Department of Biology, University of Puerto Rico at Rio Piedras, San Juan, PR 00925, USA.
Molecular Signaling and Experimental Therapy Laboratory, Comprehensive Cancer Center, University of Puerto Rico, San Juan, PR 00936, USA.
Int J Mol Sci. 2022 Nov 25;23(23):14742. doi: 10.3390/ijms232314742.
RNA-Binding Protein with Multiple Splicing (RBPMS) is a member of family 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 cisplatin sensitive 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 was 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 and A2780CP20-RBPMSC tumors compared with controls. 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. 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.
RNA 结合蛋白多剪接（RBPMS）是家族蛋白的成员，它与新生 RNA 转录本结合并调节其剪接、定位和稳定性。有证据表明，RBPMS 控制与细胞生长和增殖相关的转录因子的活性，包括 AP-1 和 Smads。文献中已经描述了三种主要的 RBPMS 蛋白剪接变体（RBPMSA、RBPMSB 和 RBPMSC）。我们之前报道过，RBPMS 水平降低会降低卵巢癌细胞对顺铂治疗的敏感性。然而，关于 RBPMS 剪接变体在卵巢癌细胞中的生物学作用知之甚少。我们通过 RT-PCR 和 Western blot 观察到，与顺铂敏感的卵巢癌细胞相比，耐顺铂的卵巢癌细胞中 RBPMSA 和 RBPMSC 的 mRNA 和蛋白水平均降低。在测试的任何卵巢癌细胞中都检测不到 RBPMSB 的 mRNA 和蛋白水平。为了更好地了解每个 RBPMSA 和 RBPMSC 的生物学作用，我们在 A2780CP20 和 OVCAR3CIS 耐顺铂的卵巢癌细胞中转染这两种剪接变体，并进行细胞增殖、细胞迁移和侵袭实验。与对照克隆相比，RBPMSA 和 RBPMSC 过表达细胞的菌落数量、菌落大小、细胞迁移和侵袭明显减少。此外，与对照克隆相比，A2780CP20-RBPMSA 和 A2780CP20-RBPMSC 克隆的衰老相关β-半乳糖苷酶（β-Gal）水平降低。与 A2780CP20-RBPMSC 克隆相比，A2780CP20-RBPMSA 克隆对顺铂治疗更敏感。与 A2780CP20-EV 对照组相比，A2780CP20-RBPMSA 和 A2780CP20-RBPMSC 克隆皮下注射到裸鼠中形成的肿瘤较小。此外，免疫组织化学分析显示，与对照组相比，A2780CP20-RBPMSA 和 A2780CP20-RBPMSC 肿瘤组织切片中的增殖（Ki67）和血管生成（CD31）染色较低。RNAseq 研究表明，在 A2780CP20-RBPMSA 和 A2780CP20-RBPMSC 克隆中，许多常见的 RNA 转录本发生改变。还观察到每个 RBPMS 变体下调的独特 RNA 转录本。Kaplan-Meier（KM）分析器数据库信息确定了与 RBPMSA 和 RBPMSC 下游效应器相关的临床相关信息。这些研究表明，RBPMSA 和 RBPMSC 水平的增加可降低卵巢癌细胞的增殖。然而，只有 RBPMSA 的表达水平与卵巢癌细胞对顺铂治疗的敏感性相关。
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