Exosome-cargoed microRNAs: Potential therapeutic molecules for diabetic wound healing

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基于TCGA数据库乳头状甲状腺癌miRNA预后风险模型的建立与分析CSCD

目的构建基于微小RNA(miRNA)表达的预测乳头状甲状腺癌(papillary thyroid carcinoma,PTC)患者预后的生存模型。方法从TCGA数据库官方网站上下载PTC miRNA测序数据和患者的临床资料,利用R3.6.0软件中的edgeR包筛选表达失调的miRNA。利用单因素Cox及Lasso回归分析筛选出与患者预后相关的miRNA(P<0.05),进一步使用多因素Cox回归分析建立预后模型的风险评分方程risk score,构建生存预后模型,使用受试者工作特征曲线(ROC)来评价模型的敏感度和特异性。结果与正常甲状腺组织相比,PTC组织中失调表达的miRNA共有75个(|log foldchange|≥2,FDR<0.05),多因素Cox回归分析最终得到基于8个miRNA(hsa-mir-6730、hsa-mir-4709、hsa-mir-196a-2、hsa-mir-146b、hsa-mir-6860、hsa-mir-509-3、hsa-mir-513c、hsa-mir-515-1)的预测患者预后的风险模型。ROC曲线下面积(AUC)分析显示,该模型具有较好的敏感度和特异性(AUC>0.8)。结论成功构建了基于miRNA表达的风险预测模型,该模型可有效预测PTC患者的预后。     

环状RNA的分子生物学功能及其在卵巢癌中的研究

环状RNA（circular RNA，circRNA）是一种在人体内广泛存在的不具有5′端帽子和3′端尾巴的共价闭合环状非编码RNA，较线性RNA更具稳定性，组织特异性强，在正常组织及肿瘤组织中的表达有显著差异，其特殊的分子生物学功能尤其是微小RNA（microRNA，miRNA）分子海绵作用，使其有望成为新的肿瘤标志物和分子靶向治疗靶标。近年研究发现circRNA可以通过多种机制调控恶性肿瘤的增殖与转移，在恶性肿瘤的早期诊断、治疗、预后以及肿瘤对放化疗敏感性等方面有指导作用。但是，目前circRNA在卵巢癌中的功能和调控机制研究尚处于初始阶段，仅有少量的报道和研究。综述circRNA的分子生物学功能及其在卵巢癌中的功能和调控机制。     

miR-105-5p在胃癌中的表达及其意义与生物学功能

目的:探讨miR-105-5p在胃癌(GC)中的表达情况、临床意义及生物学功能及其潜在的作用机制。方法:应用real-time PCR检测miR-105-5p在GC组织与癌旁组织以及不同GC细胞(MGC-803,MKN-1,SGC-7901,BGC-823和AGS)与正常胃黏膜细胞(GES-1)中的表达;分析miR-105-5p的表达与GC临床病理特征及患者预后的关系;采用Transwell迁移和侵袭实验以及MTT实验检测miR-105-5p对GC细胞迁移与侵袭能力以及增殖能力的影响;应用生物信息学工具预测miR-105-5p的下游靶点,并应用荧光素酶报告实验以及Western blot分析miR-105-5p对靶点的调节作用。结果:miR-105-5p在GC组织的表达明显高于癌旁组织,在各GC细胞中的表达均明显高于正常胃黏膜细胞(均P0.05)。miR-105-5p的表达水平与肿瘤大小(P=0.020)和远处转移(P=0.004)明显有关;miR-105-5p低表达GC患者的总体生存率明显高于miR-105-5p高表达组的患者(P=0.001 8)。选择miR-105-5p表达量相对较低的BGC-823细胞和相对较高的MKN-1细胞,分别转染miR-105-5p模拟物和miR-105-5p抑制物,转染后结果显示,BGC-823细胞的迁移、侵袭和增殖能力明显增强,而MKN-1细胞的迁移、侵袭和增殖能力明显抑制(均P0.05)。生物信息学分析发现,DIRAS家族GTP结合RAS样3(DIRAS3)可能是miR-105-5p的作用靶点;荧光素酶报告实验显示,miR-105-5p能够负向调节DIRAS3-3'UTR的荧光素酶活性;Western blot显示,转染miR-105-5p模拟物的BGC-823细胞中DIRAS3的表达明显下调,转染miR-105-5p抑制物的MKN-1细胞DIRAS3的表达明显上调(均P0.05)。结论:miR-105-5p在GC中表达升高,其可能通过靶向作用于DIRAS3增强GC细胞的迁移、侵袭及增殖,进而促进GC的发生发展。     

Different expression levels of exosomal miR-503 in peritoneal dialysis effluent from patients of different peritoneal transport characteristics and bioinformatics analysis

