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Correction: The role of three‑dimensional scaffolds based on polyglycerol sebacate/ polycaprolactone/ gelatin in the presence of Nanohydroxyapatite in promoting chondrogenic differentiation of human adipose‑derived mesenchymal stem cells. 更正:纳米羟基磷灰石存在下,基于聚甘油癸酸酯/聚己内酯/明胶的三维支架在促进人脂肪来源的间充质干细胞软骨分化中的作用。
IF 6.4 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-05-17 DOI: 10.1186/s12575-023-00209-y
Pardis Yousefi Talouki, Saeed Hesami Tackallou, Shahrokh Shojaei, Soheila Zamanlui Benisi, Vahabodin Goodarzi
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引用次数: 0
BPTF in bone marrow provides a potential progression biomarker regulated by TFAP4 through the PI3K/AKT pathway in neuroblastoma. 骨髓中的BPTF通过PI3K/AKT通路在神经母细胞瘤中提供TFAP4调控的潜在进展生物标志物。
IF 6.4 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-05-11 DOI: 10.1186/s12575-023-00200-7
Chiyi Jiang, Yeran Yang, Sidou He, Zhixia Yue, Tianyu Xing, Ping Chu, Wenfa Yang, Hui Chen, Xiaoxi Zhao, Yongbo Yu, Xuan Zhang, Yan Su, Yongli Guo, Xiaoli Ma

Background: Neuroblastoma (NB) is the most common extracranial malignant solid tumor in children, which is highly prone to bone marrow (BM) metastasis. BM can monitor early signs of mild disease and metastasis. Existing biomarkers are insufficient for the diagnosis and treatment of NB. Bromodomain PHD finger transcription factor (BPTF) is an important subunit of the chromatin-remodeling complex that is closely associated with tumors. Here, we evaluated whether BPTF in BM plays an important role in predicting NB progression, and explore the molecular mechanism of BPTF in NB.

Methods: The clinical relevance of the BPTF was predicted in the GEO (GSE62564) and TARGET database. The biological function of BPTF in NB was investigated by constructing cell lines and employing BPTF inhibitor AU1. Western blot was used to determine the changes of BPTF, TFAP4, PI3K/AKT signaling and Epithelial-mesenchymal transition (EMT) related markers. A total of 109 children with newly diagnosed NB in Beijing Children's Hospital from January 2018 to March 2021 were included in this study. RT-PCR was used to measure the BPTF and TFAP4 expression in BM. The cut-off level was set at the median value of BPTF expression levels.

Results: Databases suggested that BPTF expression was higher in NB and was significantly associated with stage and grade. Proliferation and migration of NB cells were slowed down when BPTF was silenced. Mechanistically, TFAP4 could positively regulate BPTF and promotes EMT process through activating the PI3K/AKT signaling pathway. Moreover, detection of the newly diagnosed BM specimens showed that BPTF expression was significantly higher in high-risk group, stage IV group and BM metastasis group. Children with high BPTF at initial diagnosis were considered to have high risk for disease progression and recurrence. BPTF is an independent risk factor for predicting NB progression.

Conclusions: A novel and convenient BPTF-targeted humoral detection that can prompt minimal residual and predict NB progression in the early stages of the disease were identified. BPTF inhibitor AU1 is expected to become a new targeted drug for NB therapy. It's also reveal previously unknown mechanisms of BPTF in NB cell proliferation and metastasis through TFAP4 and PI3K/AKT pathways.

背景:神经母细胞瘤(Neuroblastoma, NB)是儿童最常见的颅内外恶性实体瘤,极易发生骨髓转移。BM可以监测轻度疾病和转移的早期迹象。现有的生物标志物不足以用于NB的诊断和治疗。溴域PHD指状转录因子(Bromodomain PHD finger transcription factor, BPTF)是与肿瘤密切相关的染色质重塑复合体的重要亚基。在此,我们评估了BPTF在BM中是否在预测NB进展中发挥重要作用,并探讨了BPTF在NB中的分子机制。方法:在GEO (GSE62564)和TARGET数据库中预测BPTF的临床相关性。通过构建细胞系和使用BPTF抑制剂AU1研究BPTF在NB中的生物学功能。Western blot检测BPTF、TFAP4、PI3K/AKT信号通路及上皮间质转化(Epithelial-mesenchymal transition, EMT)相关标志物的变化。本研究共纳入2018年1月至2021年3月北京儿童医院109例新诊断NB患儿。RT-PCR检测BM中BPTF和TFAP4的表达。截断水平设为BPTF表达水平的中位数。结果:数据库显示BPTF在NB中表达较高,且与分期、分级显著相关。BPTF沉默后,NB细胞的增殖和迁移速度减慢。机制上,TFAP4可通过激活PI3K/AKT信号通路正向调节BPTF,促进EMT过程。此外,对新诊断BM标本的检测显示,BPTF在高危组、IV期组和BM转移组中表达明显升高。初始诊断时BPTF高的儿童被认为有疾病进展和复发的高风险。BPTF是预测NB进展的独立危险因素。结论:我们发现了一种新的、方便的bptf靶向体液检测方法,可以在疾病早期提示最小残留并预测NB的进展。BPTF抑制剂AU1有望成为NB治疗的新靶向药物。这也揭示了BPTF通过TFAP4和PI3K/AKT通路参与NB细胞增殖和转移的机制。
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引用次数: 1
Exosomal microRNA-342-5p secreted from adipose-derived mesenchymal stem cells mitigates acute kidney injury in sepsis mice by inhibiting TLR9. 脂肪源性间充质干细胞分泌的外泌体microRNA-342-5p通过抑制TLR9减轻脓毒症小鼠的急性肾损伤。
IF 6.4 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-04-21 DOI: 10.1186/s12575-023-00198-y
Wei Liu, Chenghuan Hu, Buyao Zhang, Mingxia Li, Fuxing Deng, Shuangping Zhao

Background: Sepsis-related acute kidney injury (AKI) is an inflammatory disease associated with extremely high mortality and health burden. This study explored the possibility of exosomes secreted by adipose-derived mesenchymal stem cells (AMSCs) serving as a carrier for microRNA (miR)-342-5p to alleviate sepsis-related AKI and investigated the possible mechanism.

