基于mRNA和miRNA芯片探索影响结直肠癌肝转移的靶基因

摘要： 目的 筛选与结直肠癌 （CRC） 肝转移相关的靶基因。方法 从Gene Expressed Omnibus （GEO） 数据库下载mRNA和miRNA的表达谱 （GSE30687和GSE44121）。通过limma函数包分析得出差异表达mRNA和差异表达 miRNA。基于DAVID在线工具进行差异表达mRNA的GO功能富集分析和KEGG通路富集分析。利用TargetScan筛选由差异表达miRNA调节的靶基因, 并构建miRNA-mRNA调节网络。结果 与原发性CRC样品相比, 在具有肝转移的CRC样品中筛选出180个差异表达mRNAs, 其中116个表达下调, 64个表达上调, 另外筛选出15个差异表达 miRNAs, 表达上调的有6个, 表达下调的有9个。差异表达mRNAs富集于32个GO terms中, 如阴离子运输、 细胞的顶端部分、 脱氧核糖核酸酶活性等。另外, 这些差异表达mRNAs主要富集在包括类固醇生物合成和霍乱弧菌感染在内的2条通路中。miRNA-mRNA调控网络包括50个miRNA-mRNA关系对, 其中具有较高节点度的基因为纤连蛋白 1 （FN1） 和骨髓嗜病毒整合位点1 （MEIS1）。结论 FN1和MEIS1基因可能是大肠癌肝转移的潜在生物标志物。     

基于生物信息学方法分析心肌梗死大鼠miRNA芯片

目的筛选大鼠梗死心肌组织microarray芯片中差异表达的miRNA,预测其互作lncRNA和靶基因,探索心肌梗死潜在的病理机制。方法结扎左前降支冠状动脉建立大鼠心梗模型。应用TRIzol法提取梗死左心室心肌区总RNA进行芯片检测。用生物信息学方法找出可能存在的lncRNA-miRNA-mRNA调控网络。结果19个明显差异表达的miRNAs中8个miRNAs(miR-21、miR-132、miR-222、miR-223-3p、miR-146a/b、miR-181b、miR-449a-5p、miR-122)已被证明是治疗心梗的候选分子,7个miRNAs(miR-365-5p、miR-490-5p、miR-6333、miR-30c-1-3p、miR-3591、miR-3596c、miR-877)是否与心肌梗死有关未知。心肌梗死发生发展中可能存在几条新的lncRNA-miRNA-mRNA作用机制。ENSRNOT00000076620-miR-146b-5p-STAT3/Rnf7/Qrsl1可能参与梗死心肌细胞的凋亡和线粒体损伤过程。ENSRNOT00000071991-miR-122-Deptor可能抑制心肌细胞自噬的发生,加剧心肌梗死的过程。NR_132625-miR-21-3P/miR-18a-5p-Coq5/Acsl1/Tmem65可能参与心肌梗死线粒体损伤过程。结论通过心肌梗死大鼠miRNA芯片分析得到的lncRNA-miRNA-mRNA三元关系,为深入研究心肌梗死分子水平的病理机制,揭示相关药物作用机制以及寻找治疗新靶标提供方向和理论依据。     

基于网络药理学探讨加味止哮平喘汤治疗支气管哮喘的作用机制

目的 基于网络药理学探讨加味止哮平喘汤治疗支气管哮喘的作用机制。方法 获取药物活性成分及关键靶点,然后运用相应数据库预测支气管哮喘的相关靶点,两者取交集后,运用STRING数据库和Cytoscape 3.9.1软件构建蛋白相互作用网络及药物-靶点网络,进行基因本体（GO）功能富集和京都基因与基因组百科全书（KEGG）通路富集分析。结果 筛选出核心药物的活性成分404个,交集靶点202个。GO功能分析得到180条分子功能,KEGG通路富集分析得到179条通路。结论 通过网络药理学分析,预测了加味止哮平喘汤治疗支气管哮喘的作用机制,为临床研究提供了参考。     

基于网络药理探讨菟藤方治疗结直肠癌的作用机制

目的 基于网络药理预测菟藤方的潜在作用机制。方法 通过中药及基因数据库平台获得核心方有效成分及药物靶点,构建中药复方调控网络;GO及KEGG富集分析药物靶涉及的主要通路以预测菟藤方应用于晚期肠癌的潜在机制。结果 菟藤方中含有的槲皮素、β-谷甾醇、甘氨酸、山柰酚及L-丙氨酸等53个潜在有效成分,可能作用于AKT1、PTGS2、VEGFA、JUN等241个潜在靶点,通过这些靶点基因表达蛋白之间的相互作用,激活HIF1信号通路、肿瘤蛋白聚糖通路及P53信号通路等,从而调控营养水平、氧化应激反应、缺氧反应、炎性反应等细胞生物学行为以治疗肠癌。结论 菟藤方为多成分、多靶点、多机制协同作用治疗结直肠癌。     