Objective To compare the expression level of exosomal miR-503 in peritoneal dialysis effluent (PDE) from patients of different peritoneal transport characteristics, predict the target genes of miR-503 and provide bioinformatic data for researches of peritoneal transport characteristics. Methods Twenty-four stable peritoneal dialysis (PD) patients were selected and divided into high transport group (H group, n=12) and low transport group (L group, n=12) according to the results of peritoneal equilibration tests (PET). The 500 ml PDE that was left on the patient's abdomen overnight was collected and concentrated using ultrafiltration cell. Exosomes in PDE were resuspended in phosphate buffered saline (PBS) after ultracentrifugation and the characteristics of PDE exosomes were identified by transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), Western blotting and fluorescent staining. MicroRNAs were extracted from PDE exosomes. The expression levels of PDE exosomal miR-503 in the two groups were detected by quantitative real-time PCR. Then the relations between the relative quantity of PDE exosomal miR-503 and PET values or 24 h ultrafiltration volume (UF) were analyzed. Targetscan and miRDB databases were used to predict the target genes of miR-503. Gene ontology (GO) functional enrichment and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway analysis were relied on DAVID (https://david.ncifcrf.gov/). Results The exosomes in PDE showed a round and cup-shaped morphology under TEM, and the diameters were approximately 100 nm measured by NTA. The specific biomarkers of exosomes, CD63, CD81 and heat shock protein -70 (HSP-70) were all detected by Western blotting. The internalization and uptake of the exosomes was observed after fluorescent staining. The relative expression level of PDE exosomal miR-503 in H group was found to be significantly higher than that in L group (P=0.002), and the relative quantity of PDE exosomal miR-503 was significantly positively correlated with PET values (r=0.547, P=0.006), but not 24 h UF (r=-0.297, P=0.159). There were 156 target genes of miR-503 in total that could be predicted by two different databases at the same time. GO analysis of these 156 target genes was mainly focused on kinase binding, regulation of protein modification and catabolic process as well as regulation of epithelial cell proliferation. KEGG enriched many tumor associated or classical signaling pathways, including transforming growth factor-β (TGF-β) signaling pathway and vascular endothelial growth factor (VEGF) signaling pathway. The prediction showed that vascular endothelial growth factor A (VEGFA) was a direct target gene of miR-503 and it was also related to many proteins involved in fibrosis mechanism. Conclusions The expression level of PDE exosomal miR-503 is significantly higher in H group, and positively correlates with PET values, which may regulate the angiogenesis of peritoneal vessels by targeting VEGFA.     

Long Noncoding RNA WEE2-AS1 Plays an Oncogenic Role in Glioblastoma by Functioning as a Molecular Sponge for MicroRNA-520f-3p

The long noncoding RNA WEE2 antisense RNA 1 (WEE2-AS1) plays an oncogenic role in hepatocellular carcinoma and triple negative breast cancer progression. In this study, we investigated the expression and roles of WEE2-AS1 in glioblastoma (GBM). Furthermore, the molecular mechanisms behind the oncogenic actions of WEE2-AS1 in GBM cells were explored in detail. WEE2-AS1 expression was detected using quantitative real-time polymerase chain reaction. The roles of WEE2-AS1 in GBM cells were evaluated by the cell counting kit-8 assay, flow cytometric analysis, Transwell cell migration and invasion assays, and tumor xenograft experiments. WEE2-AS1 expression was evidently enhanced in GBM tissues and cell lines compared with their normal counterparts. An increased level of WEE2-AS1 was correlated with the average tumor diameter, Karnofsky Performance Scale score, and shorter overall survival among GBM patients. Functionally, depleted WEE2-AS1 attenuated GBM cell proliferation, migration, and invasion in vitro, promoted cell apoptosis, and impaired tumor growth in vivo. Mechanistically, WEE2-AS1 functioned as a molecular sponge for microRNA- 520f-3p (miR-520f-3p) and consequently increased specificity protein 1 (SP1) expression in GBM cells. A series of recovery experiments revealed that the inhibition of miR-520f-3p and upregulation of SP1 could partially abrogate the influences of WEE2-AS1 downregulation on GBM cells. In conclusion, WEE2-AS1 can adsorb miR-520f-3p to increase endogenous SP1 expression, thereby facilitating the malignancy of GBM. Therefore, targeting the WEE2-AS1–miR-520f-3p–SP1 pathway might be a promising therapy for the management of GBM in the future.     

