Molecular and Cellular Probes最新文献_第3页

RCN1 affects malignant progression and macrophage M2 polarization in diffuse large B-cell lymphoma RCN1影响弥漫大b细胞淋巴瘤的恶性进展和巨噬细胞M2极化。

IF 2.3 3区生物学 Q3 BIOCHEMICAL RESEARCH METHODS

Molecular and Cellular Probes

Pub Date : 2025-07-12 DOI: 10.1016/j.mcp.2025.102042

Qiuyu Zhu , Xiafang Yang

Malignant behaviors of cancer cells and immune evasion by macrophage can lead to treatment failure in diffuse large B-cell lymphoma (DLBCL). Recent studies have revealed the aberrant expression of reticulocalbin 1 (RCN1) that is associated with malignant progression of several cancers. Nevertheless, its roles in DLBCL remain unclear. In this present study, several online bioinformatics databases substantiated high expression of RCN1 in tumor samples from DLBCL patients. Furthermore, there were obvious elevation of RCN1in tumor specimens form our collected tissues and DLBCL cells. Importantly, the shorter survival outcome was observed in patients with high levels of RCN1. Intriguingly, down-regulation of RCN1 attenuated DLBCL cell viability, invasion ability and increased cancer cell apoptosis. Moreover, TIMER database revealed the correlation between RCN1 expression and tumor-infiltrating macrophages, and M2 macrophage markers in DLBCL patients. Knockdown of RCN1 suppressed cancer cell ability to induce macrophage M2-like polarization by inhibiting the percentage of CD163⁺ macrophages and expression of M2-like markers (CD163, CD204, and IL-10). Concomitantly, M2 macrophage-induced cancer cell viability and invasion was abrogated after RCN1 down-regulation. Further assay revealed that knockdown of RCN1 suppressed activation of the PI3K/AKT signaling in DLBCL cells. Notably, reactivation this pathway via its agonist 740Y-P offset the anti-tumor efficacy of RCN1 knockdown in cancer cell malignancy and macrophage polarization towards M2 phenotype. Therefore, RCN1 may contribute to the malignant progression of DLBCL by directly regulating cancer cell behavior and indirectly affecting macrophage M2-like polarization, supporting it as a promising therapeutic target against DLBCL.

癌细胞的恶性行为和巨噬细胞的免疫逃避可导致弥漫性大b细胞淋巴瘤（DLBCL）治疗失败。最近的研究表明网状定位蛋白1 （RCN1）的异常表达与几种癌症的恶性进展有关。然而，其在DLBCL中的作用尚不清楚。在本研究中，几个在线生物信息学数据库证实了RCN1在DLBCL患者肿瘤样本中的高表达。此外，在我们收集的组织和DLBCL细胞的肿瘤标本中，rcn1明显升高。重要的是，在RCN1水平高的患者中观察到较短的生存结果。有趣的是，RCN1的下调降低了DLBCL细胞的活力、侵袭能力并增加了癌细胞的凋亡。此外，TIMER数据库揭示了RCN1表达与DLBCL患者肿瘤浸润性巨噬细胞、M2巨噬细胞标志物的相关性。RCN1的下调通过抑制CD163+巨噬细胞的百分比和m2样标志物（CD163、CD204和IL-10）的表达，抑制了癌细胞诱导巨噬细胞m2样极化的能力。同时，下调RCN1后，M2巨噬细胞诱导的癌细胞活力和侵袭能力减弱。进一步的分析显示，RCN1的敲低抑制了DLBCL细胞中PI3K/AKT信号的激活。值得注意的是，通过其激动剂740Y-P重新激活该途径抵消了RCN1敲低在癌细胞恶性和巨噬细胞向M2表型极化中的抗肿瘤作用。因此，RCN1可能通过直接调节癌细胞行为和间接影响巨噬细胞m2样极化而促进DLBCL的恶性进展，支持其作为DLBCL的有希望的治疗靶点。

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引用次数: 0

Single-cell sequencing revealed the recurrence causes of ETV6:RUNX1 fusion-positive B-ALL in children 单细胞测序揭示了儿童ETV6:RUNX1融合阳性B-ALL复发的原因

IF 2.3 3区生物学 Q3 BIOCHEMICAL RESEARCH METHODS

Molecular and Cellular Probes

Pub Date : 2025-07-12 DOI: 10.1016/j.mcp.2025.102041

Guotao Guan , Xiuxiu Wang , Xiuxin Li , Qi Wang , Xiuli Li , Xiaojun Sun , Liying Liu , Yunfeng Lu , Bingju Liu , Xinyu Li , Ping Zhao , Fei Gao , Lijun Chen , Lihua Zhao , Yunpeng Dai

Objective

The ETV6RUNX1 fusion is the most common genetic abnormality in childhood B-cell acute lymphoblastic leukemia (B-ALL), yet nearly 50 % of relapses occur in patients initially classified as low-risk with this alteration. This study aimed to unravel the underlying pathways driving relapse in ETV6RUNX1 positive B-ALL.

Methods

Single-cell RNA sequencing (scRNA-seq) was performed on a cohort of four B-ALL patients with the ETV6RUNX1 fusion (three newly diagnosed and one relapsed case, selected from 25 patients).

