-CdR可有效抑制人结肠癌裸鼠移植瘤的生长,诱导移植瘤细胞凋亡,其机制与逆转RUNX3基因的过甲基化状态,诱导移植瘤细胞RUNX3基因的重新表达相关。
OBJECTIVE To assess the inhibitory effect of 5-Aza-CdR on growth of human colon cancer xenografts in nude mice and investigate association of the inhibitory effect of 5 Aza-CdR with expression and methylation of RUNX3 in the xenografts. METHODS Human colon cancer xenograft model was established by subcutaneous inoculation of hu- man colon cancer cells SW480 in nude mice. The xenografts were randomly divided into three groups through random number table, Control group A （PBS, 2 μg/g）, Experimental group B （5-Aza-CdR, 1 μg/g）, and Experimental group C （5-Aza-CdR,2 μg/g）. All groups received intraperitoneal injection three times per week and for four weeks. The nude mice were sacrificed after the last injection. The xenograft growth and pathomorphology was observed,and the cell apop- tosis was measured through TUNEL method in three groups. The regulations of 5-Aza-CdR on RUNX3 methylation and expression were evaluated by metbylation specific PCR （MSP） ,real time RT-PCR and immunohistochemistry. RESULTS After treatment of 5-Aza-CdR or PBS for 4 weeks,the volumes of xenografts were （3.63± 1.00） cma for group A, （3.02±1.43） cm^3 for group B and （1.80±0.80） cm3 for group C,respectively. Group C got significantly smaller volume of xenografts than group B and A, F=7. 061, P = 0. 003; Group C also showed significantly higher apoptotic index [（5.66±0.70） G],compared to group B [（2. 83±0. 69）%] and group A [（1.14±0.45）G],F= 53. 631,P〈0. 001.DNA methylation detection showed group A had hypermethylation of RUNX3, while unmethylated alleles of RUNX3 got obvious amplification in group B and C. Regarding RUNX3 expression,group A showed obvious amplified expression of RUNX3 on mRNA （14.84±0.53） ,compared to group B （21.66±0.45） and C （46.72±2.64）,F=910. 128,P〈0. 001; Significant difference on protein level was also found among group A [ （6.84±2.21） % ] and group B [（ 14.54±3.98）% ] and C [（16.25±2.71）%],F=-10. 745,P=0. 004. CONCLUSIONS 5-Aza-CdR ef
出处
《中华肿瘤防治杂志》
CAS
北大核心
2015年第5期344-348,共5页
Chinese Journal of Cancer Prevention and Treatment
基金
浙江省医药卫生科技计划(2011KYA062)
