FAT10基因沉默对人食管癌细胞凋亡及肿瘤干细胞特性的影响

摘要

背景：人类白细胞抗原F介导转录因子10(human leukocyte antigen F-associated transcript 10，FAT10)在结肠癌等很多种肿瘤细胞中会发生高表达，但其与食管癌的关系报道较少。　　目的：观察siRNA干扰技术沉默类泛素蛋白(FAT10)基因表达对人食管癌细胞EC9706细胞的侵袭、凋亡及及肿瘤干细胞特性的影响。　　方法：根据FAT10 mRNA编码序列设计并合成干扰siRNA序列，瞬时转染EC9706细胞。将人食管癌细胞EC9706细胞分为3组，设FAT10 siRNA组，阴性对照组、空白对照组。用RT-PCR和Western blot法分别从mRNA水平和蛋白水平检测FAT10及bcl-2的表达；CCK-8法测定细胞体外增殖能力；流式细胞技术观察细胞凋亡及肿瘤干细胞标志物CD44+CD133+的表达；TUNEL染色检测各组细胞的的凋亡、细胞侵袭小室法检测各组细胞的体外侵袭力。　　结果与结论：①RT-PCR和Western blot显示，FAT10 siRNA组较阴性对照组和空白对照组FAT10及bcl-2 mRNA和蛋白表达都明显下降(P<0.05)；②肿瘤干细胞标志物CD44+CD133+的表达比例显著降低(P<0.05)；③与阴性对照组和空白对照组相比，FAT10 siRNA组细胞的凋亡率明显升高(P<0.01)，增殖、侵袭力显著下降(P<0.05)。④结果表明，特异性沉默FAT10基因表达能够降低食管癌细胞的侵袭力，抑制细胞的增殖，促进bcl-2表达降低，使其凋亡显著增加，使具有CD44+CD133+肿瘤干细胞特性的细胞比例降低。%BACKGROUND:Human leukocyte antigen F-associated transcription factor 10 (FAT10) is highly expressed in many tumor cel s like colon cancer cel s, but its relationship with esophageal cancer is less reported. OBJECTIVE:To investigate the effects of siRNA interference technique on the invasion, apoptosis and the characteristics of EC9706 cel s, a human esophageal cancer cel line. METHODS:siRNA sequence was designed and synthesized according to the FAT10 mRNA encoding sequence, and the EC9706 cel s were transiently transfected. EC9706 cel s were divided into three groups:siRNA FAT10 group, negative control group, and blank control group. The expression levels of bcl-2 and FAT10 were detected by RT-PCR and western blot assay, respectively. Cel counting kit-8 assay was used to measure the proliferation of cel s in vitro. Flow cytometry was used to observe the changes of cel cycle, cel apoptosis and the expression of CD44+CD133+. TUNEL staining was used to detect the apoptosis of the cel s. Cel invasion in vitro was detected by Transwel invasion assay. RESULTS AND CONCLUSION:RT-PCR and western blot findings showed that compared with the negative control group and blank control group, the expression levels of bcl-2 and FAT10 mRNA and protein were significantly decreased in the siRNA FAT10 group (P<0.05);the percentage of CD44+CD133+cel s was decreased significantly (P<0.05);and significantly increased apoptosis rate, and decreased cel proliferation and invasion were also found in the siRNA FAT10 group (P<0.05). In conclusion, the specific silencing of FAT10 gene can reduce the invasion of esophageal cancer cel s, inhibit cel proliferation, reduce bcl-2 expression, and increase the apoptosis rate. Meanwhile, the proportion of CD44+CD133+cel s is decreased.