Results

we discovered that relapsed samples exhibited a decline in T cell populations, an increase in CD8 Tex cells and B cells, and a higher proportion of malignant cells. Gene enrichment analysis demonstrated that IFN-γ response signaling pathways and inflammatory responses were significantly enriched in newly diagnosed samples. Conversely, the relapsed samples showed enrichment in the oxidative phosphorylation and glycolysis pathways. Additionally, analysis of cellular interactions revealed that malignant B cells could interact with T cells through LGALS9-HAVCR2, potentially leading to the exhaustion of effector T cells. Moreover, NPDC1, LEF1, and ERG exhibited higher activity levels in malignant B cells from relapsed patients, highlighting their roles in the progression and maintenance of leukemia.

Conclusion

In summary, our study provides valuable insights into the potential causes of relapse in B-ALL patients with ETV6RUNX1, providing a foundation for the identification of prospective therapeutic targets.

目的：ETV6RUNX1基因融合是儿童b细胞急性淋巴细胞白血病（B-ALL）中最常见的遗传异常，但近50%的复发发生在最初被分类为低风险的患者中。本研究旨在揭示驱动ETV6RUNX1阳性B-ALL复发的潜在途径。方法对4例伴有ETV6RUNX1融合的B-ALL患者进行单细胞RNA测序（scRNA-seq），其中3例为新诊断，1例为复发病例，共25例。结果我们发现，复发样本中T细胞数量下降，CD8 Tex细胞和B细胞数量增加，恶性细胞比例更高。基因富集分析表明，IFN-γ反应信号通路和炎症反应在新诊断样本中显著富集。相反，复发样品在氧化磷酸化和糖酵解途径中显示富集。此外，细胞相互作用分析显示，恶性B细胞可以通过LGALS9-HAVCR2与T细胞相互作用，可能导致效应T细胞衰竭。此外，NPDC1、LEF1和ERG在复发患者的恶性B细胞中表现出更高的活性水平，突出了它们在白血病进展和维持中的作用。综上所述，本研究为了解B-ALL患者ETV6RUNX1复发的潜在原因提供了有价值的见解，为确定前瞻性治疗靶点提供了基础。

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引用次数: 0

Identification of CPLX1 expression as a potential prognostic marker in colorectal cancer CPLX1表达作为结直肠癌潜在预后标志物的鉴定。

IF 2.3 3区生物学 Q3 BIOCHEMICAL RESEARCH METHODS

Molecular and Cellular Probes

Pub Date : 2025-07-10 DOI: 10.1016/j.mcp.2025.102040

Li Jin , Lili Qian , Xin Zhang , Ting Liu

Objectives

CPLX1 is a member of the complexin/Synaphin family. Studies have shown that CPLX1 is involved in tumor progression. However, the biological mechanism by which CPLX1 is involved in colorectal cancer (CRC) is unclear.

Methods

TIMER and TCGA database provided difference expression of CPLX1 mRNA in pan-cancer and CRC. We collected 90 cases of CRC and adjacent normal tissues for immunohistochemistry (IHC) and investigated CPLX1 protein expression and its correlation with clinical information. Cox regression and Kaplan–Meier were conducted to determine prognostic value of CPLX1 expression. We utilized Spearman analysis to explore the association between CPLX1 and immune cell infiltration, immune checkpoints. We utilized GSEA to investigate the biological molecular function for CPLX1 in CRC.

Results

CPLX1 mRNA expression was elevated in several cancers, including CRC. Elevated CPLX1 protein expression was confirmed by IHC in CRC samples, and showed a positive association with T stage. The survival analysis demonstrated that CPLX1 overexpression was linked to a poorer overall survival (OS), disease-specific survival (DSS), and progress-free interval (PFI) for CRC. Time ROC analysis suggested that the survival rate of 1-year, 2-year and 3-year for OS, DSS, and PFI was above 0.5, suggesting a certain prognostic value in CRC. Cox regression considered CPLX1 expression as a good prognostic index for predicting OS, DSS, and PFI. Furthermore, CPLX1 expression was associated with immunity in CRC. Functional analysis showed that CPLX1 related genes had participation in Notch signaling pathway.

Conclusions

CPLX1 was a latent prognostic and diagnostic marker for CRC and may also be a potential therapeutic target.

目的：CPLX1是络合蛋白/突触蛋白家族的一员。研究表明CPLX1参与肿瘤进展。然而，CPLX1参与结直肠癌（CRC）的生物学机制尚不清楚。方法：TIMER和TCGA数据库提供CPLX1 mRNA在泛癌和结直肠癌中的差异表达。我们收集90例结直肠癌及其邻近正常组织进行免疫组化（IHC），研究CPLX1蛋白表达及其与临床信息的相关性。采用Cox回归和Kaplan-Meier法确定CPLX1表达的预后价值。我们利用Spearman分析来探讨CPLX1与免疫细胞浸润、免疫检查点的关系。我们利用GSEA研究了CPLX1在CRC中的生物学分子功能。结果：CPLX1 mRNA在包括CRC在内的多种癌症中表达升高。在CRC样本中，IHC证实CPLX1蛋白表达升高，并与T期呈正相关。生存分析表明，CPLX1过表达与CRC较差的总生存期（OS）、疾病特异性生存期（DSS）和无进展间隔（PFI）有关。时间ROC分析显示，OS、DSS、PFI的1年、2年、3年生存率均在0.5以上，提示对CRC有一定的预后价值。Cox回归认为CPLX1表达是预测OS、DSS和PFI的良好预后指标。此外，CPLX1的表达与CRC的免疫有关。功能分析显示CPLX1相关基因参与Notch信号通路。结论：CPLX1是CRC的潜在预后和诊断标志物，也可能是潜在的治疗靶点。

