RNA干扰沉默DJ-1基因对Hep-2细胞裸鼠移植瘤生长的影响

Effect of DJ-1 silencing by RNA interference on growth of xenografted human laryngeal squamous cell carcinoma Hep-2 cells in nude mice

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.