Gao L, Zhang X, Dou S, Yue X, Yang J
School of Medicine, Xijing University, Xi'an 710000, China.
Department of Otolaryngology, Affiliated Hospital of Shaanxi University of Chinese Medicine, Xianyang 712000, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2021 Aug 31;41(9):1334-1341. doi: 10.12122/j.issn.1673-4254.2021.09.07.
To investigate the effects of RNA interference of long noncoding RNA FOXCUT on epithelial mesenchymal transformation and mitochondrial function in nasopharyngeal carcinoma (NPC) cells.
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.
The expression level of FOXCUT was significantly increased in NPC tissues as compared with the adjacent tissues ( < 0.001). Compared with NP69 cells, CNE1, CNE2, SUNE2, HER2 and 5-8F cells all exhibited significantly increased expressions of FOXCUT ( < 0.001). In CNE1 cells, transfection with FOXCUT shRNA significantly inhibited cell proliferation and clone formation ( < 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 ( < 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.
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.
探讨长链非编码RNA FOXCUT的RNA干扰对鼻咽癌（NPC）细胞上皮间质转化和线粒体功能的影响。
采用RT-PCR检测50例NPC患者肿瘤组织及癌旁组织以及NP69、CNE1、CNE2、SUNE2、HER2和5-8F细胞系中FOXCUT的表达水平。用靶向FOXCUT的短发夹RNA（shRNA）或阴性对照RNA构建体（shRNA-NC）转染CNE1细胞，通过CCK8法、克隆形成试验和显微镜观察评估细胞增殖和形态的变化。采用免疫荧光法检测波形蛋白阳性细胞，并检测细胞中SOD、MDA和LDH的水平。用流式细胞术检测线粒体膜电位的变化，用蛋白质免疫印迹法检测细胞中E-cad、N-cad、波形蛋白、Bax、Bcl-2、caspase-3和c-Myc的表达水平。
与癌旁组织相比，NPC组织中FOXCUT的表达水平显著升高（<0.001）。与NP69细胞相比，CNE1、CNE2、SUNE2、HER2和5-8F细胞中FOXCUT的表达均显著升高（<0.001）。在CNE1细胞中，转染FOXCUT shRNA可显著抑制细胞增殖和克隆形成（<0.001），并引起细胞形态明显改变。敲低FOXCUT可显著降低N-cad和波形蛋白的表达，增加E-cad表达以及MDA和LDH含量（<0.05），减少波形蛋白阳性细胞和SOD活性，使红色荧光细胞向绿色荧光细胞转变，绿色荧光细胞百分比增加。敲低FOXCUT还导致Bax/Bcl2和裂解的Cas3/Cas3表达显著增加，c-Myc表达降低。
干扰FOXCUT可抑制NPC细胞增殖和上皮间质转化，增强氧化应激，诱导线粒体功能损伤，促进细胞凋亡，提示干扰FOXCUT在NPC靶向治疗中的潜力。
