Epigenetic silencing of microRNA-199b-5p leads to chemoresistance via activation of JAG1 (jagged1) in ovarian cancer

Abstract

Epithelial ovarian cancer is a leading fatal malignancy in women. The high mortality rate of this cancer is due to the poor prognosis and that the majority of patients are diagnosed at late stage. Therefore, a combination of cytoreduction and adjuvant chemotherapy is the only choice for this disease at late stage. Platinum-based chemotherapy regimens are the first line treatment for ovarian cancer. However, the repetitive challenges of platinum-based agents and the frequent relapse cause acquired chemoresistance which is the major obstacle for clinical management of this disease. The underlying molecular mechanism for acquired chemoresistance remains largely unclear. Therefore, understanding the molecular mechanism in acquired chemoresistance of ovarian cancer is urgently needed. Emerging evidence has suggested that dysregulation of miRNAs is significantly involved in acquired chemoresistance in human cancers. In this study, the expression status and functional roles of miR-199b-5p were characterized in the chemoresistance of ovarian cancer. By miRCURY LNA™ miRNA array, miR-199b-5p was one of the downregulated miRNAs identified in acquired chemoresistant ovarian cancer cells. Further Q-PCR analysis found that miR-199b-5p exhibited a pattern of progressive reduction from early to advanced stage and from low to high grade ovarian cancer tissue samples (N=79). Interestingly, the expression of miR-199b-5p could be restored upon 5-Aza-2’-deoxycytidine (5-Aza-dC) treatment in ovarian cancer cells. Methylation-specific PCR and bisulfite genomic sequencing revealed that the promoter region of miR-199b-5p (Chromosome9q34) was frequently hypermethylated in chemoresistant ovarian cancer cells. Functionally, enforced expression of miR-199b-5p remarkably diminished the cisplatin-resistance in miR-199b-5p low expressing ovarian cancer cells, whereas depletion of miR-199b-5p by siRNA approach augmented cisplatin-resistance in miR-199b-5p high expressing ovarian cancer cells. A tumor xenograft mouse model further confirmed that miR-199b-5p could sensitize ovarian cancer cells to cisplatin-mediated tumor growth inhibition in vivo. On the other aspect, computational analysis plus luciferase reporter assay and western blotting identified JAG1, a key ligand of Notch1 signaling frequently associated with chemoresistance of human cancers, as a direct target of miR-199b-5p in ovarian cancer cells. Intriguingly, an inverse relationship between the downregulation of miR-199b-5p and the upregulation of JAG1 was found by in situ hybridization (ISH) and immunohistochemical (IHC) analyses in a commercial ovarian cancer tissue array (OVC1021). Functionally, enforced expression of JAG1 increased cisplatin resistance of ovarian cancer cells. In contrast, depletion of JAG1 by siRNA approach abrogated the cisplatin-resistance of ovarian cancer cells. This finding was in consistent with the effect of enforced expression of miR199b-5p in cisplatin-resistant ovarian cancer cells. Mechanistically, the Notch1 luciferase reporter assay and western blotting analysis demonstrated that the Notch1 signaling activity could be activated by JAG1 or conversely be inhibited by enforced expression of miR-199b-5p. Further investigation using the Notch specific inhibitor γ-secretase showed that JAG1-Notch1 signaling is a crucial pathway associated with chemoresistance of ovarian cancer in vitro and in vivo. Taken together, this is the first study showing that the epigenetic silencing of miR-199b-5p leads to aberrant activation of JAG1-Notch1 signaling and such signaling cascade plays a critical role in tumor progression and chemoresistance of ovarian cancer.