基于网络药理学和分子对接技术探讨温阳抗寒汤治疗支气管哮喘的作用机制

目的 采用网络药理学和分子对接技术探讨温阳抗寒汤治疗支气管哮喘的潜在作用机制。方法 基于中药系统药理学数据库与分析平台TCMSP获取温阳抗寒汤有效活性成分及其靶点,通过Gene Cards、OMIM、Pharm GKB、TTD、Drug Bank数据库获取哮喘相关靶点,将药物靶点与疾病靶点映射得到温阳抗寒汤治疗支气管哮喘的预测靶点。使用Cytoscape软件绘制药物活性成分-靶点网络图,使用String数据库构建蛋白互作网络,使用R语言进行GO富集分析和KEGG通路富集分析,预测温阳抗寒汤治疗支气管哮喘的作用机制,使用Auto Dock Tools、Autodock vina、Pymol软件进行分子对接预测和部分对接结果可视化。结果 筛选得到温阳抗寒汤作用于支气管哮喘的有效活性成分115个,检索得到支气管哮喘相关的靶点4 802个,温阳抗寒汤与支气管哮喘的交集靶点86个,关键有效成分包括槲皮素、山奈酚、木犀草素、汉黄芩素、黄芩苷、柚皮素等,涉及的核心靶点包括HIF1A、TP53、AKT1、ESR1等。通过GO分析（P <0.05）得到GO条目1 866个,其中包括生物过程（BP）...     

肺动脉高压时肺血管内皮间质转化相关miRNAs网络调控的生物信息学分析

目的探索肺动脉高压(pulmonary hypertension,PH)时肺血管内皮间质转化(End MT)相关miRNAs及其下游靶点的网络调控。方法利用文献挖掘PH相关基因及EndMT/EMT相关miRNAs。利用Biological General Repository for Interaction Datasets(Bio GRID)数据库得到基因相互作用关系。利用生物信息学(DIANA3、Miranda4、Pic Tar5、Target Scan6、miRDB7和micro T-CDS8)预测相应miRNAs的靶基因关系,超几何分析预测与肺动脉高压End MT相关miRNAs,通过对miRNAs参与功能分区中的情况筛选评分最高的miRNAs并选择部分进行实验验证,利用Cytocape 3软件构建miRNAs与其下游靶点的相互作用网络。结果根据文献挖掘出与PH有关的基因230个,与End MT/EMT相关miRNAs共189个,对应成熟体322个。其中98个miRNAs可能与PH时End MT有关。其中,仅有22个miRNAs同时参与了TGF-β/BMP、低氧和炎症3个功能通路,评分最高,分别为miR-let-7家族、miR-124、miR-130家族、miR-135、miR-144、miR-149、miR-155、miR-16-1、miR-17、miR-181家族、miR-182、miR-200家族、miR-204、miR-205、miR-21、miR-224、miR-27、miR-29家族、miR-301a、miR-31、miR-361和miR-375。对let-7g、miR-21、miR-124及miR-130家族进行实时荧光定量PCR验证发现其在低氧诱导PH大鼠肺动脉中表达均明显变化。结论利用生物学信息技术从大量miRNAs中筛选得到的22个miRNAs同时参与TGF-β/BMP、低氧和炎症信号通路,可能与PH时End MT相关,为后续深入研究PH时End MT提供了重要的理论依据。     

人脐带间充质干细胞外泌体的鉴定及其作用于肺动脉高压的生物信息学分析

目的 探讨人脐带间充质干细胞外泌体(human umbilical cord mesenchymal stem cell exosomes,hUCMSC-Exo)缓解肺动脉高压(pulmonary hypertension,PH)的潜在分子作用机制。方法 对hUCMSC-Exo进行提取、鉴定及高通量测序。利用GeneCards搜索PH作用靶点,在GEO数据库下载PH相关芯片(GSE53408、GSE113439),经过批次校正后筛选出差异基因。GeneCards和GEO数据库以及hUCMSC-Exo所含miRNAs预测的靶向调控的mRNAs取交集,构建蛋白互作网络筛选出hUCMSC-Exo作用于PH的关键靶点,并进行GO和KEGG富集分析。结果 测序得到hUCMSC-Exo中43个高表达的miRNAs,其中17个miRNAs可作用于PH,发现其作用机制可能与前列腺癌、HIF-1信号通路、PI3K-Akt信号通路、癌症的蛋白多糖和癌症通路等相关。结论 本研究可为hUCMSC-Exo防治PH的临床研究提供新思路。     