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引用次数: 0

Progress in the pathogenesis and treatment of pituitary neuroendocrine tumors (PitNETs): From molecular mechanisms to emerging therapies 垂体神经内分泌肿瘤（PitNETs）的发病机制和治疗进展：从分子机制到新兴疗法。

IF 2.3 3区生物学 Q3 BIOCHEMICAL RESEARCH METHODS

Molecular and Cellular Probes

Pub Date : 2025-06-26 DOI: 10.1016/j.mcp.2025.102039

Na Wu , Naijia Wu , Nan Wang , Yonghong Zhu

Pituitary neuroendocrine tumors (PitNETs), arising from anterior pituitary neuroendocrine cells, constitute 10–15 % of intracranial tumors. Though mostly benign, their location near critical structures like the optic chiasm and cavernous sinus causes severe neuroendocrine dysfunctions and mass effects. The 2022 WHO classification rebranded them from "pituitary adenomas" to emphasize their neuroendocrine lineage, yet challenges in prognostic prediction retain traditional terminology. Pathogenesis involves genetic mutations GNAS in GH-PitNETs, USP8 in ACTH-PitNETs), epigenetic dysregulation (METTL3-mediated m6A methylation), and signaling pathway aberrations (cAMP-PKA activation). Current treatments—surgery, dopamine agonists, somatostatin analogs, and radiation—face limitations like recurrence and side effects. Emerging strategies include targeted therapies (BET inhibitors) and immune checkpoint blockade, but tumor heterogeneity and lack of predictive biomarkers remain key obstacles. This review synthesizes pathogenesis, therapeutic advances, and research gaps to foster precision medicine and novel diagnostic/treatment paradigms for PitNETs.

垂体神经内分泌肿瘤（PitNETs）起源于垂体前叶神经内分泌细胞，占颅内肿瘤的10-15%。虽然大多数是良性的，但它们靠近关键结构，如视交叉和海绵窦，会导致严重的神经内分泌功能障碍和肿块效应。2022年世卫组织分类将其从“垂体腺瘤”重新命名，以强调其神经内分泌谱系，但在预后预测方面的挑战保留了传统术语。发病机制涉及基因突变（GH-PitNETs中的GNAS， ACTH-PitNETs中的USP8），表观遗传失调（mettl3介导的m6A甲基化）和信号通路畸变（cAMP-PKA激活）。目前的治疗方法——手术、多巴胺激动剂、生长抑素类似物和放疗——面临着复发和副作用等局限性。新兴策略包括靶向治疗（BET抑制剂）和免疫检查点阻断，但肿瘤异质性和缺乏预测性生物标志物仍然是主要障碍。本文综述了PitNETs的发病机制、治疗进展和研究空白，以促进精准医学和新的诊断/治疗范式。

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引用次数: 0

Assessment of cancer-associated fibroblast signature genes in ovarian cancer patients: impact on immunity, drug resistance, and prognosis 卵巢癌患者癌症相关成纤维细胞特征基因的评估：对免疫、耐药性和预后的影响

IF 2.3 3区生物学 Q3 BIOCHEMICAL RESEARCH METHODS

Molecular and Cellular Probes

Pub Date : 2025-06-18 DOI: 10.1016/j.mcp.2025.102038

Shunjin Zhang , Jiazhuo Yan , Wenjing Pan , Chaoyang Jia , Wei Liu , Sijia Liu , Zhao Wang , Yujie Liu , Yunyan Zhang

Ovarian cancer (OC) is women's third most common gynecologic tumor and is highly lethal. Cancer-associated fibroblasts (CAFs) are associated with cancer at all stages of disease progression and are involved in biological processes, including inflammatory processes, tumor development occurrence, and immune rejection. This study aimed to construct prognosis-related CAFs regulatory factors to predict the survival of OC patients. Datasets of OC patients with complete clinical information were collected from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) databases. First, we identified potential regulator factors of CAFs in OC based on the xCell algorithm and weighted gene co-expression analysis (WGCNA). Further screening using one-way cox regression analysis and LASSO regression models yielded 22 prognosis-related CAFs regulatory factors, using which a model was constructed. Subsequently, the diagnostic effectiveness of the model was assessed using receiver operating characteristic (ROC) curves, and the validity of the CAFs regulatory factors survival model was verified in three additional independent datasets and single cell data. Meanwhile, experimental validation was conducted using immunohistochemistry and Western blot. The results showed that GAS1 (Growth arrest specific 1) exhibited a higher expression pattern in fibroblasts from ovarian cancer patients.

The assessment of resistance and immune checkpoint differences across various risk score groups indicates that the CAFs regulatory factor survival model is practical for guiding systemic treatment. In summary, this study establishes a prognostic model composed of 22 CAFs regulatory factors to predict the prognosis of ovarian cancer (OC), providing new perspectives for the clinical treatment of OC.

