University of Puerto Rico Comprehensive Cancer Center, San Juan, PR 00935, USA.
Department of Biology, University of Puerto Rico, Rio Piedras Campus, San Juan, PR 00925, USA.
Int J Mol Sci. 2022 Jan 4;23(1):535. doi: 10.3390/ijms23010535.
Worldwide, the number of cancer-related deaths continues to increase due to the ability of cancer cells to become chemotherapy-resistant and metastasize. For women with ovarian cancer, a staggering 70% will become resistant to the front-line therapy, cisplatin. Although many mechanisms of cisplatin resistance have been proposed, the key mechanisms of such resistance remain elusive. The RNA binding protein with multiple splicing (RBPMS) binds to nascent RNA transcripts and regulates splicing, transport, localization, and stability. Evidence indicates that RBPMS also binds to protein members of the AP-1 transcription factor complex repressing its activity. Until now, little has been known about the biological function of RBPMS in ovarian cancer. Accordingly, we interrogated available Internet databases and found that ovarian cancer patients with high RBPMS levels live longer compared to patients with low RBPMS levels. Similarly, immunohistochemical (IHC) analysis in a tissue array of ovarian cancer patient samples showed that serous ovarian cancer tissues showed weaker RBPMS staining when compared with normal ovarian tissues. We generated clustered regularly interspaced short palindromic repeats (CRISPR)-mediated RBPMS knockout vectors that were stably transfected in the high-grade serous ovarian cancer cell line, OVCAR3. The knockout of RBPMS in these cells was confirmed via bioinformatics analysis, real-time PCR, and Western blot analysis. We found that the RBPMS knockout clones grew faster and had increased invasiveness than the control CRISPR clones. RBPMS knockout also reduced the sensitivity of the OVCAR3 cells to cisplatin treatment. Moreover, β-galactosidase (β-Gal) measurements showed that RBPMS knockdown induced senescence in ovarian cancer cells. We performed RNAseq in the RBPMS knockout clones and identified several downstream-RBPMS transcripts, including non-coding RNAs (ncRNAs) and protein-coding genes associated with alteration of the tumor microenvironment as well as those with oncogenic or tumor suppressor capabilities. Moreover, proteomic studies confirmed that RBPMS regulates the expression of proteins involved in cell detoxification, RNA processing, and cytoskeleton network and cell integrity. Interrogation of the Kaplan-Meier (KM) plotter database identified multiple downstream-RBPMS effectors that could be used as prognostic and response-to-therapy biomarkers in ovarian cancer. These studies suggest that RBPMS acts as a tumor suppressor gene and that lower levels of RBPMS promote the cisplatin resistance of ovarian cancer cells.
由于癌细胞能够产生化疗耐药性并转移，全球因癌症死亡的人数不断增加。对于患有卵巢癌的女性来说，令人震惊的是 70%的人会对一线治疗药物顺铂产生耐药性。虽然已经提出了许多顺铂耐药的机制，但这种耐药的关键机制仍然难以捉摸。具有多个剪接的 RNA 结合蛋白 (RBPMS) 与新生 RNA 转录本结合，并调节剪接、运输、定位和稳定性。有证据表明，RBPMS 还与 AP-1 转录因子复合物的蛋白质成员结合，抑制其活性。到目前为止，人们对 RBPMS 在卵巢癌中的生物学功能知之甚少。因此，我们查询了现有的互联网数据库，发现 RBPMS 水平较高的卵巢癌患者的存活时间长于 RBPMS 水平较低的患者。同样，在卵巢癌患者组织阵列的免疫组织化学 (IHC) 分析中，与正常卵巢组织相比，浆液性卵巢癌组织的 RBPMS 染色较弱。我们生成了簇状规则间隔短回文重复序列 (CRISPR) 介导的 RBPMS 敲除载体，该载体稳定转染于高级别浆液性卵巢癌细胞系 OVCAR3 中。通过生物信息学分析、实时 PCR 和 Western blot 分析证实了这些细胞中 RBPMS 的敲除。我们发现 RBPMS 敲除克隆的生长速度比对照 CRISPR 克隆快，侵袭性也增强。RBPMS 敲除还降低了 OVCAR3 细胞对顺铂治疗的敏感性。此外，β-半乳糖苷酶 (β-Gal) 测量表明，RBPMS 敲低诱导卵巢癌细胞衰老。我们在 RBPMS 敲除克隆中进行了 RNAseq，并鉴定了几个下游-RBPMS 转录本，包括与肿瘤微环境改变以及致癌或肿瘤抑制能力相关的非编码 RNA (ncRNA) 和蛋白编码基因。此外，蛋白质组学研究证实，RBPMS 调节参与细胞解毒、RNA 加工以及细胞骨架网络和细胞完整性的蛋白质的表达。对 Kaplan-Meier (KM) 绘图器数据库的查询确定了多个下游-RBPMS 效应子，可作为卵巢癌的预后和治疗反应生物标志物。这些研究表明，RBPMS 作为一种肿瘤抑制基因发挥作用，较低水平的 RBPMS 促进了卵巢癌细胞对顺铂的耐药性。
