The Inactivation of Hippo Signaling Pathway Promotes the Development of Adenomyosis by Regulating EMT, Proliferation, and Apoptosis of Cells

Abstract

Adenomyosis is a benign gynecological disease. The pathogenesis of adenomyosis is still unclear. The Hippo signaling pathway is highly conserved in vivo and associated with endometriosis and various cancers. Our objective was to study the expression of Hippo signaling pathway-related proteins in the uterus of mice with and without adenomyosis. We also sought to determine the relationship between the Hippo signaling pathway and cell migration, invasion, proliferation, and apoptosis in adenomyosis. The inactivation of Hippo signaling pathway and abnormal expression of EMT-related proteins were observed in mice with adenomyosis. In vitro, the YAP inhibitor verteporfin can inhibit the proliferation and migration of Ishikawa cells and promote apoptosis, while inhibiting the EMT process. In addition, intraperitoneal injection of verteporfin inhibits EMT process and proliferation and promotes apoptosis of cells in the uterus of adenomyosis mice. It suggests that the Hippo signaling pathway participates in the EMT, proliferation, and apoptosis of cells in adenomyosis. In conclusion, these results suggest that Hippo signaling pathway may be involved in the development of adenomyosis by regulating EMT, proliferation, and apoptosis of cells, which provide a potential target for the treatment of adenomyosis.

Keywords: Adenomyosis (ADM); Epithelial-mesenchymal transition (EMT); Hippo signaling pathway; YAP (Yes-associated protein).

Fig. 1

Inactivation of Hippo signaling pathway and occurrence of EMT in adenomyosis mice. a H&E staining of the successful establishment of adenomyosis mice. b Protein expression of Mst1, p-Lats1, YAP, p-YAP, Tead, and Cyr61 in uterine tissue was determined by western blot. c Protein expression of E-cadherin, N-cadherin, Vimentin, Twist, Snail, MMP-2, and MMP-9 in uterine tissue was determined by western blot. d The expression levels of E-cadherin, N-cadherin, Vimentin, and Twist in uterine tissue were detected by IHC assay. Data were presented as mean ± SD. n = 5. *P < 0.05; **P < 0.01; ns, no significance

Fig. 2

Verteporfin inhibits proliferation and promotes apoptosis of Ishikawa cells. a, b, and d CCK-8, colony formation, and EdU assay of the proliferation of verteporfin-treated Ishikawa cells. c Protein expression of bcl2 and bax in verteporfin-treated Ishikawa cells was determined by western blot. e Flow cytometry analysis of the apoptosis of verteporfin-treated Ishikawa cells. Data were presented as mean ± SD. ***P < 0.001; ****P < 0.0001

Fig. 3

Verteporfin inhibits the migration ability of Ishikawa cells in vitro. a Wound healing assay of the migration ability of verteporfin-treated Ishikawa cells. b Transwell assay of the migration ability of verteporfin-treated Ishikawa cells. Data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

Verteporfin inhibits EMT process in Ishikawa cells. a Protein expression of Lats1, YAP, p-YAP, Tead, CTGF, and Cyr61 in the verteporfin-treated Ishikawa cells was determined by western blot. b Protein expression of E-cadherin, N-cadherin, Vimentin, Slug, Snail, and Twist in the verteporfin-treated Ishikawa cells was determined by western blot

Fig. 5

Verteporfin inhibits EMT process in mice with adenomyosis. a Protein expression of Mst1, p-Lats1, YAP, p-YAP, Tead, and Cyr61 was determined by western blot. b Protein expression of E-cadherin, N-cadherin, Twist, Snail, MMP-2, and MMP-9 was determined by western blot. c The expression levels of E-cadherin, N-cadherin, Vimentin, and Twist in uterine tissue were detected by IHC assay. Data were presented as mean ± SD. n = 5. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance

Fig. 6

Verteporfin inhibits cell proliferation and promotes apoptosis in mice with adenomyosis. a Protein expression of Bcl2 and Bax was determined by western blot. b The expression level of PCNA in uterine tissue was detected by IHC assay. c The cell apoptosis in uterine tissue was detected by TUNEL assay. Data were presented as mean ± SD. n = 5. *P < 0.05; **P < 0.01; ns, no significance