外泌体miRNA在卵巢癌中的研究进展

卵巢癌(ovarian cancer,OC)致死率在女性生殖系统肿瘤中位居第一,因其早期无典型症状,大多数患者在晚期才得到诊断。此外,由于癌细胞易对化疗药物产生耐药,多数患者会出现复发,导致患者预后很差。因此寻找有效的早期诊断及治疗方法对OC的早期预测、疗效评估、预后及复发判断等具有重要意义。外泌体(exosome)是近年来研究的热点,其富含脂质、蛋白质、RNA和DNA,通过与细胞膜融合将内容物释放到细胞外环境,影响相邻或远处细胞的生物学行为。研究认为外泌体可参与肿瘤微环境中细胞间通讯,通过传递癌或抑癌信息分子,影响肿瘤的进程。微小RNA(miRNA)可被包裹在外泌体中传递至受体细胞,影响肿瘤细胞的增殖、转移、耐药等,在肿瘤进展中发挥重要作用。本文对外泌体miRNA在OC中的研究进行综述。     

微小RNA在新生儿缺血缺氧性脑病中的表达

微小RNA(miRNA)是一类高度保守的内源性小分子RNA。miRNA主要通过选择性结合mRNA调控基因表达。目前研究结果表明，中枢神经系统存在大量miRNA，并参与神经细胞的正常生长、发育，以及组织损伤修复、肿瘤发生、神经退行性变等多种病理、生理过程。笔者拟就新生儿缺血缺氧性脑病(HIE) miRNA谱系的最新研究进展进行阐述，探讨其miRNA特异性表达，对新生儿HIE诊断和预后判断的意义，旨在为该病的相关诊治研究提供参考。     

妊娠期高血压患者血清微小RNA-210、-204-5p、-376c与其血液流变学指标的相关性及其诊断价值研究

目的探讨妊娠期高血压患者血清微小RNA(miRNA)-210、-204-5p、-376c与其血液流变学指标的相关性，以及其诊断价值。 方法采用简单随机抽样方法，随机抽取2016年10月至2018年10月，于河北省黄骅市人民医院收治的妊娠期高血压患者80例为研究对象，并纳入研究组(n＝80)。采用相同抽样方法，随机抽取同期于本院接受定期产前检查的80例中孕期正常妊娠妇女为对照，纳入对照组(n＝80)。采用实时荧光定量PCR，检测2组受试者血清miRNA-210、-204-5p及-376c的相对表达量。采用全自动血细胞分析仪，检测2组受试者的血液流变学指标。2组受试者血清miRNA-210、-204-5p、-376c相对表达量及血液流变学指标比较，采用成组t检验。采用受试者工作曲线下面积(ROC-AUC)，分析血清miRNA-210、-204-5p及-376c的相对表达量，对妊娠期高血压的诊断价值。采用Pearson相关系数分析妊娠期高血压患者血清miRNA-210、-204-5p及-376c的相对表达量与其血液流变学指标的相关性。本研究获得河北省黄骅市人民医院伦理委员会批准(批准文号：18101523)，并且受试者均签署临床研究知情同意书。2组受试者的年龄、入组时孕龄、孕前人体质量指数、孕次、产次等一般临床资料分别比较，差异均无统计学意义(P>0.05)。 结果①研究组患者血清miRNA-210、-204-5p的相对表达量分别为(1.56±0.35)与(1.78±0.40)，均显著高于对照组的(0.59±0.11)与(0.72±0.15)，研究组患者血清miRNA-376c的相对表达量为(0.33±0.09)，却显著低于对照组的(1.24±0.32)，2组上述指标分别比较，差异均有统计学意义(t＝23.648、22.193、24.485，均为P<0.001)。②血清miRNA-210、-204-5p及-376c的相对表达量诊断妊娠期高血压的ROC-AUC分别为0.824(95%CI：0.738～0.902，P<0.001)，0.871(95%CI：0.810～0.943，P<0.001)，0.833(95%CI：0.746～0.908，P<0.001)。根据约登指数最大原则，血清miRNA-210、-204-5p及-376c的相对表达量，对于诊断妊娠期高血压的最佳临界值分别为0.696、1.512及0.712，此时其诊断妊娠期高血压的敏感度分别为83.3%、93.1%及85.0%，特异度分别为85.0%、75.0%及85.0%。③研究组患者的全血低切黏度(WRV)、低切还原黏度(LSRV)、血浆黏度(PV)分别为(7.9±1.5) mPa·s、(17.6±3.6) mPa·s、(1.6±0.3) mPa·s，分别显著高于对照组的(7.2±1.3) mPa·s、(15.6±3.4) mPa·s、(1.2±0.2) mPa·s，2组上述指标比较，差异均有统计学意义(t＝3.154、P＝0.002，t＝3.613、P<0.001，t＝9.923、P<0.001)。④妊娠期高血压患者血清miRNA-210相对表达量与其WRV、LSRV、PV，均呈正相关关系(r＝0.343、P＝0.002，r＝0.415、P<0.001，r＝0.287，P＝0.001)；血清miRNA-204-5p相对表达量与PV亦呈正相关关系(r＝0.326、P＝0.003)；miRNA-376c相对表达量与WRV、LSRV，均呈正相关关系(r＝0.317、P＝0.004，r＝0.351、P＝0.001)。 结论妊娠期高血压患者血清miRNA-210、-204-5p、-376c相对表达量与其血液流变学指标存在相关性，并且有望成为诊断妊娠期高血压的潜在生物标志物。