Methods: Serum was obtained from 30 patients with sepsis-associated AKI and 30 healthy volunteers for the measurement of miR-342-5p, blood urea nitrogen (BUN), and serum creatinine (SCr) levels. For in vitro experiments, AMSCs were transfected with LV-miR-342-5p or LV-miR-67 to acquire miR-342-5p-modified AMSCs and miR-67-modified AMSCs, from which the exosomes (AMSC-Exo-342 and AMSC-Exo-67) were isolated. The human renal proximal tubular epithelial cell line HK-2 was induced by lipopolysaccharide (LPS) to construct a cellular model of sepsis. The expression of Toll-like receptor 9 (TLR9) was also detected in AKI cells and mouse models. The interaction between miR-342-5p and TLR9 was predicted by dual luciferase reporter gene assay.

Results: Detection on clinical serum samples showed that BUN, SCr, and TLR9 were elevated and miR-342-5p level was suppressed in the serum of patients with sepsis-associated AKI. Transfection with LV-miR-342-5p reinforced miR-342-5p expression in AMSCs and AMSC-secreted exosomes. miR-342-5p negatively targeted TLR9. LPS treatment enhanced TLR9 expression, reduced miR-342-5p levels, suppressed autophagy, and increased inflammation in HK-2 cells, while the opposite trends were observed in LPS-induced HK-2 cells exposed to AMSC-Exo-342, Rapa, miR-342-5p mimic, or si-TLR9. Additionally, the effects of AMSC-Exo-342 on autophagy and inflammation in LPS-induced cells could be weakened by 3-MA or pcDNA3.1-TLR9 treatment. Injection of AMSC-Exo-342 enhanced autophagy, mitigated kidney injury, suppressed inflammation, and reduced BUN and SCr levels in sepsis-related AKI mouse models.

Conclusion: miR-342-5p transferred by exosomes from miR-342-5p-modified AMSCs ameliorated AKI by inhibiting TLR9 to accelerate autophagy.

背景:败血症相关性急性肾损伤(AKI)是一种具有极高死亡率和健康负担的炎症性疾病。本研究探讨了脂肪源性间充质干细胞(AMSCs)分泌的外泌体作为microRNA (miR)-342-5p载体减轻脓毒症相关AKI的可能性,并探讨了其可能的机制。方法:采集30例败血症相关性AKI患者和30名健康志愿者的血清,测定miR-342-5p、血尿素氮(BUN)和血清肌酐(SCr)水平。在体外实验中,用LV-miR-342-5p或LV-miR-67转染AMSCs,获得mir -342-5p修饰的AMSCs和mir -67修饰的AMSCs,并从中分离外泌体(msc - exo -342和msc - exo -67)。采用脂多糖(LPS)诱导人肾近端小管上皮细胞系HK-2建立脓毒症细胞模型。在AKI细胞和小鼠模型中也检测到toll样受体9 (TLR9)的表达。通过双荧光素酶报告基因测定预测miR-342-5p与TLR9之间的相互作用。结果:临床血清样本检测显示败血症相关性AKI患者血清BUN、SCr、TLR9升高,miR-342-5p水平被抑制。转染LV-miR-342-5p可增强AMSCs和amsc分泌外泌体中miR-342-5p的表达。miR-342-5p负性靶向TLR9。LPS处理增强了TLR9表达,降低了miR-342-5p水平,抑制了自噬,并增加了HK-2细胞的炎症,而LPS诱导的HK-2细胞暴露于AMSC-Exo-342、Rapa、miR-342-5p mimic或si-TLR9时,观察到相反的趋势。此外,AMSC-Exo-342对lps诱导的细胞自噬和炎症的作用可以通过3-MA或pcDNA3.1-TLR9处理而减弱。在败血症相关AKI小鼠模型中,注射AMSC-Exo-342增强自噬,减轻肾损伤,抑制炎症,降低BUN和SCr水平。结论:miR-342-5p通过外泌体从miR-342-5p修饰的AMSCs中转移,通过抑制TLR9加速自噬来改善AKI。
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引用次数: 3
The role of three-dimensional scaffolds based on polyglycerol sebacate/ polycaprolactone/ gelatin in the presence of Nanohydroxyapatite in promoting chondrogenic differentiation of human adipose-derived mesenchymal stem cells. 纳米羟基磷灰石存在下的聚甘油脂酸酯/聚己内酯/明胶三维支架在促进人脂肪源间充质干细胞软骨分化中的作用。
IF 6.4 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-03-24 DOI: 10.1186/s12575-023-00197-z
Pardis Yousefi Talouki, Saeed Hesami Tackallou, Shahrokh Shojaei, Soheila Zamanlui Benisi, Vahabodin Goodarzi

Background: Tissue engineering for cartilage regeneration has made great advances in recent years, although there are still challenges to overcome. This study aimed to evaluate the chondrogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) on three-dimensional scaffolds based on polyglycerol sebacate (PGS) / polycaprolactone (PCL) / gelatin(Gel) in the presence of Nanohydroxyapatite (nHA).

