雌激素相关受体α介导的脂噬对鼻咽癌细胞增殖和迁移能力的影响

Effect of estrogen-related receptorα-mediated lipophagy on proliferation and migration abilities of nasopharyngeal cancer cells

目的探讨雌激素相关受体α(ESRRA)介导的脂噬对鼻咽癌细胞增殖、迁移能力的影响。方法选取2021年至2023年南通大学附属医院经病理确诊的临床样本16例,其中正常鼻咽黏膜8例,鼻咽癌8例;选择永生化的正常鼻咽上皮细胞株NP69和鼻咽癌细胞C666-1、CNE2、TW03-EBV、TW03。将鼻咽癌C666-1、CNE2细胞株分别分为siR-NC组(转染小干扰RNA阴性对照序列)和siR-ESRRA组(转染针对ESRRA基因的小干扰RNA)。采用蛋白质印迹法和免疫组织化学法检测ESRRA蛋白相对表达水平;EdU实验检测C666-1、CNE2细胞增殖能力;Transwell实验检测C666-1、CNE2细胞迁移能力;实时荧光定量聚合酶链反应(qRT-PCR)检测ESRRA、围脂滴蛋白3(PLIN3)mRNA的相对表达水平;透射电镜观察C666-1、CNE2细胞脂噬的形成;使用氟硼荧染料(Bodipy)标记脂滴后,通过细胞免疫荧光实验观察脂滴与LC3、PLIN3、LAMP2的共定位情况;双荧光素酶报告基因实验验证ESRRA与PLIN3的靶向关系。结果鼻咽癌组织中ESRRA蛋白相对表达量(1.15±0.75)高于正常鼻咽黏膜组织(0.32±0.21),差异有统计学意义(t=3.02,P=0.009)。鼻咽癌细胞株C666-1、CNE2、TW03-EBV、TW03中ESRRA蛋白相对表达量分别为1.539±0.044、1.420±0.030、2.867±0.044、1.323±0.022,均高于正常鼻咽上皮细胞NP69(0.094±0.002),差异有统计学意义(F=34.08,P<0.001)。EdU实验结果显示,siR-NC组和siR-ESRRA组CNE2细胞中EdU标记阳性细胞比例分别为(70.44±4.06)%和(51.51±0.92)%(t=7.88,P=0.001),C666-1细胞中分别为(62.25±3.89)%和(54.91±0.27)%(t=3.26,P=0.031)。Transwell实验结果显示,CNE2、C666-1细胞中siR-ESRRA组迁移细胞数均少于siR-NC组[CNE2细胞:(181±7)个比(261±21)个;C666-1细胞:(201±16)个比(256±7)个],差异均有统计学意义(t=6.30,P=0.003;t=5.43,P=0.006)。qRT-PCR检测结果显示,siR-ESRRA组PLIN3 mRNA相对表达量较siR-NC组高(CNE2细胞:1.58±0.16比0.83±0.17,t=5.59,P=0.005;C666-1细胞:1.37±0.12比1.06±0.06,t=3.86,P=0.018)。双荧光素酶报告基因实验结果显示ESRRA与PLIN3有靶向结合关系。透射电镜观察敲低ESRRA后细胞中脂滴含量增加,与自噬小体结合减少;免疫荧光实验结果显示,与siR-NC组比较,siR-ESRRA组LC3和LAMP2与脂滴的共定位减少、PLIN3与脂滴的共定位增加。结论ESRRA在鼻咽癌组织和细胞中高表达,作为转录抑制子抑制PLIN3转录,降低脂滴稳定性,介导脂噬,促进鼻咽癌增殖、迁移。

Objective To investigate the effect of estrogen-related receptorα(ESRRA)-mediated lipophagy on the proliferation and migration abilities of nasopharyngeal carcinoma cells.Methods A total of 16 clinical samples diagnosed by pathology in the Affiliated Hospital of Nantong University from 2021 to 2023 were selected,including 8 normal nasopharyngeal mucosa tissues and 8 nasopharyngeal carcinoma tissues.Immortalized normal nasopharyngeal epithelial cell line NP69 and nasopharyngeal carcinoma cell lines C666-1,CNE2,TW03-EBV and TW03 were selected.The cell lines C666-1 and CNE2 were divided into the siR-NC group(transfected with small interfering RNA negative control sequence)and siR-ESRRA group(transfected with small interfering RNA against ESRRA gene).The relative expression levels of ESRRA were detected by Western blotting and immunohistochemical assay.EdU assay was used to detect the proliferation ability of C666-1 and CNE2 cells,and Transwell assay was used to detect the migration ability.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the relative expression levels of ESRRA and perilipin 3(PLIN3)mRNA.The formation of lipophagy in C666-1 and CNE2 cells was observed by transmission electron microscopy.The co-localization of LC3,PLIN3 and LAMP2 with lipid droplets labeling with Bodipy was detected by immunofluorescence assay.Dual-luciferase reporter gene assay was used to verify the targeting relationship between ESRRA and PLIN3.Results The relative expression level of ESRRA in nasopharyngeal carcinoma tissues was higher than that in normal nasopharyngeal mucosa tissues(1.15±0.75 vs.0.32±0.21,t=3.02,P=0.009).The relative expression level of ESRRA in nasopharyngeal carcinoma cell lines C666-1(1.539±0.044),CNE2(1.420±0.030),TW03-EBV(2.867±0.044),and TW03(1.323±0.022)were higher than that in normal nasopharyngeal epithelial cell line NP69(0.094±0.002),and the difference was statistically significant(F=34.08,P<0.001).The results of EdU assay showed that the proportions of EdU labeled positive cells in CNE2 cells of siR-NC group and siR-ESRRA group were(70.44±4.06)%and(51.51±0.92)%(t=7.88,P=0.001),and the proportions in C666-1 cells were(62.25±3.89)%and(54.91±0.27)%(t=3.26,P=0.031).The results of Transwell assay showed that the number of migrating cells in CNE2 and C666-1 cells was less than that in siR-NC group[CNE2 cells:(181±7)cells vs.(261±21)cells;C666-1 cells:(201±16)cells vs.(256±7)cells],and the differences were statistically significant(t=6.30,P=0.003;t=5.43,P=0.006).According to qRT-PCR results,the relative expression level of PLIN3 mRNA in the siR-ESRRA group was higher than that in the siR-NC group(CNE2 cells:1.58±0.16 vs.0.83±0.17,t=5.59,P=0.005;C666-1 cells:1.37±0.12 vs.1.06±0.06,t=3.86,P=0.018).The dual-luciferase reporter gene assay results indicated a targeted binding interaction between PLIN3 and ESRRA.Transmission electron microscopy observation showed that the lipid droplets in nasopharyngeal carcinoma cells increased and the binding to autophagosomes decreased after knockdown of ESRRA.The results of immunofluorescence assay demonstrated that,in contrast to the siR-NC group,there was a decrease in the co-localization of LC3 and LAMP2 and an increase in the co-localization of lipid droplets with PLIN3.Conclusions ESRRA is highly expressed in nasopharyngeal carcinoma tissues and cells.As a transcription repressor,ESRRA may work to prevent PLIN3 from being transcribed,decrease lipid droplet stability,mediate lipophagy,and promote proliferation and migration of nasopharyngeal carcinoma cells.