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

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

目的探讨干扰长链编码RNAFOXCUT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RNAFOXCUT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RNAFOXCUT对CNE1细胞抑制效果显著,具有靶向治疗鼻咽癌的潜力。

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.