Materials and methods: In this study, a series of nHA-nanocomposite scaffolds were fabricated using 100:0:0, 60:40:0, and 60:20:20 weight ratios of PGS to PCL: Gel copolymers through salt leaching method. The morphology and porosity of prepared samples was characterized by SEM and EDX mapping analysis. Also, the dynamic contact angle and PBS adsorption tests are used to identify the effect of copolymerization and nanoparticles on scaffolds' hydrophilicity. The hydrolytic degradation properties were also analyzed. Furthermore, cell viability and proliferation as well as cell adhesion are evaluated to find out the biocompatibility. To determine the potential ability of nHA-nanocomposite scaffolds in chondrogenic differentiation, RT-PCR assay was performed to monitor the expression of collagen II, aggrecan, and Sox9 genes as markers of cartilage differentiation.

Results: The nanocomposites had an elastic modulus within a range of 0.71-1.30 MPa and 0.65-0.43 MPa, in dry and wet states, respectively. The PGS/PCL sample showed a water contact angle of 72.44 ± 2.2°, while the hydrophilicity significantly improved by adding HA nanoparticles. It was found from the hydrolytic degradation study that HA incorporation can accelerate the degradation rate compared with PGS and PGS/PCL samples. Furthermore, the in vitro biocompatibility tests showed significant cell attachment, proliferation, and viability of adipose-derived mesenchymal stem cells (ADMSCs). RT-PCR also indicated a significant increase in collagen II, aggrecan and Sox9 mRNA levels.

Conclusions: Our findings demonstrated that these nanocomposite scaffolds promote the differentiation of hADSCs into chondrocytes possibly by the increase in mRNA levels of collagen II, aggrecan, and Sox9 as markers of chondrogenic differentiation. In conclusion, the addition of PCL, Gelatin, and HA into PGS is a practical approach to adjust the general features of PGS to prepare a promising scaffold for cartilage tissue engineering.

背景:软骨再生的组织工程近年来取得了很大的进展,但仍有许多挑战需要克服。本研究旨在评估在纳米羟基磷灰石(nHA)存在下,人脂肪源性间充质干细胞(hADSCs)在聚甘油癸二酸酯(PGS) /聚己内酯(PCL) /明胶(Gel)三维支架上的软骨分化。材料与方法:本研究采用盐浸法制备PGS与PCL:凝胶共聚物的重量比分别为100:0:0、60:40:0和60:20:20,制备了一系列nha -纳米复合材料支架。通过SEM和EDX图谱分析表征了制备样品的形貌和孔隙度。同时,通过动态接触角和PBS吸附试验,确定了共聚和纳米颗粒对支架亲水性的影响。并对其水解降解性能进行了分析。通过细胞活力、细胞增殖和细胞粘附性的测定来确定其生物相容性。为了确定nha -纳米复合支架在软骨分化中的潜在能力,我们采用RT-PCR检测II型胶原蛋白、聚集蛋白和Sox9基因作为软骨分化的标志物的表达。结果:纳米复合材料在干、湿状态下的弹性模量分别为0.71 ~ 1.30 MPa和0.65 ~ 0.43 MPa。PGS/PCL样品的水接触角为72.44±2.2°,而HA纳米粒子的加入显著改善了PGS/PCL样品的亲水性。水解降解研究发现,与PGS和PGS/PCL样品相比,HA掺入能加快降解速度。此外,体外生物相容性测试显示,脂肪源性间充质干细胞(ADMSCs)具有显著的细胞附着、增殖和活力。RT-PCR也显示II型胶原、聚集蛋白和Sox9 mRNA水平显著升高。结论:我们的研究结果表明,这些纳米复合支架可能通过增加II型胶原、聚集蛋白和Sox9的mRNA水平(作为软骨分化的标志物)来促进hscs向软骨细胞的分化。综上所述,在PGS中添加PCL、明胶和透明质酸是一种调整PGS一般特性的实用方法,可以制备出一种有前景的软骨组织工程支架。
{"title":"The role of three-dimensional scaffolds based on polyglycerol sebacate/ polycaprolactone/ gelatin in the presence of Nanohydroxyapatite in promoting chondrogenic differentiation of human adipose-derived mesenchymal stem cells.","authors":"Pardis Yousefi Talouki,&nbsp;Saeed Hesami Tackallou,&nbsp;Shahrokh Shojaei,&nbsp;Soheila Zamanlui Benisi,&nbsp;Vahabodin Goodarzi","doi":"10.1186/s12575-023-00197-z","DOIUrl":"https://doi.org/10.1186/s12575-023-00197-z","url":null,"abstract":"<p><strong>Background: </strong>Tissue engineering for cartilage regeneration has made great advances in recent years, although there are still challenges to overcome. This study aimed to evaluate the chondrogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) on three-dimensional scaffolds based on polyglycerol sebacate (PGS) / polycaprolactone (PCL) / gelatin(Gel) in the presence of Nanohydroxyapatite (nHA).</p><p><strong>Materials and methods: </strong>In this study, a series of nHA-nanocomposite scaffolds were fabricated using 100:0:0, 60:40:0, and 60:20:20 weight ratios of PGS to PCL: Gel copolymers through salt leaching method. The morphology and porosity of prepared samples was characterized by SEM and EDX mapping analysis. Also, the dynamic contact angle and PBS adsorption tests are used to identify the effect of copolymerization and nanoparticles on scaffolds' hydrophilicity. The hydrolytic degradation properties were also analyzed. Furthermore, cell viability and proliferation as well as cell adhesion are evaluated to find out the biocompatibility. To determine the potential ability of nHA-nanocomposite scaffolds in chondrogenic differentiation, RT-PCR assay was performed to monitor the expression of collagen II, aggrecan, and Sox9 genes as markers of cartilage differentiation.</p><p><strong>Results: </strong>The nanocomposites had an elastic modulus within a range of 0.71-1.30 MPa and 0.65-0.43 MPa, in dry and wet states, respectively. The PGS/PCL sample showed a water contact angle of 72.44 ± 2.2°, while the hydrophilicity significantly improved by adding HA nanoparticles. It was found from the hydrolytic degradation study that HA incorporation can accelerate the degradation rate compared with PGS and PGS/PCL samples. Furthermore, the in vitro biocompatibility tests showed significant cell attachment, proliferation, and viability of adipose-derived mesenchymal stem cells (ADMSCs). RT-PCR also indicated a significant increase in collagen II, aggrecan and Sox9 mRNA levels.</p><p><strong>Conclusions: </strong>Our findings demonstrated that these nanocomposite scaffolds promote the differentiation of hADSCs into chondrocytes possibly by the increase in mRNA levels of collagen II, aggrecan, and Sox9 as markers of chondrogenic differentiation. In conclusion, the addition of PCL, Gelatin, and HA into PGS is a practical approach to adjust the general features of PGS to prepare a promising scaffold for cartilage tissue engineering.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"25 1","pages":"9"},"PeriodicalIF":6.4,"publicationDate":"2023-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10039520/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9478036","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
Macrophage-Derived MMP-9 and MMP-2 are Closely Related to the Rupture of the Fibrous Capsule of Hepatocellular Carcinoma Leading to Tumor Invasion. 巨噬细胞来源的MMP-9和MMP-2与肝细胞癌纤维囊破裂导致肿瘤侵袭密切相关。
IF 6.4 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-03-14 DOI: 10.1186/s12575-023-00196-0
Quanwei Cui, Xuben Wang, Yongwei Zhang, Yiqing Shen, Yeben Qian

