干扰长链编码RNA FOXCUT能抑制鼻咽癌细胞上皮间质转化及诱导线粒体损伤

目的 探讨干扰长链编码RNA FOXCUT对鼻咽癌细胞上皮间质转化及线粒体功能的影响。方法 RT-PCR检测50例鼻咽癌患者癌组织及癌旁组织和NP69、CNE1、CNE2、SUNE2、HER2和5-8F细胞株中FOXCUT表达水平；将shRNA FOXCUT转染至CNE1细胞，随机分组为Control组、shRNA-NC组和FOXCUT-shRNA3组。采用CCK8法和克隆形成实验检测细胞增殖；显微镜观察细胞形态；免疫荧光检测Vimentin+含量；试剂盒检测氧化应激标记物SOD、MDA、LDH的水平；流式检测线粒体膜电位的变化；Western blot法检测E-cad、N-cad、Vimentin、Bax、Bcl-2、caspase-3和c-Myc蛋白表达水平。结果 与癌旁组织相比，鼻咽癌组织中FOXCUT表达水平均升高（P<0.001）。与NP69细胞相比，CNE1、CNE2、SUNE2、HER2和5-8F细胞FOXCUT表达水平均升高（P<0.001）。与Control组相比较，FOXCUT-shRNA3组细胞增殖倍数及克隆形成率降低（P<0.001），细胞形态呈短梭形、扁平型或圆形，细胞间的连接较为紧密，具有铺路石样特征；N-cad、Vimentin的表达水平降低，E-cad的表达水平升高（P<0.05），Vimentin+含量降低（P<0.001），MDA、LDH的含量明显升高（P<0.05），SOD的活性明显降低（P<0.05），红色荧光细胞向绿色荧光细胞转变较明显，绿色荧光细胞百分比增加，Bax/Bcl2、Cleaved cas3/cas3表达显著上升，c-Myc表达显著降低（P<0.001）。结论 干扰长链非编码RNA FOXCUT能够抑制鼻咽癌细胞增殖，减少上皮间质转化，增强氧化应激、降低膜电位，诱导CNE1细胞线粒体功能损伤，促进凋亡；干扰长链非编码RNA FOXCUT对CNE1细胞抑制效果显著，具有靶向治疗鼻咽癌的潜力。

Interference of long noncoding RNA FOXCUT inhibits epithelial-mesenchymal transformation and induces mitochondrial injury in nasopharyngeal carcinoma cells

Objective To investigate the effects of RNA interference of long noncoding RNA FOXCUT on epithelial mesenchymal transformation and mitochondrial function in nasopharyngeal carcinoma (NPC) cells. Methods FOXCUT expression levels were detected by RT-PCR in tumor tissues and adjacent tissues from 50 patients with NPC and in NP69, CNE1, CNE2, SUNE2, HER2 and 5-8F cell lines. CNE1 cells were transfected with a short hairpin RNA (shRNA) targeting FOXCUT or a negative control RNA construct (shRNA-NC), and the changes in cell proliferation and morphology were assessed with CCK8 assay, clone formation assay and microscopic observation. An immunofluorescence assay was used to examine the vimentin-positive cells, and the levels of SOD, MDA and LDH in the cells were detected. The changes of mitochondrial membrane potential were detected with flow cytometry, and the expression levels of E-cad, N-cad, vimentin, Bax, Bcl-2, caspase-3 and c-Myc in the cells were detected with Western blotting. Results The expression level of FOXCUT was significantly increased in NPC tissues as compared with the adjacent tissues (P<0.001). Compared with NP69 cells, CNE1, CNE2, SUNE2, HER2 and 5-8F cells all exhibited significantly increased expressions of FOXCUT (P<0.001). In CNE1 cells, transfection with FOXCUT shRNA significantly inhibited cell proliferation and clone formation (P<0.001), and caused obvious changes in cell morphology. FOXCUT knockdown significantly decreased the expressions of N-cad and vimentin, increased E- cad expression and the contents of MDA and LDH (P<0.05), reduced vimentin- positive cells and the activity of SOD, and caused a shift of red fluorescent cells to green fluorescent cells and an increased percentage of green fluorescent cells. FOXCUT knockdown also resulted in significantly increased expressions of Bax/Bcl2 and cleaved Cas3/Cas3 and a lowered expression of c-Myc. Conclusions Interference of FOXCUT can inhibit the proliferation and epithelial-mesenchymal transformation, enhance oxidative stress, induce mitochondrial function injury, and promote apoptosis in NPC cells, suggesting the potential of FOXCUT interference for targeted treatment of NPC.