不同转移潜能肝癌细胞株miRNAs表达谱的差异性研究

目的筛选不同转移潜能肝癌细胞株的microRNA(miRNA)差异表达谱,并预测分析差异表达miRNAs调控的靶基因与功能。方法提取总RNA进行miRNA芯片分析,通过分析MHCC-97H(高转移)、Hep3B(不转移)两种不同转移潜能肝癌细胞株与正常肝细胞L02比较,以及两种肝癌细胞株相互比较的miRNA差异表达谱,筛选出差异表达明显的miRNAs进行qRT-PCR验证,利用4个靶基因预测软件(Target Scan、miRanda、miRWalk、miRDB)进行靶基因预测,并通过生物信息学分析来探讨候选靶基因的生物学功能。结果与正常肝细胞L02相比,肝癌细胞株MHCC-97H、Hep3B中的miR-192-5p、miR-215-5p表达均明显上调,而miR-130a-3p、miR-196a-5p则明显降低;与不转移肝癌细胞株Hep3B相比,miR-224-5p在肝癌细胞株MHCC-97H中明显上调,而miR-146a-5p、miR-483-3p、miR-200b-3p则明显降低。qRT-PCR验证结果与芯片结果相一致。结论 MHCC-97H及Hep3B两种转移能力不同的肝癌细胞株的miRNAs存在差异表达,这些差异表达的miRNAs与肝细胞癌的发生发展、转移侵袭密切相关。     

基于miRNAs的知母水提物体外抑制胃癌细胞SGC7901增殖作用机制

目的 从微小核苷酸（miRNAs）的角度研究知母水提物（aqueous extract from Anemarrhenae Rhizoma,ARAE）体外抑制SGC7901细胞增殖的作用机制。方法 采用MTT法检测ARAE对SGC7901细胞增殖的影响;采用流式细胞术检测ARAE对SGC7901细胞凋亡的影响;采用高通量测序法检测细胞中miRNAs的表达丰度差异;采用miranda、mirbase和targetscan 3个数据库信息比对,预测差异miRNAs的靶基因,并通过KEGG分析靶基因的相关功能;采用实时荧光定量PCR法验证主要靶基因的表达变化。结果 ARAE能明显抑制SGC7901细胞的增殖并诱导其凋亡,调节细胞miR-16-5p、miR-20a-5p、miR-26b-5p和miR-15b-5p的表达水平;并提示这些miRNAs所调控的靶基因主要富集于PI3K-Akt、JAK-STAT和MAPK等信号通路。结论 ARAE可能通过调节SGC7901细胞内miRNAs的表达从而抑制SGC7901细胞增殖,并诱导其凋亡。     

急性氯化汞中毒后mmu-miR-429-5p靶向调控Aqp1致肾损伤的研究

目的本研究通过小鼠急性氯化汞(Mercury dichloride, HgCl_2)中毒模型,研究肾水通道蛋白-1(Aquaporin-1, Aqp1)及mmu-miR-429-5p的表达变化,从而探究其可能发挥作用的分子机制,为急性HgCl_2中毒后的临床治疗提供更多的靶点以及为司法鉴定提供更多思路。方法全自动生化分析仪测定肾功能指标水平,苏木素-伊红(HE)染色观察肾组织结构病理改变。免疫组织化学实验及Western blot检测Aqp1蛋白定量及定位。生物信息学网站预测能与Aqp1靶向结合的微小RNA(MicroRNAS, miRNAS)。结合双荧光素酶报告基因实验检测mmu-miR-429-5p与Aqp1之间是否存在结合靶点,实时荧光定量PCR(quantitative Real-Time PCR, qRT-PCR)检测Aqp1及mmu-miR-429-5p在肾组织中的表达水平。结果急性HgCl_2中毒后肾小管上皮细胞水肿及变性坏死,Aqp1在肾小管上皮细胞膜、肾小球毛细血管内皮细胞膜上表达降低。双荧光素酶报告基因实验结果显示mmu-miR-429-5p与Aqp1之间存在靶点结合。QRT-PCR结果显示Aqp1基因表达降低而mmu-miR-429-5p表达升高,两者之间存在负相关关系。结论急性HgCl_2中毒后mmu-miR-429-5p表达升高并可能通过靶向结合Aqp1致Aqp1蛋白表达降低,从而引起肾损伤。     

