RAD51B-AS1 promotes the malignant biological behavior of ovarian cancer through upregulation of RAD51B

Long non-coding RNAs(lncRNAs)play an indispensable role in the occurrence and development of ovarian cancer(OC).However,the potential involvement of lncRNAs in the progression of OC is largely unknown.To investigate the detailed roles and mechanisms of RAD51 homolog B-antisense 1(RAD51B-AS1),a novel lncRNA in OC,reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was performed to verify the expression of RAD51B-AS1.Cellular proliferation,metastasis,and apoptosis were detected using the cell counting kit-8(CCK-8),colony-formation,transwell,and flow cytometry assays.Mouse xenograft models were established for the detection of tumorigenesis.The results revealed that RAD51B-AS1 was significantly upregulated in a highly metastatic human OC cell line and OC tissues.RAD51B-AS1 significantly increased the proliferation and metastasis of OC cells and enhanced their resistance to anoikis.Biogenetics prediction analysis revealed that the only target gene of RAD51B-AS1 was RAD51B.Subsequent gene function experiments revealed that RAD51B exerts the same biological effects as RAD51B-AS1.Rescue experiments demonstrated that the malignant biological behaviors promoted by RAD51B-AS1 overexpression were partially or completely reversed by RAD51B silencing in vitro and in vivo.Thus,RAD51B-AS1 promotes the malignant biological behaviors of OC and activates the protein kinase B(Akt)/B cell lymphoma protein-2(Bcl-2)signaling pathway,and these effects may be associated with the positive regulation of RAD51B expression.RAD51B-AS1 is expected to serve as a novel molecular biomarker for the diagnosis and prediction of poor prognosis in OC,and as a potential therapeutic target for disease management.

Single-cell transcriptomics reveals tumor landscape in ovarian carcinosarcoma

Objective:The present study used single-cell RNA sequencing(scRNA-seq)to characterize the cellular composition of ovarian carcinosarcoma(OCS)and identify its molecular characteristics.Methods:scRNA-seq was performed in resected primary OCS for an in-depth analysis of tumor cells and the tumor microenvironment.Immunohistochemistry staining was used for validation.The scRNA-seq data of OCS were compared with those of high-grade serous ovarian carcinoma(HGSOC)tumors and other OCS tumors.Results:Both malignant epithelial and malignant mesenchymal cells were observed in the OCS patient of this study.We identified four epithelial cell subclusters with different biological roles.Among them,epithelial subcluster 4 presented high levels of breast cancer type 1 susceptibility protein homolog(BRCA1)and DNA topoisomerase 2-α(TOP2A)expression and was related to drug resistance and cell cycle.We analyzed the interaction between epithelial and mesenchymal cells and found that fibroblast growth factor(FGF)and pleiotrophin(PTN)signalings were the main pathways contributing to communication between these cells.Moreover,we compared the malignant epithelial and mesenchymal cells of this OCS tumor with our previous published HGSOC scRNA-seq data and OCS data.All the epithelial subclusters in the OCS tumor could be found in the HGSOC samples.Notably,the mesenchymal subcluster C14 exhibited specific expression patterns in the OCS tumor,characterized by elevated expression of cytochrome P450 family 24 subfamily A member 1(CYP24A1),collagen type XXIIIα1 chain(COL23A1),cholecystokinin(CCK),bone morphogenetic protein 7(BMP7),PTN,Wnt inhibitory factor 1(WIF1),and insulin-like growth factor 2(IGF2).Moreover,this subcluster showed distinct characteristics when compared with both another previously published OCS tumor and normal ovarian tissue.Conclusions:This study provides the single-cell transcriptomics signature of human OCS,which constitutes a new resource for elucidating OCS diversity.

Nanoengineered Gallium Ion Incorporated Formulation for Safe and Efficient Reversal of PARP Inhibition and Platinum Resistance in Ovarian Cancer

Platinum-based chemotherapy remains the main systemic treatment of ovarian cancer(OC).However,the inevitable development of platinum and poly(adenosine diphosphate-ribose)polymerase inhibitor(PARPi)resistance is associated with poor outcomes,which becomes a major obstacle in the management of this disease.The present study developed“all-in-one”nanoparticles that contained the PARPi olaparib and gallium(Ga)(III)(olaparib-Ga)to effectively reverse PARPi resistance in platinum-resistant A2780-cis and SKOV3-cis OC cells and in SKOV3-cis tumor models.Notably,the olaparib-Ga suppressed SKOV3-cis tumor growth with negligible toxicity.Moreover,the suppression effect was more evident when combining olaparib-Ga with cisplatin or carboplatin,as evaluated in A2780-cis and SKOV3-cis cells.Mechanistically,the combined treatment induced DNA damage,which elicited the activation of ataxia telangiectasia mutated(ATM)/AMT-and Rad3-related(ATR)checkpoint kinase 1(Chk1)/Chk2 signal transduction pathways.This led to the arrest of cell cycle progression at S and G2/M phases,which eventually resulted in apoptosis and cell death due to unrepairable DNA damage.In addition,effective therapeutic responses to olaparib-Ga and cisplatin combination or olaparib-Ga and carboplatin combination were observed in SKOV3-cis tumor-bearing animal models.Altogether,the present findings demonstrate that olaparib-Ga has therapeutic implications in platinum-resistant OC cells,and the combination of olaparib-Ga with cisplatin or carboplatin may be promising for treating patients with OC who exhibit resistance to both PARPi and platinum.

