摘要
目的:观察沉默DJ-1基因对人喉癌Hep-2细胞株裸鼠移植瘤的作用及其机制。方法:建立人喉癌Hep-2裸鼠移植瘤模型,24只裸鼠随机分为三组(每组8只):对照组(5%葡萄糖溶液)、小剂量组(20μg 的DJ-1基因特异性小干扰RNA)、大剂量组(40μg的DJ-1基因特异性小干扰RNA)。各组瘤体内分别注射相应液体50μL,2次/周,共6次,每次测量瘤体质量及大小。停药后48 h脱颈椎法处死裸鼠,剥离瘤体,测瘤体质量,计算抑瘤率。采用免疫组织化学法分别检测剥离瘤体组织Caspase-3、Ki-67的表达情况;实时定量RT-PCR和蛋白质印迹法分别检测各组移植瘤 DJ-1、PTEN、survivin 基因和蛋白的表达。结果:小剂量组[(0.66±0.15) g]和大剂量组[(0.48±0.11) g]的瘤体质量均小于对照组[(0.83±0.16)g,均P <0.05],且抑瘤率分别为(20.48±0.18)%和(42.16±0.13)%。小剂量组和大剂量组Caspase-3表达增高, Ki-67表达减少,与对照组差异均有统计学意义(均P <0.05);与对照组比较,小剂量组和大剂量组DJ-1、survivin的mRNA和蛋白表达均减少(均P<0.05),而PTEN的mRNA和蛋白表达均增加(均P<0.05)。结论:大剂量的DJ-1小干扰RNA可能通过下调DJ-1和促进PTEN的表达,从而阻断PI3 K-PKB/Akt信号通路并调节survivin表达来促进肿瘤细胞凋亡,抑制人喉癌Hep-2裸鼠移植瘤的生长。
Objective: To investigate the effect of silencing DJ-1 on xenografted human laryngeal squamous cell carcinoma ( LSCC ) Hep-2 cells in nude mice. Methods: Xenograft model of human LSCC was established by subcutaneous transplantation of Hep-2 cells in 24 nude mice.The LSCC-bearing nude mice were randomly divided into 3 groups ( n=8 in each):DJ-1 siRNA low dose group and DJ-1 siRNA high dose group were injected in tumors with 20 μg of DJ-1 siRNA or 40 μg of DJ-1 siRNA in 50 μL, respectively; control group was injected with 5% glucose solution in 50 μL, twice a week for 3 weeks.The weight and size of tumors were measured before injection.The animals were sacrificed 48 h after the final treatment, and the tumors were harvested and weighed.The apoptosis and proliferation of tumor cells were determined;the expressions of Caspase-3 and Ki-67 in tumor specimens were detected with immunohistochemistry.The expression of DJ-1, PTEN, survivin mRNA and protein in tumor tissues were detected by RT-PCR and Western blotting, respectively.Results:Tumor weight in low dose group [(0.66 ±0.15) g] and high dose group [(0.48 ±0.11) g] were significantly lower than that in control group [(0.83 ±0.16)g, all P〈0.05].The inhibition rates of low dose group and high dose group were ( 20.48 ± 0.18 )% and ( 42.16 ± 0.13 )%, respectively. Immunohistochemistry showed that the expression of Caspase-3 was increased and Ki-67 was reduced in tumor specimens, compared with the control group ( all P 〈0.05 ) . RT-PCR and Western blot results showed that in low dose group and high dose group the mRNA and protein expression of DJ-1 and survivin significantly decreased ( all P 〈0.05 ) , while PTEN mRNA and protein content increased ( all P 〈0.05 ) . Conclusion: High dose DJ-1 siRNA can inhibit the tumor growth in human LSCC xenograft nude mouse model, which indicates that down-regulating DJ-1 and survivin, and up-regulating PTEN expression may lead to blockage of PI3K-PKB/Akt signaling pathway and promoting tumor cell apoptosis.
出处
《浙江大学学报(医学版)》
CAS
CSCD
北大核心
2016年第4期349-355,共7页
Journal of Zhejiang University(Medical Sciences)
基金
浙江省自然科学基金(LY14H160003)
浙江省医药卫生省部培育计划(2014PYA017)
浙江省医药卫生科技计划项目(2012ZDA042)
宁波市科技创新团队(2012B82019,2015B11050)
宁波市自然科学基金(2012A610208)
浙江省医学会临床科研资金项目(2015ZYC-A49)