卵巢癌（OC）是女性第三大常见妇科肿瘤，具有高致死率。癌症相关成纤维细胞（CAFs）在疾病进展的所有阶段都与癌症相关，并参与生物过程，包括炎症过程、肿瘤发生和免疫排斥。本研究旨在构建与预后相关的cas调节因子来预测OC患者的生存。具有完整临床信息的OC患者数据集来自基因表达Omnibus （GEO）和癌症基因组图谱（TCGA）数据库。首先，我们基于xCell算法和加权基因共表达分析（weighted gene co-expression analysis， WGCNA）确定了OC中cas的潜在调控因子。进一步使用单向cox回归分析和LASSO回归模型筛选得到22个与预后相关的cas调节因子，并以此构建模型。随后，使用受试者工作特征（ROC）曲线评估该模型的诊断有效性，并在另外三个独立数据集和单细胞数据中验证CAFs调节因子生存模型的有效性。同时采用免疫组织化学和Western blot方法进行实验验证。结果显示，GAS1 （Growth arrest specific 1）在卵巢癌患者的成纤维细胞中有较高的表达模式。不同风险评分组的耐药性和免疫检查点差异评估表明，CAFs调节因子生存模型可用于指导全身治疗。综上所述，本研究建立了由22个cas调节因子组成的预测卵巢癌（OC）预后的预后模型，为卵巢癌的临床治疗提供了新的视角。

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引用次数: 0

MicroRNA-130 as a critical modulator of cardiac remodeling: interplay between autophagic flux and ferroptotic pathways in acute myocardial infarction MicroRNA-130作为心脏重构的关键调节剂：急性心肌梗死中自噬通量和铁沉降途径之间的相互作用

IF 2.3 3区生物学 Q3 BIOCHEMICAL RESEARCH METHODS

Molecular and Cellular Probes

Pub Date : 2025-06-14 DOI: 10.1016/j.mcp.2025.102037

Liang Chen , Dongyang Jiang , Wenxin Kou , Yawei Xu

Background

Acute myocardial infarction (AMI) continues to be a leading cause of morbidity and death.

Objective

This study investigates miR-130's regulatory mechanisms in AMI progression.

Methods

Bioinformatics analysis of miR-130 conservation and differential expression was performed using miRbase and Gene Expression Omnibus datasets. Angiotensin II (Ang II)-treated H9C2 cardiomyocytes modeled AMI in vitro. miR-130 inhibitor/mimic transfection, combined with autophagy inhibitor Spautin-1 or ferroptosis inhibitor Ferrostatin-1, were assessed via qPCR, ELISA (PC III, HA, CTnT, CK-MB), Western blot (LC3-II/LC3-I, p62, SLC7A11, GPX4), and flow cytometry.

Results

Our results demonstrate that Ang II stimulation significantly elevates miR-130 expression in H9C2 cells, concomitant with increased secretion of myocardial injury and fibrosis markers. Inhibition of miR-130 markedly improved cell viability and reduced apoptosis, accompanied by decreased expression of fibrosis markers such as α-SMA and Collagen I, and a rebalancing of autophagy dynamics, as indicated by an increased LC3-II/LC3-I ratio and elevated p62 levels. Conversely, miR-130 overexpression via a synthetic mimic reduced cell viability and enhanced apoptosis, with a corresponding rise in fibrosis markers (PC III, HA, CTnT, CK-MB) and disruption of both autophagy and ferroptosis pathways, evidenced by decreased levels of SLC7A11 and GPX4. Notably, the adverse effects induced by miR-130 mimic were effectively reversed by Spautin-1 and Ferrostatin-1 co-treatment, suggesting that miR-130 modulates AMI-related cellular responses through intertwined autophagy and ferroptosis mechanisms.

Conclusion

These findings reveal that miR-130 is a pivotal regulator of myocardial injury in AMI, mediating its effects via the modulation of autophagy and ferroptosis pathways. Targeting miR-130 may therefore represent a promising therapeutic strategy for mitigating myocardial damage and improving cardiac function following AMI.

背景：急性心肌梗死（AMI）仍然是发病和死亡的主要原因。目的：探讨miR-130在AMI进展中的调控机制。方法：使用miRbase和Gene expression Omnibus数据集对miR-130的保守性和差异表达进行生物信息学分析。血管紧张素II （Ang II）处理的H9C2心肌细胞体外模型AMI。通过qPCR、ELISA （PC III、HA、CTnT、CK-MB）、Western blot （LC3-II/LC3-I、p62、SLC7A11、GPX4）和流式细胞术评估miR-130抑制剂/模拟物转染，联合自噬抑制剂Spautin-1或Ferrostatin-1。结果：我们的研究结果表明，Ang II刺激显著提高H9C2细胞中miR-130的表达，同时心肌损伤和纤维化标志物的分泌增加。抑制miR-130可显著提高细胞活力，减少细胞凋亡，同时α-SMA和胶原I等纤维化标志物的表达降低，自噬动力学的再平衡，LC3-II/LC3-I比值升高，p62水平升高。相反，通过合成模拟物过表达miR-130会降低细胞活力，增强细胞凋亡，纤维化标志物（PC III、HA、CTnT、CK-MB）相应升高，自噬和铁死亡途径均被破坏，SLC7A11和GPX4水平下降证明了这一点。值得注意的是，miR-130 mimic诱导的不良反应可以通过Spautin-1和Ferrostatin-1共同处理有效逆转，这表明miR-130通过相互交织的自噬和铁死亡机制调节ami相关的细胞反应。结论：这些研究结果表明，miR-130是AMI心肌损伤的关键调节因子，通过调节自噬和铁凋亡途径介导其作用。因此，靶向miR-130可能是减轻AMI后心肌损伤和改善心功能的一种有希望的治疗策略。

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引用次数: 0

DNMT3A triggers tumorigenesis of non-small cell lung cancer through regulation of SLIT2 methylation and SLIT2-mediated macrophage M1/M2 polarization DNMT3A通过调控SLIT2甲基化和SLIT2介导的巨噬细胞M1/M2极化，触发非小细胞肺癌的肿瘤发生。

IF 2.3 3区生物学 Q3 BIOCHEMICAL RESEARCH METHODS

Molecular and Cellular Probes

Pub Date : 2025-05-28 DOI: 10.1016/j.mcp.2025.102033

Tian-xing Ni , Jia-bo Shen

Background

Increased DNA methylation is prevalent in human cancers and is one of the important characteristics of tumors. This research aims to investigate the molecular mechanisms that involve DNMT3A and DNA methylation modification of SLIT2 in non-small cell lung cancer (NSCLC).

