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Study Question: Is chloride channel-3 (ClC-3) involved in regulating the biological behavior of endometrial stromal cells (ESCs)?Summary Answer: ClC-3 promotes endometriotic cell migration and invasion.What Is Known Already: ClC-3 plays a significant role in the migration and invasion of various kinds of cells.Study Design, Size, Duration: An ITALIC! in vitro investigation of the effect of ClC-3 on the migration and invasion of ectopic ESCs from patients with endometriosis.Participants/materials, Setting, Methods: The ectopic and eutopic endometrial samples from 43 female patients with endometriosis and the endometrial samples from 39 non-endometriotic female patients were collected. Primary cells from these samples were isolated and cultured. Real-time RT-PCR, immunohistochemistry and western blot were used to detect the expression of ClC-3 and matrix metalloproteinase 9 (MMP-9). Small interfering RNA (siRNA) technology was employed to knock down ClC-3 expression. The migration and invasion ability of ESCs was measured by the transwell assay with uncoated or Matrigel-coated membranes.Main Results and the Role Of Chance: The expression of ClC-3 mRNA and proteins was significantly up-regulated in the ectopic tissues from endometriotic patients, while that in the eutopic endometrial tissues of the same patients did not significantly differ from that in non-endometriotic patients. The migration and invasion ability and MMP-9 expression was increased in the ESCs from ectopic endometrial tissues. The knockdown of ClC-3 expression by ClC-3 siRNA inhibited ESC migration and invasion and attenuated the expression of MMP-9. ClC-3 expression level was well-correlated to the clinical characteristics and symptoms of endometriosis patients, including infertility, dysmenorrhea, chronic pelvic pain, dyspareunia and diameter of endometriosis lesion.Limitations, Reasons For Caution: Further studies are needed to examine the regulatory mechanism of estrogen on ClC-3 expression of ESCs.Wider Implications Of the Findings: ClC-3 is involved in the migration and invasion processes of ESCs and can regulate MMP-9 expression. Up-regulation of ClC-3 expression may contribute to endometriosis development by regulating MMP-9 expression.Study Funding/competing Interests: This work was supported by the National Natural Science Foundation of China (81173064, 81272223, 81273539), the Ministry of Education of China (20124401110009), the Natural Science Foundation of Guangdong Province (S2011010001589) and the Science and Technology Programs of Guangdong (2013B051000059), Guangzhou (2013J500015) and Dongguan (2011108102006). The authors have no conflict of interest. [ABSTRACT FROM AUTHOR]