Background: Hepatocellular carcinoma (HCC) is an aggressive tumor with a poor clinical prognosis. Rupture of the fibrous capsule (FC) is a very important clinical phenomenon in the invasion and metastasis of HCC. FC is mainly composed of type I collagen (COL1A1). However, it is not clear what caused the FC rupture. In this study, we aimed to determine whether the rupture of FC in HCC patients was related to macrophage-derived MMP-9 and MMP-2, and their clinical diagnostic value for FC rupture.

Results: By performing immunohistochemical and immunofluorescence staining of ruptured FC and intact FC, the results showed that the ruptured area of FC aggregated a large number of macrophages with MMP-9 and MMP-2. Western blot analysis and Quantitative real-time PCR were used to assess the expression of MMP-9 and MMP-2 in the ruptured and relatively intact area of FC in ruptured FC patients, and the results revealed a significantly different expression of MMP-9 and MMP-2. ELISA experiments show that we could discriminate effectively between ruptured FC and intact FC by MMP-9 and MMP-2.

Conclusions: Taken together, macrophage-derived MMP-9 and MMP-2 were closely related to the rupture of the FC of HCC and subsequently led to the migration and invasion of the tumor cells through the ruptured area of FC to the para cancer. It is suggested that when performing surgical resection, it is necessary to expand the range of tumor resection for patients with ruptured FC and hence reduce the possibility of recurrence and metastasis in HCC patients.