Methods

Gene expression was examined using Western blot assay, immunohistochemistry and RT-qPCR. Cell viability and motility were measured by CCK-8, colony formation, Transwell and wound healing assays. Macrophage M1/M2 polarization was assessed through a flow cytometry assay. Using ELISA, the secretion levels of inflammatory factors by macrophage M1/M2 polarization were determined. ChIP, qMSP and dual-luciferase reporter assays confirmed the relationship between DNMT3A and SLIT2.

Results

High expression of DNMT3A was observed in NSCLC patients, enhancing NSCLC cell viability and metastasis. Mechanically, DNMT3A was identified to target SLIT2. DNMT3A inhibited SLIT2 expression through DNA methylation modification in NSCLC. Further, overexpression of SLIT2 impeded M2 polarization of macrophages in NSCLC. And SLIT2 overexpression hindered NSCLC tumor growth in vivo by affecting macrophage M2 polarization. Finally, DNMT3A was found to promote the progression of NSCLC by downregulating SLIT2.

Conclusion

DNMT3A promotes the progression of NSCLC via regulating methylation modification of SLIT2 and SLIT2-mediated macrophage M1/M2 polarization.

背景：DNA甲基化增加在人类癌症中普遍存在，是肿瘤的重要特征之一。本研究旨在探讨DNMT3A和SLIT2 DNA甲基化修饰在非小细胞肺癌（NSCLC）中的分子机制。方法：采用Western blot、免疫组化、RT-qPCR检测基因表达。采用CCK-8法、菌落形成法、Transwell法和创面愈合法测定细胞活力和运动性。通过流式细胞术检测巨噬细胞M1/M2极化。ELISA法检测巨噬细胞M1/M2极化后炎性因子的分泌水平。ChIP、qMSP和双荧光素酶报告基因检测证实了DNMT3A和SLIT2之间的关系。结果：DNMT3A在NSCLC患者中高表达，增强了NSCLC细胞活力和转移。机械地，DNMT3A被鉴定为靶向SLIT2。DNMT3A通过DNA甲基化修饰抑制NSCLC中SLIT2的表达。此外，SLIT2的过表达阻碍了NSCLC中巨噬细胞的M2极化。SLIT2过表达通过影响巨噬细胞M2极化抑制体内NSCLC肿瘤生长。最后，我们发现DNMT3A通过下调SLIT2促进NSCLC的进展。结论：DNMT3A通过调节SLIT2的甲基化修饰和SLIT2介导的巨噬细胞M1/M2极化促进NSCLC的进展。

{"title":"DNMT3A triggers tumorigenesis of non-small cell lung cancer through regulation of SLIT2 methylation and SLIT2-mediated macrophage M1/M2 polarization","authors":"Tian-xing Ni , Jia-bo Shen","doi":"10.1016/j.mcp.2025.102033","DOIUrl":"10.1016/j.mcp.2025.102033","url":null,"abstract":"<div><h3>Background</h3><div>Increased DNA methylation is prevalent in human cancers and is one of the important characteristics of tumors. This research aims to investigate the molecular mechanisms that involve DNMT3A and DNA methylation modification of SLIT2 in non-small cell lung cancer (NSCLC).</div></div><div><h3>Methods</h3><div>Gene expression was examined using Western blot assay, immunohistochemistry and RT-qPCR. Cell viability and motility were measured by CCK-8, colony formation, Transwell and wound healing assays. Macrophage M1/M2 polarization was assessed through a flow cytometry assay. Using ELISA, the secretion levels of inflammatory factors by macrophage M1/M2 polarization were determined. ChIP, qMSP and dual-luciferase reporter assays confirmed the relationship between DNMT3A and SLIT2.</div></div><div><h3>Results</h3><div>High expression of DNMT3A was observed in NSCLC patients, enhancing NSCLC cell viability and metastasis. Mechanically, DNMT3A was identified to target SLIT2. DNMT3A inhibited SLIT2 expression through DNA methylation modification in NSCLC. Further, overexpression of SLIT2 impeded M2 polarization of macrophages in NSCLC. And SLIT2 overexpression hindered NSCLC tumor growth <em>in vivo</em> by affecting macrophage M2 polarization. Finally, DNMT3A was found to promote the progression of NSCLC by downregulating SLIT2.</div></div><div><h3>Conclusion</h3><div>DNMT3A promotes the progression of NSCLC via regulating methylation modification of SLIT2 and SLIT2-mediated macrophage M1/M2 polarization.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"82 ","pages":"Article 102033"},"PeriodicalIF":2.3,"publicationDate":"2025-05-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144188363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Gastrodin promotes osteogenic differentiation by stimulating the Wnt/β-catenin signaling pathway 天麻素通过刺激Wnt/β-catenin信号通路促进成骨分化。

IF 2.3 3区生物学 Q3 BIOCHEMICAL RESEARCH METHODS

Molecular and Cellular Probes

Pub Date : 2025-05-26 DOI: 10.1016/j.mcp.2025.102035

Wei Jiang , Lifeng Zhang , Wenshan Shan , Zuomeng Wu , Jiaqi Wang , Cailiang Shen

Background

Osteoporosis is a common disease that can lead to fracture as well as various skeletal symptoms and is a global health problem. Traditional Chinese medicine (TCM) may offer novel approaches for treating osteoporosis. Our study aimed to investigate the osteogenic potential and underlying mechanisms of gastrodin in promoting osteogenic differentiation.

