Yamamoto Akiko, Tanaka Yosuke, Ishibashi Sachiko, Ikeda Masumi, Sugita Keisuke, Ono Masanori, Nishi Hirotaka, Kurata Morito
Department of Obstetrics and Gynecology, Tokyo Medical University, 6-7-1Shinjuku-Ku, Tokyo , Nishishinjuku, 1600023, Japan.
Department of Comprehensive Pathology, Graduate School of Institute of Science Tokyo, Tokyo, Japan.
J Ovarian Res. 2025 Apr 2;18(1):68. doi: 10.1186/s13048-025-01644-z.
Ovarian cancer remains one of the most lethal gynecological malignancies owing to its high recurrence rate and chemotherapeutic resistance. MYC is a well-known proto-oncogene that is frequently amplified in ovarian cancer and has been implicated in drug resistance. Previously, we established a new promoter-reporter system combined with a CRISPR activation library to identify unknown MYC regulators, and M1AP was identified as a novel MYC regulator. However, considering the insufficient explanation for the absence of guide RNA (gRNA) of MYC, this present study explored methods to prevent the gRNA of MYC itself from binding. This study first modified the promoter-reporter vector to improve its quality, then conducted CRISPR screening and analyzed candidate genes as MYC promoter regulators using next-generation sequencing in OVSAHO ovarian cancer cells. Eighty-six genes had ≥ 1000 reads, and Pearson's correlation coefficient analysis was performed on the cBioPortal of the Cancer Genomics database. Fourteen genes were identified as candidate MYC regulators with positive and significant correlations with MYC. Seven genes, including CYP4v2, ASPH, ANP32D, PCED1A, ABI1, FUZ, and HOOK2, demonstrated significantly higher luciferase activity than the control genes. Four genes, including ABI1, PCED1A, HOOK2, and CYP4v2, activated the MYC promoter, which showed over twofold higher activity than the control when overexpressed using a vector. In conclusion, four genes that activate MYC promoters were identified in an ovarian cancer cell line using the CRISPR library system with a modified promoter-reporter tool. These results will prove helpful in the development of novel treatment strategies for ovarian cancer.
由于卵巢癌的高复发率和化疗耐药性，它仍然是最致命的妇科恶性肿瘤之一。MYC是一种著名的原癌基因，在卵巢癌中经常扩增，并与耐药性有关。此前，我们建立了一种新的启动子-报告基因系统，并结合CRISPR激活文库来鉴定未知的MYC调节因子，M1AP被鉴定为一种新的MYC调节因子。然而，考虑到对MYC的引导RNA（gRNA）缺失的解释不足，本研究探索了防止MYC自身的gRNA结合的方法。本研究首先对启动子-报告基因载体进行修饰以提高其质量，然后在OVSAHO卵巢癌细胞中进行CRISPR筛选，并使用下一代测序分析作为MYC启动子调节因子的候选基因。86个基因的读数≥1000，并在癌症基因组数据库的cBioPortal上进行了Pearson相关系数分析。14个基因被鉴定为与MYC呈正相关且显著相关的候选MYC调节因子。包括CYP4v2、ASPH、ANP32D、PCED1A、ABI1、FUZ和HOOK2在内的7个基因显示出比对照基因显著更高的荧光素酶活性。包括ABI1、PCED1A、HOOK2和CYP4v2在内的4个基因激活了MYC启动子，当使用载体过表达时，其活性比对照高出两倍以上。总之，使用带有修饰的启动子-报告工具的CRISPR文库系统在卵巢癌细胞系中鉴定出了4个激活MYC启动子的基因。这些结果将有助于卵巢癌新治疗策略的开发。