背景:肝细胞癌是一种侵袭性肿瘤,临床预后较差。纤维囊破裂(FC)是HCC侵袭转移的重要临床现象。FC主要由I型胶原(COL1A1)组成。然而,目前还不清楚是什么导致了FC破裂。在本研究中,我们旨在确定HCC患者FC破裂是否与巨噬细胞来源的MMP-9和MMP-2有关,以及它们对FC破裂的临床诊断价值。结果:对破裂的FC和完整的FC进行免疫组化和免疫荧光染色,结果显示FC破裂区聚集了大量具有MMP-9和MMP-2的巨噬细胞。采用Western blot和Quantitative real-time PCR检测FC患者FC破裂区和相对完整区MMP-9和MMP-2的表达,结果显示MMP-9和MMP-2的表达存在显著差异。ELISA实验表明,MMP-9和MMP-2可以有效区分破裂FC和完整FC。结论:综上所述,巨噬细胞来源的MMP-9和MMP-2与HCC FC破裂密切相关,并导致肿瘤细胞通过FC破裂区向癌旁迁移和侵袭。提示在进行手术切除时,有必要扩大FC破裂患者的肿瘤切除范围,从而降低HCC患者复发转移的可能性。
{"title":"Macrophage-Derived MMP-9 and MMP-2 are Closely Related to the Rupture of the Fibrous Capsule of Hepatocellular Carcinoma Leading to Tumor Invasion.","authors":"Quanwei Cui,&nbsp;Xuben Wang,&nbsp;Yongwei Zhang,&nbsp;Yiqing Shen,&nbsp;Yeben Qian","doi":"10.1186/s12575-023-00196-0","DOIUrl":"https://doi.org/10.1186/s12575-023-00196-0","url":null,"abstract":"<p><strong>Background: </strong>Hepatocellular carcinoma (HCC) is an aggressive tumor with a poor clinical prognosis. Rupture of the fibrous capsule (FC) is a very important clinical phenomenon in the invasion and metastasis of HCC. FC is mainly composed of type I collagen (COL1A1). However, it is not clear what caused the FC rupture. In this study, we aimed to determine whether the rupture of FC in HCC patients was related to macrophage-derived MMP-9 and MMP-2, and their clinical diagnostic value for FC rupture.</p><p><strong>Results: </strong>By performing immunohistochemical and immunofluorescence staining of ruptured FC and intact FC, the results showed that the ruptured area of FC aggregated a large number of macrophages with MMP-9 and MMP-2. Western blot analysis and Quantitative real-time PCR were used to assess the expression of MMP-9 and MMP-2 in the ruptured and relatively intact area of FC in ruptured FC patients, and the results revealed a significantly different expression of MMP-9 and MMP-2. ELISA experiments show that we could discriminate effectively between ruptured FC and intact FC by MMP-9 and MMP-2.</p><p><strong>Conclusions: </strong>Taken together, macrophage-derived MMP-9 and MMP-2 were closely related to the rupture of the FC of HCC and subsequently led to the migration and invasion of the tumor cells through the ruptured area of FC to the para cancer. It is suggested that when performing surgical resection, it is necessary to expand the range of tumor resection for patients with ruptured FC and hence reduce the possibility of recurrence and metastasis in HCC patients.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"25 1","pages":"8"},"PeriodicalIF":6.4,"publicationDate":"2023-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10012540/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9131299","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation. 在转录组文库制备过程中用高亲和力寡核苷酸阻断丰富的RNA转录。
IF 3.7 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-03-08 DOI: 10.1186/s12575-023-00193-3
Celine Everaert, Jasper Verwilt, Kimberly Verniers, Niels Vandamme, Alvaro Marcos Rubio, Jo Vandesompele, Pieter Mestdagh

Background: RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species.

Results: We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3' end sequencing and long-read sequencing, and MALAT1 in single-cell 3' end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity.

Conclusion: Our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.

背景:RNA测序已成为转录组分析的金标准,但对低丰度转录物的定量具有挑战性的固有局限性。与微阵列技术相反,RNA测序读数是按转录物丰度的函数按比例划分的。因此,低丰度的RNA与高丰度的(有时是无信息的)RNA物种竞争。结果:我们开发了一种基于高亲和力RNA结合寡核苷酸的易于使用的策略,以阻断特定RNA转录物的逆转录和PCR扩增,从而显著降低其在最终测序库中的丰度。为了证明我们的方法的广泛应用潜力,我们将其应用于不同的转录物和文库制备策略,包括人血浆小RNA测序中的YRNA、3’端测序和长读测序中的线粒体rRNA,以及单细胞3’端排序中的MALAT1。我们证明了阻断策略是高效、可重复、特异的,通常会导致更好的转录组覆盖率和复杂性。结论:除了简单地在RT反应中添加阻断寡核苷酸外,我们的方法不需要修改文库制备程序,因此可以很容易地整合到几乎任何RNA测序文库制备方案中。
{"title":"Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation.","authors":"Celine Everaert, Jasper Verwilt, Kimberly Verniers, Niels Vandamme, Alvaro Marcos Rubio, Jo Vandesompele, Pieter Mestdagh","doi":"10.1186/s12575-023-00193-3","DOIUrl":"10.1186/s12575-023-00193-3","url":null,"abstract":"<p><strong>Background: </strong>RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species.</p><p><strong>Results: </strong>We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3' end sequencing and long-read sequencing, and MALAT1 in single-cell 3' end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity.</p><p><strong>Conclusion: </strong>Our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"25 1","pages":"7"},"PeriodicalIF":3.7,"publicationDate":"2023-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9996952/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9092589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
YTHDF2 exerts tumor-suppressor roles in gastric cancer via up-regulating PPP2CA independently of m6A modification. YTHDF2通过不依赖m6A修饰而上调PPP2CA在胃癌中发挥抑瘤作用。
IF 6.4 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-03-04 DOI: 10.1186/s12575-023-00195-1
Ying Zhou, Kailing Fan, Ning Dou, Li Li, Jialin Wang, Jingde Chen, Yandong Li, Yong Gao

Background: YTHDF2 is one of important readers of N6-methyladenosine (m6A) modification on RNA. Growing evidence implicates that YTHDF2 takes an indispensable part in the regulation of tumorigenesis and metastasis in different cancers, but its biological functions and underlying mechanisms remain elusive in gastric cancer (GC).

Aim: To investigate the clinical relevance and biological function of YTHDF2 in GC.

Results: Compared with matched normal stomach tissues, YTHDF2 expression was markedly decreased in gastric cancer tissues. The expression level of YTHDF2 was inversely associated with gastric cancer patients' tumor size, AJCC classification and prognosis. Functionally, YTHDF2 reduction facilitated gastric cancer cell growth and migration in vitro and in vivo, whereas YTHDF2 overexpression exhibited opposite phenotypes. Mechanistically, YTHDF2 enhanced expression of PPP2CA, the catalytic subunit of PP2A (Protein phosphatase 2A), in an m6A-independent manner, and silencing of PPP2CA antagonized the anti-tumor effects caused by overexpression of YTHDF2 in GC cells.

Conclusion: These findings demonstrate that YTHDF2 is down-regulated in GC and its down-regulation promotes GC progression via a possible mechanism involving PPP2CA expression, suggesting that YTHDF2 may be a hopeful biomarker for diagnosis and an unrevealed treatment target for GC.