Methods

By combining network pharmacology and bioinformatics, we conducted experiments to inspect cell viability, Alkaline Phosphatase (ALP) viability, and Alizarin red staining (ARS) and investigate the expression profiles of genes and proteins relevant to osteogenesis, including β-catenin, Runt-related transcription factor 2 (Runx2), Low Density Lipoprotein Receptor-Related Protein 5 (LRP5) and Glycogen synthase kinase-3 beta (GSK-3β). Statistical analysis was used for validation.

Results

Network pharmacology and bioinformatics analyses revealed that gastrodin might influence osteogenic differentiation. The experimental results revealed that gastrodin had no toxic effects and was able to promote ALP activity and stimulate osteogenic differentiation of Mouse Calvaria-derived Osteoblastic Cell Line.

(MC3T3-E1) cells. Subsequent network pharmacology and bioinformatics studies revealed that gastrodin might affect osteogenic differentiation through the Wnt/β-catenin signaling pathway. The results revealed that gastrodin influenced osteogenic differentiation genes and protein expression, including the upregulation of β-catenin, Runx2, and LRP5 and the downregulation of GSK-3β, and Dickkopf-1 (DKK-1) inhibited its promotion.

Conclusion

Gastrodin enhances the Wingless (Wnt)/β-catenin signaling pathway by increasing β-catenin accumulation and nuclear migration as well as decreasing GSK-3β, which increases Runx2 expression, consequently encouraging MC3T3-E1 cell osteogenic differentiation, and may be applied as a potential drug for osteoporosis therapy and prevention.

背景：骨质疏松症是一种常见疾病，可导致骨折和各种骨骼症状，是一个全球性的健康问题。中医药可能为治疗骨质疏松症提供新的途径。本研究旨在探讨天麻素促进成骨分化的潜能及其机制。方法：采用网络药理学和生物信息学相结合的方法，检测细胞活力、碱性磷酸酶（ALP）活力和茜素红染色（ARS），研究成骨相关基因和蛋白的表达谱，包括β-catenin、runt相关转录因子2 （Runx2）、低密度脂蛋白受体相关蛋白5 （LRP5）和糖原合成酶激酶3β (GSK-3β)。采用统计分析进行验证。结果：网络药理学和生物信息学分析显示天麻素可能影响成骨分化。实验结果表明天麻素对小鼠骨源性成骨细胞系（MC3T3-E1）细胞无毒性作用，且能促进ALP活性，促进成骨分化。随后的网络药理学和生物信息学研究表明天麻素可能通过Wnt/β-catenin信号通路影响成骨分化。结果显示天麻素影响成骨分化基因及蛋白表达，包括β-catenin、Runx2、LRP5上调，GSK-3β下调，Dickkopf-1 （DKK-1）抑制其促进作用。结论：天麻素通过增加β-catenin的积累和核迁移，降低GSK-3β，增加Runx2的表达，从而促进MC3T3-E1细胞成骨分化，从而增强Wnt /β-catenin信号通路，可能成为治疗和预防骨质疏松症的潜在药物。

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引用次数: 0

Transcriptional regulation of tumor suppressor gene RASSF1A by HBx HBx对肿瘤抑制基因RASSF1A的转录调控。

IF 2.3 3区生物学 Q3 BIOCHEMICAL RESEARCH METHODS

Molecular and Cellular Probes

Pub Date : 2025-05-24 DOI: 10.1016/j.mcp.2025.102034

Yanhong Kang , Wei Li , Junfeng Wei , Lin Yang , Yi Kang

Introduction

The occurrence of liver cancer in China is primarily attributed to chronic hepatitis B virus (HBV) infection. HBV X protein (HBx) has emerged as a significant carcinogenic driver in HBV-related liver cancer. However, the underlying mechanism by which HBx contributes to liver cancer development is not fully understood.

Methods

This study investigated HBx's role in regulating the tumor-suppressor gene RASSF1A. Firstly, the RASSF1A plasmid was constructed using a luciferase reporter system. The dual luciferase assay system detected HBx's effect on RASSF1A promoter activity. Western blotting and quantitative PCR methods measured HBx's impact on RASSF1A protein and mRNA expression. Chip was used to test the binding of HBx and SP1. CCK8, transwell, flow cytometry were used to detect the effect of RASSF1A on HCC proliferation. Methylation-specific PCR analyzed HBx's effect on RASSF1A methylation.

Results

Our results show that HBx significantly enhances RASSF1A promoter activity in an SP1 binding site-dependent manner. When only one SP1 binding site remained, HBx's effect was abolished. RASSF1A can inhibit HCC proliferation. Both mRNA and protein expression levels of RASSF1A were lower in HBx-expressing THLE-2 cells than in control cells, correlating with higher RASSF1A promoter methylation.

