Liu Yang, Liu Guoyan
Departments of Obstetrics and Gynecology, Tianjin Medical University General Hospital, Tianjin, 300052, China.
Correspondence to: Dr. Guoyan Liu, Key Laboratory of Cancer Prevention and Therapy of Tianjin, Department of Gynecologic Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin's Clinical Research Center for Cancer, Huanhuxi Road, Hexi District, Tianjin, 300060, China.
J Cancer. 2024 Jan 20;15(5):1397-1413. doi: 10.7150/jca.91896. eCollection 2024.
Patients initially sensitive to PARPi (PARP inhibitor) regain resistance because of homologous recombination (HR) restoration, although PARPi has a synthetic lethality effect on serous ovarian cancer cells with BRCA1/2 mutations. This study aimed to investigate the role of NEAT1 in HR function and whether targeting NEAT1 in serous ovarian cancer cells could increase PARPi sensitivity. Ovarian cancer patients' clinical information and the expression of NEAT1 were collected from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). Ovarian cancer (OC) cells HeyA8 and SKOV3 were silenced by transfecting NEAT1 ASO. QRT-PCR confirmed the mRNA expression of RAD51, FOXM1, NEAT1_1 and NEAT1_2. We assessed the expression of RAD51, FOXM1, and γ-H2AX by Western blotting and Immunofluorescence. Comet Assays were used to detect DNA double-strand damage levels. In OC cells transfected with NEAT1 ASO or co-transfected overexpression RAD51/empty vector and si-NEAT1/si-ctrl, the sensitivity to Olaparib was determined using CCK8 assay. The Kaplan-Meier survival curves assessed the prognostic and predictive roles of NEAT1 in OC. NEAT1 was an independent prognostic marker of ovarian cancer. NEAT1 knockdown reduced the expression of NEAT1_1, NEAT1_2, RAD51, and FOXM1 and increased the expression of γ-H2AX. In addition, Olaparib increased the expression of RAD51, representing HR repair efficiency, which was inhibited by NEAT1 knockdown. Moreover, the knockdown of NEAT1 increased the DNA damage caused by Olaparib, demonstrated by increased nuclear γ-H2AX foci, DNA in the tail, and expression of γ-H2AX. NEAT1 knockdown sensitized ovarian cancer cells to Olaparib by targeting RAD51-HR. NEAT1 expression could predict response to chemotherapy for ovarian cancer. NEAT1 knockdown inhibited HR capacity and increased DNA damage caused by Olaparib in serous ovarian cancer cells, making them more sensitive to Olaparib and providing a crucial therapeutic advantage of increasing sensitivity to Olaparib.
尽管PARP抑制剂（PARPi）对具有BRCA1/2突变的浆液性卵巢癌细胞具有合成致死效应，但最初对PARPi敏感的患者会因同源重组（HR）功能的恢复而重新产生耐药性。本研究旨在探讨NEAT1在HR功能中的作用，以及在浆液性卵巢癌细胞中靶向NEAT1是否能增加对PARPi的敏感性。从癌症基因组图谱（TCGA）和国际癌症基因组联盟（ICGC）收集卵巢癌患者的临床信息和NEAT1的表达情况。通过转染NEAT1反义寡核苷酸（ASO）使卵巢癌（OC）细胞HeyA8和SKOV3沉默。实时定量聚合酶链反应（QRT-PCR）检测RAD51、FOXM1、NEAT1_1和NEAT1_2的mRNA表达。通过蛋白质免疫印迹法和免疫荧光法评估RAD51、FOXM1和γ-H2AX的表达。彗星试验用于检测DNA双链损伤水平。在转染了NEAT1 ASO或共转染过表达RAD51/空载体以及si-NEAT1/si-对照的OC细胞中，使用细胞计数试剂盒8（CCK8）检测对奥拉帕利的敏感性。Kaplan-Meier生存曲线评估NEAT1在OC中的预后和预测作用。NEAT1是卵巢癌的独立预后标志物。敲低NEAT1可降低NEAT1_1、NEAT1_2、RAD51和FOXM1的表达，并增加γ-H2AX的表达。此外，奥拉帕利增加了代表HR修复效率的RAD51的表达，而敲低NEAT1可抑制这一作用。而且，敲低NEAT1增加了奥拉帕利引起的DNA损伤，表现为细胞核内γ-H2AX焦点增加、尾部DNA增加以及γ-H2AX表达增加。敲低NEAT1通过靶向RAD51-HR使卵巢癌细胞对奥拉帕利敏感。NEAT1的表达可预测卵巢癌对化疗的反应。敲低NEAT1可抑制浆液性卵巢癌细胞的HR能力，并增加奥拉帕利引起的DNA损伤，使其对奥拉帕利更敏感，为提高对奥拉帕利的敏感性提供了关键的治疗优势。
