Objective: To investigate the role of adenosine diphosphate ribosylation factor 6 (Arf6) in the pathogenesis of endometriosis. Methods: Endometrial tissues were sampled from women who were hospitalized in the Affiliated Hospital of Medical School of Ningbo University and Ningbo Women and Children's Hospital from November 2020 to May 2021 with endometriosis (n=44, endometriosis group) and without endometriosis (n=17, control group). The expression of Arf6 protein in the endometrial tissues was detected by western blot. Endometrial epithelial cells from both groups were primary cultured and the distribution of intracellular mitochondria was detected by immunofluorescence. The expression of Arf6 protein was down-regulated by small interference RNA (siRNA), the distribution of mitochondria in cells with decreased Arf6 protein expression was observed, and the expression of mitochondria-related proteins development and differentiation enhancing factor 1 (DDEF1, also called AMAP1), reactive oxygen species 1 (ROS1) and epithelial-mesenchymal transition (EMT)-related proteins E-cadherin, vimentin were detected. Transwell assay was used to detect the changes in the migration ability of the cells. Results: Compared with the control group, ectopic endometrial tissue of endometriosis group showed high expression of Arf6 protein (0.174±0.019 vs 0.423±0.033; t=29.630, P<0.01); and in ectopic endometrial epithelial cells, mitochondria were distributed near the edge of the cell membrane. While Arf6 expression was down-regulated by siRNA, the distribution of mitochondria in ectopic cells returned to natural, close to the control level. In addition, the expression levels of AMAP1 and ROS1 in ectopic cells after Arf6 protein knockdown were significantly decreased. Transwell assay results indicated that knockdown of Arf6 could reduce the migration ability of ectopic epithelial cells [migration cell count: (34.3±7.5) cells]; and immunofluorescence verified low expression of E-cadherin but high expression of vimentin in ectopic epithelial cells, whereas knockdown of Arf6 protein E-cadherin expression increased but vimentin expression decreased. Conclusions: High expression of Arf6 protein in ectopic endometrial epithelial cells leads to the distribution of mitochondria tending to membrane marginalization, while inducing EMT, which are involved in the mechanism of endoheterosis pathogenesis.
目的： 研究二磷酸腺苷核糖基化因子6（Arf6）在子宫内膜异位症（内异症）发病中的作用。 方法： 收集从2020年11月至2021年5月在宁波大学医学院附属医院和宁波市妇女儿童医院住院手术并经病理确诊为卵巢型内异症患者（n=44，内异症组）的异位子宫内膜组织，同期收集因子宫肌瘤行子宫全切除术患者（n=17，对照组）的子宫内膜组织，采用蛋白印迹法检测子宫内膜组织中Arf6蛋白的表达；分离两组的子宫内膜上皮细胞进行原代培养，应用免疫荧光方法检测上皮细胞的线粒体分布；采用RNA干扰技术下调Arf6蛋白的表达，观察Arf6蛋白表达下调后细胞的线粒体分布情况，并用蛋白印迹法检测Arf6基因抑制前后线粒体相关蛋白发育分化增强因子1（DDEF1，又称AMAP1）、活性氧簇1（ROS1）表达的变化、用免疫荧光方法检测上皮-间质细胞转化（EMT）相关蛋白E-钙黏蛋白（E-cad）、波形蛋白（Vim）的表达变化，细胞迁移实验检测细胞迁移能力的变化。 结果： 与对照组子宫内膜组织（0.174±0.019）相比，异位子宫内膜组织Arf6蛋白呈现高表达（0.423±0.033；t=29.630，P<0.01）；线粒体在异位子宫内膜上皮细胞中靠近细胞膜边缘分布。当RNA干扰技术使Arf6基因抑制后，在异位上皮细胞中线粒体分布在细胞膜边缘明显减少，接近对照组的上皮细胞（贴近细胞核分布）；当Arf6蛋白表达下调后，转染后的异位上皮细胞中AMAP1和ROS1蛋白的表达减少。细胞迁移实验结果显示，Arf6蛋白表达下调后，异位上皮细胞的迁移能力降低，迁移细胞数量为（34.3±7.5）个。异位上皮细胞中低表达E-cad而高表达Vim，但Arf6蛋白表达下调后，转染后的异位上皮细胞中E-cad表达增加，Vim表达下降。 结论： 异位子宫内膜上皮细胞高表达Arf6蛋白可导致线粒体分布趋向细胞膜边缘化，诱导EMT，参与内异症的发病机制。.