Osteopontin Regulates Endometrial Stromal Cell Migration in Endometriosis through the PI3K Pathway : Osteopontin Regulates Endometrial Cell Migration in Endometriosis

Abstract

Endometriosis is generally characterized as a tumor-like disease because of its potential for distant metastasis and local tissue invasion, while whether osteopontin (OPN) plays a role in the pathogenesis of endometriosis has not been thoroughly investigated. We investigated the expression of OPN, urokinase plasminogen activator (uPA), phosphatidylinositol 3 kinase (PI3K), and phospho-PI3 kinase (p-PI3K) in endometrial stromal cells (ESCs). The serum concentration of OPN was determined by enzyme-linked immunosorbent assays (ELISA). OPN was downregulated to explore the corresponding change of uPA, p-PI3K, F-actin, and α-tubulin. The expression of OPN, uPA, PI3K, and p-PI3K was evaluated by western blot and quantitative real-time PCR (RT-qPCR) and the expression of F-actin and α-tubulin was confirmed by immunofluorescence assay. The proliferation and migration abilities of ESCs were investigated by CCK8, transwell, and wound scratch assays. Endometrial OPN, p-PI3K, and uPA expressions and serum OPN levels were increased in patients with endometriosis compared with the control. The expressions of p-PI3K, uPA, and α-tubulin were decreased by siRNA-OPN interference in ectopic ESCs. Activation and inhibition of the PI3K pathway apparently upregulate and downregulate uPA expression. Knockdown of OPN and inhibition of the PI3K pathway remarkably inhibited cell migration in ectopic ESCs. Meanwhile, activation of the PI3K pathway promoted the migration ability of ectopic ESCs. OPN may regulate the expression of uPA through the PI3K signal pathway to affect the migration ability of ESCs, indicating that OPN, uPA, and the PI3K pathway may be potential targets for interrupting development of endometriosis.

Keywords: Osteopontin; PI3K; cell migration; endometriosis; uPA.

Fig. 1

Identification of primary cultured ESCs and expression of OPN, uPA, and PI3K. a The serum OPN level was detected by ELISA (control n = 18; endometriosis n = 46). b, c Primary cultured ESCs were identified by immunohistochemical staining in EcESC (200 × scale, bar = 100 μm). d, e The protein (control n = 4; eutopic n = 6; ectopic n = 7) expression of OPN, uPA, PI3K, and p-PI3K in eutopic ESCs (EuESCs), ectopic ESCs (EcESCs), and control ESCs (CoESCs) were examined by western blot. f The mRNA (control n = 5; eutopic n = 6; ectopic n = 8) expression of OPN and uPA in EuESCs, EcESCs, and CoESCs were examined by RT-qPCR. g The correlation analysis of OPN level between in ectopic endometrium tissue and in serum was displayed (n = 7). Results are presented as the mean ± SEM (*p < 0.05, **p < 0.01)

Fig. 2

Knockdown of osteopontin (OPN) downregulate the expression of uPA and p-PI3K in EcESCs (si-Ctrl represents control; si-Scr represents siRNA scrambled; si-OPN represents siRNA-OPN; n = 4). a, b, c The protein (a and b) and mRNA (c) levels of OPN were detected by western blot and RT-qPCR at 48 h after transfection. d, e Protein expression of uPA, PI3K, and p-PI3K in untreated EcESCs, siRNA-scrambled treated EcESCs, and siRNA-OPN-treated EcESCs were examined by western blot. Results are presented as the mean ± SEM (*p < 0.05, **p < 0.01)

Fig. 3

Activation and inhibition of p-PI3K significantly increased and decreased uPA protein expression respectively (n = 4). a, c The protein level of p-PI3K was detected by western blot after 24-h treatment. b, d Protein expression of uPA and OPN in untreated EcESCs, ly294002-treated EcESCs, and SF1670-treated EcESCs was examined by western blot. Results are presented as the mean ± SEM (*p < 0.05, **p < 0.01)

Fig. 4

Knockdown of osteopontin (OPN) affected cytomorphology and induced α-tubulin reorganization in EcESCs (si-Scr represents siRNA scrambled; si-OPN represents siRNA-OPN). a EcESCs were triple stained with DAPI (for nucleus, blue), FITC-phalloidin (for F-actin, green), and Alexa Fluor 594-DM1A (for α-tubulin, red) at 48 h after siRNA-OPN transfection(1000 × scale, bar = 10 μm, n = 4). b Color pixels (px represents pixels) of F-actin and α-tubulin were quantified based on more than 200 cells for each experiment and four independent experiments were performed. Results are presented as the mean ± SEM (*p < 0.05)

Fig. 5

Knockdown of osteopontin (OPN) attenuated cell migration while exhibited no effect on cell proliferation in EcESCs (si-Ctrl represents control; si-Scr represents siRNA scrambled; si-OPN represents siRNA-OPN). a, c Cell migration was detected by transwell assay (200 × scale, n = 8). b, d Cell migration was detected by wound scratch assay (100 × scale, n = 10). e Cell proliferation was examined by CCK8 assay (n = 7) at 0 h, 24 h, and 48 h after transfection. Results are presented as the mean ± SEM (*p < 0.05, **p < 0.01)

Fig. 6

Migration of EcESCs was increased by p-PI3K activation and decreased by p-PI3K inhibition. a, b Cell migration was detected by transwell assay (200 × scale, n = 8). c, d Cell migration was detected by wound scratch assay (100 × scale, n = 8). Results are presented as the mean ± SEM (*p < 0.05, **p < 0.01)