奥拉帕利联合IL-1β抑制剂对HRD阴性卵巢上皮性癌细胞生长的抑制作用

目的:探讨聚二磷酸腺苷核糖聚合酶(PARP)抑制剂联合白细胞介素1β(IL-1β)抑制剂对同源重组修复缺陷(HRD)阴性卵巢上皮性癌(卵巢癌)细胞的抑制作用。方法:(1)使用HRD阴性的卵巢癌细胞系OVCAR3、CAOV3细胞,先分别给予PARP抑制剂奥拉帕利(122 μmol/L)和二甲基亚砜(作为对照)处理OVCAR3细胞,RNA测序及筛选差异表达基因;随后采用酶联免疫吸附试验(ELISA)和蛋白印迹(western blot)法验证奥拉帕利处理后OVCAR3、CAOV3细胞中IL-1β蛋白的表达。(2)根据不同浓度(0、1.25、2.5、5、10、20、40、80、160、320 μmol/L)IL-1β抑制剂diacerein(为蒽醌类化合物)在OVCAR3和CAOV3细胞中的药物剂量反应曲线,确定两种细胞IL-1β抑制剂的50%抑制浓度(IC 50)。细胞实验分为4组,对照组、奥拉帕利组、IL-1β抑制剂组、奥拉帕利+IL-1β抑制剂组,活细胞计数(CCK-8)法检测4组OVCAR3、CAOV3细胞的存活率。(3)将稳定表达荧光素酶基因luciferase的OVCAR3细胞(OVCAR3-Luc细胞)按照1×10 7个/只接种于裸鼠腹腔中,建立裸鼠腹腔移植瘤模型。动物实验将16只成瘤裸鼠采用随机表法随机分为4组,对照组、奥拉帕利组、IL-1β抑制剂组、奥拉帕利+IL-1β抑制剂组,每组4只。采用荧光素酶小动物活体成像测定4组裸鼠移植瘤的荧光信号强度,免疫组化法检测移植瘤组织中增殖相关核抗原(Ki-67)蛋白的表达。(4)药物毒副反应:腹腔注射后第29天,裸鼠称重后取其外周血进行血常规和肝肾功能检测;随后处死裸鼠,取裸鼠重要器官进行HE染色评估药物对器官的损害情况。 结果:(1)RNA测序及差异表达基因筛选,共获得25个显著差异表达基因,尤以IL-1β mRNA的表达差异最显著。ELISA法检测显示,奥拉帕利处理后OVCAR3、CAOV3细胞分泌的IL-1β蛋白含量分别为(36.2±3.5)和(49.5±3.5)pg/ml,均显著高于对照OVCAR3、CAOV3细胞[分别为(5.3±0.7)和(14.7±0.7)pg/ml;P均<0.001];western blot检测显示,奥拉帕利处理后OVCAR3和CAOV3细胞中IL-1β蛋白的相对表达水平分别为2.87±0.37、2.05±0.08,均显著高于对照OVCAR3、CAOV3细胞(均设为1.00;P均<0.001)。(2)剂量反应曲线确定IL-1β抑制剂在OVCAR3细胞中的IC 50为75 μmol/L,CAOV3细胞中为100 μmol/L。CCK-8法检测显示,对照组、奥拉帕利组、IL-1β抑制剂组、奥拉帕利+IL-1β抑制剂组OVCAR3细胞的存活率分别为(100.0±0.4)%、(63.1±6.2)%、(61.6±4.7)%、(32.9±5.2)%,CAOV3细胞分别为(100.0±3.5)%、(63.3±3.8)%、(63.8±3.5)%、(30.0±1.3)%,奥拉帕利+IL-1β抑制剂组OVCAR3、CAOV3细胞的存活率均低于对照组、奥拉帕利组、IL-1β抑制剂组( P均<0.01)。(3)腹腔注射后第29天,荧光素酶小动物活体成像显示,奥拉帕利+IL-1β抑制剂组裸鼠移植瘤的荧光信号强度为(0.5±0.4)×10 10 p/s,显著低于对照组[(4.2±1.0)×10 10 p/s]、奥拉帕利组[(3.1±0.9)×10 10 p/s]、IL-1β抑制剂组[(2.2±0.9)×10 10 p/s;P均<0.05];奥拉帕利+IL-1β抑制剂组裸鼠瘤重为(0.09±0.03)g,显著低于对照组[(0.25±0.05)g]、奥拉帕利组[(0.17±0.03)g]、IL-1β抑制剂组[(0.19±0.04)g;P均<0.05]。免疫组化法检测显示,奥拉帕利+IL-1β抑制剂组裸鼠移植瘤组织中Ki-67蛋白的表达水平为0.33±0.10,显著低于对照组(1.00±0.20)、奥拉帕利组(0.76±0.07)、IL-1β抑制剂组(0.77±0.12;P均<0.05)。(4)药物毒副反应:腹腔注射后第29天,奥拉帕利+IL-1β抑制剂组裸鼠的体重、血常规和肝肾功能,分别与对照组、奥拉帕利组、IL-1β抑制剂组比较,差异均无明显统计学意义( P均>0.05)。重要器官包括心、肝、脾、肺和肾,镜下观察均未见明显致死性损害。 结论:PARP抑制剂奥拉帕利联合IL-1β抑制剂在HRD阴性的卵巢癌细胞中显示出显著的肿瘤抑制作用,且无明显的药物毒副反应。