背景:YTHDF2是RNA上n6 -甲基腺苷(m6A)修饰的重要读者之一。越来越多的证据表明,YTHDF2在不同癌症的肿瘤发生和转移调控中发挥着不可或缺的作用,但其在胃癌中的生物学功能和潜在机制尚不清楚。目的:探讨YTHDF2在胃癌中的临床意义及生物学功能。结果:与匹配的正常胃组织相比,胃癌组织中YTHDF2的表达明显降低。YTHDF2表达水平与胃癌患者肿瘤大小、AJCC分型及预后呈负相关。在功能上,YTHDF2的减少促进了胃癌细胞在体外和体内的生长和迁移,而YTHDF2的过表达则表现出相反的表型。在机制上,YTHDF2以不依赖m6a的方式增强PP2A(蛋白磷酸酶2A)的催化亚基PPP2CA的表达,而沉默PPP2CA可拮抗GC细胞中YTHDF2过表达引起的抗肿瘤作用。结论:这些研究结果表明,YTHDF2在胃癌中下调,其下调可能通过PPP2CA表达的机制促进胃癌的进展,提示YTHDF2可能是一种有希望的胃癌诊断生物标志物和尚未发现的治疗靶点。
{"title":"YTHDF2 exerts tumor-suppressor roles in gastric cancer via up-regulating PPP2CA independently of m<sup>6</sup>A modification.","authors":"Ying Zhou,&nbsp;Kailing Fan,&nbsp;Ning Dou,&nbsp;Li Li,&nbsp;Jialin Wang,&nbsp;Jingde Chen,&nbsp;Yandong Li,&nbsp;Yong Gao","doi":"10.1186/s12575-023-00195-1","DOIUrl":"https://doi.org/10.1186/s12575-023-00195-1","url":null,"abstract":"<p><strong>Background: </strong>YTHDF2 is one of important readers of N6-methyladenosine (m<sup>6</sup>A) modification on RNA. Growing evidence implicates that YTHDF2 takes an indispensable part in the regulation of tumorigenesis and metastasis in different cancers, but its biological functions and underlying mechanisms remain elusive in gastric cancer (GC).</p><p><strong>Aim: </strong>To investigate the clinical relevance and biological function of YTHDF2 in GC.</p><p><strong>Results: </strong>Compared with matched normal stomach tissues, YTHDF2 expression was markedly decreased in gastric cancer tissues. The expression level of YTHDF2 was inversely associated with gastric cancer patients' tumor size, AJCC classification and prognosis. Functionally, YTHDF2 reduction facilitated gastric cancer cell growth and migration in vitro and in vivo, whereas YTHDF2 overexpression exhibited opposite phenotypes. Mechanistically, YTHDF2 enhanced expression of PPP2CA, the catalytic subunit of PP2A (Protein phosphatase 2A), in an m<sup>6</sup>A-independent manner, and silencing of PPP2CA antagonized the anti-tumor effects caused by overexpression of YTHDF2 in GC cells.</p><p><strong>Conclusion: </strong>These findings demonstrate that YTHDF2 is down-regulated in GC and its down-regulation promotes GC progression via a possible mechanism involving PPP2CA expression, suggesting that YTHDF2 may be a hopeful biomarker for diagnosis and an unrevealed treatment target for GC.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"25 1","pages":"6"},"PeriodicalIF":6.4,"publicationDate":"2023-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9985201/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10847149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Influence of Exosomes on Astrocytes in the Pre-Metastatic Niche of Lung Cancer Brain Metastases. 外泌体对肺癌脑转移前生态位星形胶质细胞的影响。
IF 6.4 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-03-01 DOI: 10.1186/s12575-023-00192-4
Lingyun Ye, Yinfei Wu, Juan Zhou, Mengqing Xie, Zhemin Zhang, Chunxia Su

Background: Lung cancer is the most common cause of cancer-related death globally. There are several reasons for this high mortality rate, including metastasis to multiple organs, especially the brain. Exosomes play a pivotal role in tumor metastasis by remodeling the microenvironment of remote target organs and promoting the pre-metastatic niche's formation. Since astrocytes are indispensable for maintaining the homeostasis of brain microenvironment, it's of great interest to explore the influence of lung cancer cell-derived exosomes on astrocytes to further understand the mechanism of lung cancer brain metastasis.

Results: Twenty four h after co-culture of H1299 cell-derived exosomes and SVG P12 cells, the viability of astrocytes decreased and the apoptosis increased. The levels of cytokines in the supernatant including GROα/CXCL1, IFN-γ, IL-3, IL-5, IL-15, LIF, M-CSF, NGF, PDGF, and VEGF were significantly enhanced, while IL-7 secretion was significantly reduced. Meanwhile, apoptosis-related proteins MAP2K1, TUBA1C, RELA, and CASP6 were up-regulated. And the differentially expressed proteins were involved in regulating metabolic pathways.

Conclusion: Exosomes of H1299 could induce apoptosis of astrocytes as well as promote their secretion of cytokines that were conducive to the formation of the inflammatory microenvironment and immunosuppressive microenvironment, and affect their metabolic pathways, thus facilitating the formation of pre-metastatic niche in lung cancer brain metastases.

