RNA干扰TRAP1表达抑制CD133^+CD44^+喉癌干细胞生长并促进凋亡

RNA interference targeting inhibition of TRAP1 suppresses cell growth and promotes apoptosis in CD133^+CD44^+ laryngeal carcinoma stem cells

背景:肿瘤坏死因子受体相关蛋白1(TRAP1)是线粒体特异性热休克蛋白90家族的同源基因。大量研究表明其过量表达与多种癌症的发生密切相关。但其在喉鳞癌发生中的作用及作用机制目前还不清楚。目的:探讨RNA干扰能否靶向抑制TRAP1过表达以及对人喉癌干细胞增殖和凋亡的作用效果。方法:采用免疫磁珠技术从人喉癌Hep-2细胞系中分选出CD133^+CD44^+喉癌干细胞。设计及合成TRAP1的sh RNA序列,Lipofectamine^(TM) 2000转染CD133^+CD44^+喉癌干细胞。CCK-8增殖实验、集落形成实验、流式细胞术检测干扰TRAP1基因表达对CD133^+CD44^+喉癌干细胞增殖和凋亡的影响,采用分光光度法检测细胞内caspase-3,caspase-8,caspase-9活性。结果与结论:(1)TRAP1 sh RNA转染的CD133^+CD44^+喉癌干细胞内TRAP1基因和蛋白表达明显下调(P<0.01);(2)与空白对照组和转染空质粒阴性对照组比较,TRAP1表达下调抑制了CD133^+CD44^+喉癌干细胞增殖和集落形成能力(P<0.05);(3)与空白对照组和转染空质粒阴性对照组比较,TRAP1表达下调增加CD133^+CD44^+喉癌干细胞凋亡率(P<0.05);(4)TRAP1 sh RNA介导细胞凋亡与caspase-3,caspase-8和caspase-9激活有关;(5)结果提示RNA干扰TRAP1表达可抑制CD133^+CD44^+喉癌干细胞生长并促进细胞凋亡。TRAP1可能为喉鳞癌治疗的基因靶点。

BACKGROUND： Tumor necrosis factor-associated protein 1 （TRAP1） is a heat-shock protein 90-related mitochondrial chaperone. Accumulative evidence has demonstrated that TRAP1 overexpression is closely related to carcinogenesis. However, the exact function and mechanism of TRAP1 in the occurrence of laryngeal carcinoma remains unclear. OBJECTIVE： To investigate whether RNA interference can inhibit TRAP1 overexpression and to explore its effects on growth and apoptosis of CD133＋CD44＋ laryngeal carcinoma stem cells. METHODS： CD133＋CD44＋ laryngeal carcinoma stem cells were sorted from human laryngeal carcinoma Hep-2 cellsusing immunomagnetic beads. The shRNA sequence of TRAP1 was designed and synthesized and CD133＋CD44＋ laryngeal carcinoma stem cells were transfected with LipofectamineTM 2000. Cell counting kit-8 assay, colony formation assay and flow cytometry were used to investigate the effects of interference of TRAP1 expression on growth and apoptosis of CD133＋CD44＋ laryngeal carcinoma stem cells. Spectrophotometric method was used to detect the activity of caspase-3, -8 and -9. RESULTS AND CONCLUSION： TRAP1 mRNA and protein expression levels were significantly decreased in TRAP1 shRNA-transfected CD133＋CD44＋ laryngeal carcinoma stem cells （P 〈 0.01）. Compared with the blank control and negative control groups, the growth and colony formation of CD133＋CD44＋ laryngeal carcinoma stem cells were significantly inhibited in the TRAP1 shRNA-transfected group （P 〈 0.05）. Apoptosis of CD133＋CD44＋ laryngeal carcinoma stem cells was significantly inhibited in the TRAP1 shRNA-transfected group as compared with the blank control and negative control groups （P 〈 0.05）. TRAP1 shRNA-mediated cell apoptosis was associated with the activation of caspase-3, -8 and -9. These results suggest that RNA interference targeting inhibition of TRAP1 suppresses cell growth but promotes apoptosis in CD133＋CD44＋ aryngeal carcinoma stem cells. TRAP1 is likely to be a gene target for treatment of laryngeal carcinoma.