■硫化氢(H2S)已被确立为气体传送器家族的关键成员,该家族最近在包括癌症在内的各种病理状况中显示出关键作用。
■这项研究调查了H2S在乳腺癌(BC)发病机制中的作用,关于BC免疫识别能力和使用非编码RNA靶向H2S的结果。
■本研究招募了80名BC患者。培养BC细胞系并使用经验证的寡核苷酸递送系统转染。使用qRT-PCR进行基因和蛋白质表达分析,蛋白质印迹和流式细胞术。使用MTT进行BC标志的体外分析,BrdU,改良的Boyden室,迁移和菌落形成测定。通过分光光度法测量H2S和一氧化氮(NO)水平。使用适当的试剂盒进行原代自然杀伤细胞(NK细胞)和T细胞分离和嵌合抗原受体转导(CART细胞)。测量NK和T细胞的细胞毒性。最后,使用不同的软件和双荧光素酶测定试剂盒进行计算靶标预测分析和结合确认分析,分别。
■H2S合成酶,胱硫醚β-合酶(CBS)和胱硫醚γ-裂解酶(CSE),在与肿瘤增殖指数相关的临床样品中表现出升高的水平。在HER2+BC和三阴性BC(TNBC)细胞中CBS和CSE的敲除导致BC恶性肿瘤的显著减弱。除了增加HER2+BC和TNBC对HER2靶向CAR-T细胞和NK细胞的细胞毒活性的敏感性外,分别。转录组学和磷蛋白分析显示,H2S信号通过MCF7中的Akt,MDA-MB-231中的STAT3和两种细胞系中的miR-155/NOS2/NO信号介导。最后,发现miR-4317作为CBS和CSE的上游调节因子协同地消除BC细胞的恶性。
■这些发现证明了H2S信号传导在BC发病机制中的潜在作用以及其靶向缓解疾病的潜力。
UNASSIGNED: Hydrogen sulphide (H2S) has been established as a key member of the gasotransmitters family that recently showed a pivotal role in various pathological conditions including cancer.
UNASSIGNED: This study investigated the role of H2S in breast cancer (BC) pathogenesis, on BC immune recognition capacity and the consequence of targeting H2S using non-coding RNAs.
UNASSIGNED: Eighty BC patients have been recruited for the study. BC cell lines were cultured and transfected using validated oligonucleotide delivery system. Gene and protein expression analysis was performed using qRT-PCR, western blot and flow-cytometry. In-vitro analysis for BC hallmarks was performed using MTT, BrdU, Modified Boyden chamber, migration and colony forming assays. H2S and nitric oxide (NO) levels were measured spectrophotometrically. Primary natural killer cells (NK cells) and T cell isolation and chimeric antigen receptor transduction (CAR T cells) were performed using appropriate kits. NK and T cells cytotoxicity was measured. Finally, computational target prediction analysis and binding confirmation analyses were performed using different software and dual luciferase assay kit, respectively.
UNASSIGNED: The H2S synthesizing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), exhibited elevated levels in the clinical samples that correlated with tumor proliferation index. Knock-down of CBS and CSE in the HER2+ BC and triple negative BC (TNBC) cells resulted in significant attenuation of BC malignancy. In addition to increased susceptibility of HER2+ BC and TNBC to the cytotoxic activity of HER2 targeting CAR T cells and NK cells, respectively. Transcriptomic and phosphoprotein analysis revealed that H2S signaling is mediated through Akt in MCF7, STAT3 in MDA-MB-231 and miR-155/ NOS2/NO signaling in both cell lines. Lastly, miR-4317 was found to function as an upstream regulator of CBS and CSE synergistically abrogates the malignancy of BC cells.
UNASSIGNED: These findings demonstrate the potential role of H2S signaling in BC pathogenesis and the potential of its targeting for disease mitigation.