背景:肺癌是全球癌症相关死亡的最常见原因。造成这种高死亡率的原因有很多,包括转移到多个器官,尤其是大脑。外泌体通过重塑远处靶器官的微环境和促进转移前生态位的形成,在肿瘤转移中发挥关键作用。由于星形胶质细胞在维持脑微环境的稳态中不可或缺,因此探索肺癌细胞源性外泌体对星形胶质细胞的影响,以进一步了解肺癌脑转移的机制具有重要意义。结果:H1299细胞源性外泌体与SVG P12细胞共培养24 h后,星形胶质细胞活力下降,凋亡增加。上清液中GROα/CXCL1、IFN-γ、IL-3、IL-5、IL-15、LIF、M-CSF、NGF、PDGF、VEGF等细胞因子水平显著升高,IL-7分泌显著降低。同时,凋亡相关蛋白MAP2K1、TUBA1C、RELA、CASP6上调。差异表达蛋白参与调节代谢途径。结论:H1299外泌体可诱导星形胶质细胞凋亡,促进星形胶质细胞分泌有利于炎症微环境和免疫抑制微环境形成的细胞因子,影响其代谢途径,促进肺癌脑转移灶转移前生态位的形成。
{"title":"Influence of Exosomes on Astrocytes in the Pre-Metastatic Niche of Lung Cancer Brain Metastases.","authors":"Lingyun Ye,&nbsp;Yinfei Wu,&nbsp;Juan Zhou,&nbsp;Mengqing Xie,&nbsp;Zhemin Zhang,&nbsp;Chunxia Su","doi":"10.1186/s12575-023-00192-4","DOIUrl":"https://doi.org/10.1186/s12575-023-00192-4","url":null,"abstract":"<p><strong>Background: </strong>Lung cancer is the most common cause of cancer-related death globally. There are several reasons for this high mortality rate, including metastasis to multiple organs, especially the brain. Exosomes play a pivotal role in tumor metastasis by remodeling the microenvironment of remote target organs and promoting the pre-metastatic niche's formation. Since astrocytes are indispensable for maintaining the homeostasis of brain microenvironment, it's of great interest to explore the influence of lung cancer cell-derived exosomes on astrocytes to further understand the mechanism of lung cancer brain metastasis.</p><p><strong>Results: </strong>Twenty four h after co-culture of H1299 cell-derived exosomes and SVG P12 cells, the viability of astrocytes decreased and the apoptosis increased. The levels of cytokines in the supernatant including GROα/CXCL1, IFN-γ, IL-3, IL-5, IL-15, LIF, M-CSF, NGF, PDGF, and VEGF were significantly enhanced, while IL-7 secretion was significantly reduced. Meanwhile, apoptosis-related proteins MAP2K1, TUBA1C, RELA, and CASP6 were up-regulated. And the differentially expressed proteins were involved in regulating metabolic pathways.</p><p><strong>Conclusion: </strong>Exosomes of H1299 could induce apoptosis of astrocytes as well as promote their secretion of cytokines that were conducive to the formation of the inflammatory microenvironment and immunosuppressive microenvironment, and affect their metabolic pathways, thus facilitating the formation of pre-metastatic niche in lung cancer brain metastases.</p>","PeriodicalId":8960,"journal":{"name":"Biological Procedures Online","volume":"25 1","pages":"5"},"PeriodicalIF":6.4,"publicationDate":"2023-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9976367/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10826181","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
A 3D human co-culture to model neuron-astrocyte interactions in tauopathies. 一个三维人类共培养模型神经元-星形胶质细胞相互作用在牛头病变。
IF 6.4 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-02-23 DOI: 10.1186/s12575-023-00194-2
Kevin L Batenburg, Claudia Sestito, Paulien Cornelissen-Steijger, Jan R T van Weering, Leo S Price, Vivi M Heine, Wiep Scheper

Background: Intraneuronal tau aggregation is the major pathological hallmark of neurodegenerative tauopathies. It is now generally acknowledged that tau aggregation also affects astrocytes in a cell non-autonomous manner. However, mechanisms involved are unclear, partly because of the lack of models that reflect the situation in the human tauopathy brain. To accurately model neuron-astrocyte interaction in tauopathies, there is a need for a model that contains both human neurons and human astrocytes, intraneuronal tau pathology and mimics the three-dimensional architecture of the brain.

Results: Here we established a novel 100-200 µm thick 3D human neuron/astrocyte co-culture model of tau pathology, comprising homogenous populations of hiPSC-derived neurons and primary human astrocytes in microwell format. Using confocal, electron and live microscopy, we validate the procedures by showing that neurons in the 3D co-culture form pre- and postsynapses and display spontaneous calcium transients within 4 weeks. Astrocytes in the 3D co-culture display bipolar and stellate morphologies with extensive processes that ensheath neuronal somas, spatially align with axons and dendrites and can be found perisynaptically. The complex morphology of astrocytes and the interaction with neurons in the 3D co-culture mirrors that in the human brain, indicating the model's potential to study physiological and pathological neuron-astrocyte interaction in vitro. Finally, we successfully implemented a methodology to introduce seed-independent intraneuronal tau aggregation in the 3D co-culture, enabling study of neuron-astrocyte interaction in early tau pathogenesis.

Conclusions: Altogether, these data provide proof-of-concept for the utility of this rapid, miniaturized, and standardized 3D model for cell type-specific manipulations, such as the intraneuronal pathology that is associated with neurodegenerative disorders.