Conclusion

These findings suggest HBx enhances RASSF1A promoter activity and upregulates transcription via SP1, potentially preceding RASSF1A promoter methylation. This study provides new insights into HBx's regulation of the tumor suppressor gene RASSF1A in HBV-related liver cancer.

在中国，肝癌的发生主要归因于慢性乙型肝炎病毒（HBV）感染。HBV X蛋白（HBx）已成为HBV相关肝癌的重要致癌驱动因素。然而，HBx促进肝癌发展的潜在机制尚不完全清楚。方法：本研究探讨HBx在调节肿瘤抑制基因RASSF1A中的作用。首先，利用荧光素酶报告系统构建RASSF1A质粒。双荧光素酶测定系统检测HBx对RASSF1A启动子活性的影响。Western blotting和定量PCR方法检测HBx对RASSF1A蛋白和mRNA表达的影响。采用芯片检测HBx与SP1的结合情况。采用CCK8、transwell、流式细胞术检测RASSF1A对HCC增殖的影响。甲基化特异性PCR分析HBx对RASSF1A甲基化的影响。结果：我们的研究结果表明，HBx以SP1结合位点依赖的方式显著增强RASSF1A启动子活性。当仅保留一个SP1结合位点时，HBx的作用被消除。RASSF1A可以抑制HCC的增殖。在表达hbx的THLE-2细胞中，RASSF1A的mRNA和蛋白表达水平均低于对照细胞，这与RASSF1A启动子甲基化程度较高有关。结论：这些发现表明HBx增强RASSF1A启动子活性并通过SP1上调转录，可能先于RASSF1A启动子甲基化。本研究为HBx在hbv相关肝癌中调控肿瘤抑制基因RASSF1A提供了新的见解。

{"title":"Transcriptional regulation of tumor suppressor gene RASSF1A by HBx","authors":"Yanhong Kang , Wei Li , Junfeng Wei , Lin Yang , Yi Kang","doi":"10.1016/j.mcp.2025.102034","DOIUrl":"10.1016/j.mcp.2025.102034","url":null,"abstract":"<div><h3>Introduction</h3><div>The occurrence of liver cancer in China is primarily attributed to chronic hepatitis B virus (HBV) infection. HBV X protein (HBx) has emerged as a significant carcinogenic driver in HBV-related liver cancer. However, the underlying mechanism by which HBx contributes to liver cancer development is not fully understood.</div></div><div><h3>Methods</h3><div>This study investigated HBx's role in regulating the tumor-suppressor gene RASSF1A. Firstly, the RASSF1A plasmid was constructed using a luciferase reporter system. The dual luciferase assay system detected HBx's effect on RASSF1A promoter activity. Western blotting and quantitative PCR methods measured HBx's impact on RASSF1A protein and mRNA expression. Chip was used to test the binding of HBx and SP1. CCK8, transwell, flow cytometry were used to detect the effect of RASSF1A on HCC proliferation. Methylation-specific PCR analyzed HBx's effect on RASSF1A methylation.</div></div><div><h3>Results</h3><div>Our results show that HBx significantly enhances RASSF1A promoter activity in an SP1 binding site-dependent manner. When only one SP1 binding site remained, HBx's effect was abolished. RASSF1A can inhibit HCC proliferation. Both mRNA and protein expression levels of RASSF1A were lower in HBx-expressing THLE-2 cells than in control cells, correlating with higher RASSF1A promoter methylation.</div></div><div><h3>Conclusion</h3><div>These findings suggest HBx enhances RASSF1A promoter activity and upregulates transcription via SP1, potentially preceding RASSF1A promoter methylation. This study provides new insights into HBx's regulation of the tumor suppressor gene RASSF1A in HBV-related liver cancer.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"82 ","pages":"Article 102034"},"PeriodicalIF":2.3,"publicationDate":"2025-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144152636","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Exosomal lncRNA profiles in patients with HFrEF: Evidence for KLF3-AS1 as a novel diagnostic biomarker HFrEF患者外泌体lncRNA谱：KLF3-AS1作为一种新的诊断生物标志物的证据

IF 2.3 3区生物学 Q3 BIOCHEMICAL RESEARCH METHODS

Molecular and Cellular Probes

Pub Date : 2025-05-20 DOI: 10.1016/j.mcp.2025.102032

Lei Wang , Yanan Zhang , Jiapu Wang , Xiao Jiang , Gang Wang , Haixiong Wang , Yan Shu , Han Huiyuan

Background

Serum exosomal long noncoding RNAs (lncRNAs) have not been studied extensively as biomarkers in heart failure (HF) with reduced ejection fraction (HFrEF). We compared lncRNA expression in patients with HFrEF hospitalized for acute HF with that in healthy individuals to identify differentially expressed exosomal lncRNAs. Furthermore, we explored the clinical value of exosomal KLF3-AS1 in diagnosing HF and investigated its role in cardiac hypertrophy.

Method

Exosomes were isolated from patients with HFrEF and healthy individuals. We performed microarray analysis of differentially expressed lncRNAs and genes (DELs and DEGs, respectively) associated with HF. Protein-protein interaction (PPI), lncRNA-mRNA-KEGG pathway, and interaction networks between lncRNAs and RNA-binding proteins (RBPs) were developed. Expression patterns were verified using qRT-PCR. The diagnostic applicability of exosomal lncRNAs in HF was quantified by plotting receiver operating characteristic (ROC) curves. The size of the cardiomyocytes was evaluated using α-actinin immunostaining.

