Unraveling the role of RNA Binding Protein with Multiple Splicing (RBPMS) in ovarian cancer cells

Abstract

RNA-Binding Protein with Multiple Splicing (RBPMS) is member of a family of proteins that bind to nascent RNA transcripts and regulate their splicing, localization, and stability. Evidence indicates that RBPMS controls the activity of transcription factors associated with cell growth and proliferation, including AP-1 and Smads. Three major RBPMS protein splice variants (RBPMSA, RBPMSB, and RBPMSC) have been described in the literature. We previously reported that reduced RBPMS levels decreased the sensitivity of ovarian cancer cells to cisplatin treatment. However, little is known about the biological role of the RBPMS splice variants in ovarian cancer cells. We performed RT-PCR and Western blots and observed that both RBPMSA and RBPMSC are reduced at the mRNA and protein levels in cisplatin-resistant as compared with cisplatinsensitive ovarian cancer cells. The mRNA and protein levels of RBPMSB were not detectable in any of the ovarian cancer cells tested. To better understand the biological role of each RBPMSA and RBPMSC, we transfected these two splice variants in the A2780CP20 and OVCAR3CIS cisplatin resistant ovarian cancer cells and performed cell proliferation, cell migration, and invasion assays. Compared with control clones, a significant reduction in the number of colonies, colony size, cell migration, and invasion were observed with RBPMSA and RBPMSC overexpressed cells. Moreover, A2780CP20-RBPMSA and A2780CP20-RBPMSC clones showed reduced senescence-associated β-galactosidase (β-Gal)-levels when compared with control clones. A2780CP20-RBPMSA clones were more sensitive to cisplatin treatment as compared with A2780CP20-RBPMSC clones. The A2780CP20-RBPMSA and A2780CP20-RBPMSC clones subcutaneously injected into athymic nude mice formed smaller tumors as compared with A2780CP20-EV control group. Additionally, immunohistochemical analysis showed lower proliferation (Ki67) and angiogenesis (CD31) staining in tissue sections of A2780CP20-RBPMSA IX and A2780CP20-RBPMSC tumors compared with controls. In addition, Western blot analysis made with Immunoprecipitation (IP) samples suggested that Smad 2/3/4, c-Fos and c-Jun interact with RBPMS A and C splice variants when compared with A2780CP20-EV. Luciferase reporter assays identify RBPMS as miR-21-3p target gene. Real-time PCR confirmed that pri-mir-21 was significantly down-regulated in RBPMSA and RBPMSC when compared with control cell lines. RNAseq studies revealed many common RNA transcripts altered in A2780CP20-RBPMSA and A2780CP20-RBPMSC clones. Unique RNA transcripts deregulated by each RBPMS variant were also observed. Kaplan–Meier (KM) plotter database information identified clinically relevant RBPMSA and RBPMSC downstream effectors. Immunoprecipitation (IP) coupled to mass spectrometry (MS) revealed that RBPMSA and RBPMSC binds to proteins including the metastasis inhibition factor (NME1) and the immunoglobulin kappa locus (IGK). These studies suggest that increased levels of RBPMSA and RBPMSC reduce cell proliferation in ovarian cancer cells. However, only RBPMSA expression levels were associated with the sensitivity of ovarian cancer cells to cisplatin treatment. Also, our findings showed that RBPMS regulates the Smad- 2,3,4/c-Fos/c-Jun/miR-21 pathway in ovarian cancer cells. Overall, our findings indicate that RBPMSA and RBPMSC acts as tumor suppressor gene in ovarian cancer cells.