背景:神经元内tau聚集是神经退行性tau病的主要病理标志。现在普遍认为tau聚集也以细胞非自主的方式影响星形胶质细胞。然而,所涉及的机制尚不清楚,部分原因是缺乏反映人类脑损伤情况的模型。为了准确地模拟tau病变中神经元与星形胶质细胞的相互作用,需要一个既包含人类神经元又包含人类星形胶质细胞、神经元内tau病理和模拟大脑三维结构的模型。结果:在这里,我们建立了一个新的100-200µm厚的3D人神经元/星形胶质细胞共培养的tau病理学模型,包括hipsc来源的神经元和微孔格式的原代人星形胶质细胞。使用共聚焦显微镜、电子显微镜和活体显微镜,我们通过显示3D共培养中的神经元在4周内形成突触前和突触后并显示自发钙瞬变来验证这些程序。三维共培养的星形胶质细胞显示双极和星状形态,具有广泛的突起,包住神经元体,在空间上与轴突和树突排列,并且可以在突触周围发现。三维共培养中星形胶质细胞的复杂形态和与神经元的相互作用反映了人脑中的情况,表明该模型具有体外研究生理和病理神经元-星形胶质细胞相互作用的潜力。最后,我们成功地实施了一种方法,在3D共培养中引入了不依赖种子的神经元内tau聚集,从而研究了早期tau发病机制中的神经元-星形胶质细胞相互作用。结论:总的来说,这些数据为这种快速、小型化和标准化的3D模型在细胞类型特异性操作中的应用提供了概念验证,例如与神经退行性疾病相关的神经元内病理。
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引用次数: 4
Ultrasound and microbubble-mediated delivery of miR-424-5p has a therapeutic effect in preeclampsia. 超声和微泡介导的miR-424-5p递送对子痫前期有治疗作用。
IF 6.4 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2023-02-15 DOI: 10.1186/s12575-023-00191-5
Xudong Wang, Yan Wu, Qinliang Sun, Zhonghui Jiang, Guoying Che, Yangyang Tao, Jiawei Tian

Objective: To determine the influence of ultrasound/microbubble-mediated miR-424-5p delivery on trophoblast cells and the underlying mechanism.

Methods: Blood pressure and 24-h proteinuria of patients with preeclampsia (PE) were measured as well as the levels of miR-424-5p and amine oxidase copper containing 1 (AOC1) in placental tissues. HTR-8/Svneo and TEV-1 cells were subjected to cell transfection or ultrasonic microbubble transfection for determination of the expression of miR-424-5p, AOC1, β-catenin and c-Myc as well as cell proliferation, apoptosis, migration and invasiveness. The concentrations of placental growth factor (PLGF), human chorionic gonadotropin (β-hCG) and tumor necrosis factor-α (TNF-α) were measured in HTR-8/Svneo and TEV-1 cells. RNA immunoprecipitation (RIP) and dual luciferase reporter assay detected the binding of miR-424-5p to AOC1. A PE mouse model was induced by subcutaneous injection of L-NAME, where the influence of ultrasound/microbubble-mediated miR-424-5p delivery was evaluated.

Results: miR-424-5p was downregulated while AOC1 was upregulated in the placental tissues from PE patients. Overexpression of miR-424-5p activated Wnt/β-catenin signaling pathway and promoted the proliferation of HTR-8/Svneo and TEV-1 cells as well as enhanced the migratory and invasive behaviors. AOC1 overexpression partly eliminated the effects of miR-424-5p on HTR-8/Svneo and TEV-1 cells. Ultrasound and microbubble mediated gene delivery enhanced the transfection efficiency of miR-424-5p and further promoted the effects of miR-424-5p in trophoblast cells. Ultrasound/microbubble-mediated miR-424-5p delivery alleviated experimental PE in mice.

Conclusion: Ultrasound and microbubble-mediated miR-424-5p delivery targets AOC1 and activates Wnt/β-catenin signaling pathway, thus promoting the aggressive phenotype of trophoblast cells, which indicating that miR-424-5p/AOC1 axis might be involved with PE pathogenesis.

目的:探讨超声/微泡介导的miR-424-5p传递对滋养细胞的影响及其机制。方法:测定子痫前期(PE)患者的血压、24小时蛋白尿以及胎盘组织中miR-424-5p和含铜胺氧化酶1 (AOC1)的水平。对HTR-8/Svneo和TEV-1细胞进行细胞转染或超声微泡转染,检测miR-424-5p、AOC1、β-catenin和c-Myc的表达以及细胞增殖、凋亡、迁移和侵袭性。测定HTR-8/Svneo和TEV-1细胞中胎盘生长因子(PLGF)、人绒毛膜促性腺激素(β-hCG)和肿瘤坏死因子-α (TNF-α)的浓度。RNA免疫沉淀(RIP)和双荧光素酶报告基因检测检测miR-424-5p与AOC1的结合。通过皮下注射L-NAME诱导PE小鼠模型,评估超声/微泡介导miR-424-5p传递的影响。结果:PE患者胎盘组织中miR-424-5p下调,AOC1上调。过表达miR-424-5p激活Wnt/β-catenin信号通路,促进HTR-8/Svneo和TEV-1细胞的增殖,增强其迁移和侵袭行为。AOC1过表达部分消除了miR-424-5p对HTR-8/Svneo和TEV-1细胞的影响。超声和微泡介导的基因传递提高了miR-424-5p的转染效率,进一步促进了miR-424-5p在滋养细胞中的作用。超声/微泡介导的miR-424-5p递送减轻了小鼠实验性PE。结论:超声和微泡介导的miR-424-5p传递靶向AOC1,激活Wnt/β-catenin信号通路,从而促进滋养细胞的侵袭性表型,提示miR-424-5p/AOC1轴可能参与PE发病。
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引用次数: 1
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Biological Procedures Online
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