Results

In total, 138 DELs and 1132 DEGs were identified. PPI network analysis identified INS, CTNNB1, and CAT as the most prominent hub genes, whereas MDM2, MYH6, ENAH, and KLF3-AS1 were significantly enriched in the RBP interaction network. In the validation phase, patients with HFrEF exhibited a significant increase in KLF3-AS1 expression compared with healthy individuals. Exosomal KLF3-AS1 had an area under the ROC curve of 0.861. Functionally, KLF3-AS1 overexpression reduced Ang II-induced cardiac hypertrophy in vitro.

Conclusion

Our results elucidated the exact patterns of circulating exosomal mRNAs and lncRNA expression in patients with HFrEF hospitalized for acute HF. Moreover, the high expression of exosomal KLF3-AS1 is a potential diagnostic biomarker for HFrEF.

血清外泌体长链非编码rna （lncRNAs）作为心力衰竭（HF）伴射血分数降低（HFrEF）的生物标志物尚未得到广泛研究。我们比较了因急性HF住院的HFrEF患者与健康个体的lncRNA表达，以确定外泌体lncRNA的差异表达。此外，我们还探讨了外泌体KLF3-AS1在诊断HF中的临床价值，并探讨了其在心肌肥厚中的作用。方法分别从HFrEF患者和健康人体内分离酶体。我们对与HF相关的差异表达lncrna和基因（分别为DELs和deg）进行了微阵列分析。建立了蛋白-蛋白相互作用（PPI）、lncRNA-mRNA-KEGG通路以及lncrna与rna结合蛋白（rbp）的相互作用网络。使用qRT-PCR验证表达模式。通过绘制受试者工作特征（ROC）曲线，量化外泌体lncrna在HF诊断中的适用性。采用α-肌动素免疫染色法观察心肌细胞大小。结果共鉴定出138个DELs和1132个deg。PPI网络分析发现INS、CTNNB1和CAT是最突出的枢纽基因，而MDM2、MYH6、ENAH和KLF3-AS1在RBP相互作用网络中显著富集。在验证阶段，与健康个体相比，HFrEF患者的KLF3-AS1表达显著增加。外泌体KLF3-AS1的ROC曲线下面积为0.861。在功能上，KLF3-AS1过表达可减少Ang ii诱导的体外心肌肥大。结论我们的研究结果阐明了急性HF住院的HFrEF患者循环外泌体mrna和lncRNA表达的确切模式。此外，外泌体KLF3-AS1的高表达是HFrEF的潜在诊断生物标志物。

{"title":"Exosomal lncRNA profiles in patients with HFrEF: Evidence for KLF3-AS1 as a novel diagnostic biomarker","authors":"Lei Wang , Yanan Zhang , Jiapu Wang , Xiao Jiang , Gang Wang , Haixiong Wang , Yan Shu , Han Huiyuan","doi":"10.1016/j.mcp.2025.102032","DOIUrl":"10.1016/j.mcp.2025.102032","url":null,"abstract":"<div><h3>Background</h3><div>Serum exosomal long noncoding RNAs (lncRNAs) have not been studied extensively as biomarkers in heart failure (HF) with reduced ejection fraction (HFrEF). We compared lncRNA expression in patients with HFrEF hospitalized for acute HF with that in healthy individuals to identify differentially expressed exosomal lncRNAs. Furthermore, we explored the clinical value of exosomal KLF3-AS1 in diagnosing HF and investigated its role in cardiac hypertrophy.</div></div><div><h3>Method</h3><div>Exosomes were isolated from patients with HFrEF and healthy individuals. We performed microarray analysis of differentially expressed lncRNAs and genes (DELs and DEGs, respectively) associated with HF. Protein-protein interaction (PPI), lncRNA-mRNA-KEGG pathway, and interaction networks between lncRNAs and RNA-binding proteins (RBPs) were developed. Expression patterns were verified using qRT-PCR. The diagnostic applicability of exosomal lncRNAs in HF was quantified by plotting receiver operating characteristic (ROC) curves. The size of the cardiomyocytes was evaluated using α-actinin immunostaining.</div></div><div><h3>Results</h3><div>In total, 138 DELs and 1132 DEGs were identified. PPI network analysis identified <em>INS</em>, <em>CTNNB1</em>, and <em>CAT</em> as the most prominent hub genes, whereas <em>MDM2</em>, <em>MYH6</em>, <em>ENAH</em>, and KLF3-AS1 were significantly enriched in the RBP interaction network. In the validation phase, patients with HFrEF exhibited a significant increase in KLF3-AS1 expression compared with healthy individuals. Exosomal KLF3-AS1 had an area under the ROC curve of 0.861. Functionally, KLF3-AS1 overexpression reduced Ang II-induced cardiac hypertrophy <em>in vitro</em>.</div></div><div><h3>Conclusion</h3><div>Our results elucidated the exact patterns of circulating exosomal mRNAs and lncRNA expression in patients with HFrEF hospitalized for acute HF. Moreover, the high expression of exosomal KLF3-AS1 is a potential diagnostic biomarker for HFrEF.</div></div>","PeriodicalId":49799,"journal":{"name":"Molecular and Cellular Probes","volume":"82 ","pages":"Article 102032"},"PeriodicalIF":2.3,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144115680","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0