Identification of miR-3182 and miR-3143 target genes involved in the cell cycle as a novel approach in TNBC treatment: A systems biology approach

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis, lacking therapeutic targets. miRNAs play crucial roles in TNBC through regulating various mechanisms, including cellular growth and proliferation. This study aims to identify critical target genes of two novel miRNAs (miR-3143 and miR-3182) involved in the cell cycle of TNBC as possible therapeutic targets and investigates their regulatory and therapeutic roles through a systems biology approach and in vitro experiment. Datasets related to the TNBC cell line (MDA-MB-231) were screened and retrieved, and Gene regulatory networks were constructed. Significant regulatory motifs were detected and analyzed using the FANMOD and Cytoscape analyzer, and the clusters and seeds were identified using the MCODE. Functional enrichment analysis was also performed using DAVID and STRING. The most critical genes were determined using the analysis of GRN motifs and PPI clusters. The essential genes involved in the cell cycle were selected and verified using the bc-GenExMiner v4.7. We overexpressed miR-3143 and miR-3182 in the MDA-MB-231 cell line using human umbilical cord mesenchymal stem cell (HUCMSC)-miRNA loaded exosomes, and the expression of the critical target genes was investigated using RT-qPCR. We identified eight critical genes as potential therapeutic targets. Their expression decreased by overexpression of miR-3143 and miR-3182 in RT-qPCR. The identified critical genes have probably significant roles in the pathogenesis of TNBC through the cell cycle. We suggest that the overexpression of miR-3143 and miR-3182 could be a new therapeutic candidate in TNBC and is worth more investigation.     

Aberrant expression of microRNAs in gastric cancer and biological significance of miR-574-3p

The discovery of microRNAs (miRNAs) provides a new and powerful tool for studying the mechanisms, diagnosis and treatments of cancer. In this study, we employed AFFX miRNA expression chips to search for miRNAs that may be aberrantly expressed in gastric cancer tissues and to investigate the potential roles that miRNAs may play in the development and progression of gastric cancer. 14 miRNAs were found to be down-regulated and 2 miRNAs up-regulated in gastric cancer tissues compared to the normal gastric tissues. Among the aberrantly expressed miRNAs, miR-574-3p was selected to further study its expression features and functional roles. Interestingly, the reduced expression of miR-574-3p occurred mainly in the early stages of gastric cancer or in cancers with high level of differentiation, suggesting that it can be used as a marker for a mild case of gastric cancer. Functional study revealed that cell proliferation, migration and invasion were significantly inhibited in miR-574-3p-transfected gastric cancer SGC7901 cells. Computational prediction and experimental validation suggest that Cullin2 may be one of the targets of miR-574-3p. Overall our study suggests that the aberrantly expressed miRNAs may play regulatory and functional roles in the development and progression of gastric cancer.     

The upregulation of miR-98-5p affects the glycosylation of IgA1 through cytokines in IgA nephropathy

ObjectiveIncreases in galactose-deficient IgA1 (Gd-IgA1) play a crucial role in the pathogenesis of IgA nephropathy (IgAN), and several recent experiments have shown that microRNAs (miRNAs) are involved in regulating the development and physiological function of the kidney. The aims of this study were to identify miRNAs that can affect the pathogenesis of IgAN and reveal the underlying regulatory mechanism of IgA1 glycosylation in peripheral blood.MethodsThe differentially expressed miRNAs in peripheral blood mononuclear cells (PBMCs) between IgAN patients and healthy controls were screened by high-throughput sequencing, and the targets of these miRNAs were predicted and verified by dual-luciferase reporter assays. We also explored the miRNA regulation of Gd-IgA1 through the transfection of miRNA mimics and related plasmids.ResultsThe high-throughput sequencing results showed that miR-98-5p was more highly expressed in the PBMCs of IgAN patients compared with healthy controls, and the luciferase reporter gene system confirmed that miR-98-5p might target chemokine ligand 3 (CCL3). The transfection of si-CCL3 confirmed that a decrease in CCL3 can affect the expression of interleukin-6 (IL-6) and C1GALT1. The overexpression of miR-98-5p in PBMCs through the transfection of miR-98-5p mimic reduced the CCL3 and C1GALT1 levels and increased the IL-6 levels, and these changes in PBMCs were attenuated by cotransfection with the CCL3 plasmid.ConclusionThe results showed that in PBMCs, miR-98-5p can target CCL3 to decrease its expression and thereby increase the IL-6 levels, and the resulting increase in IL-6 can decrease C1GALT1 expression. Therefore, miR-98-5p might be involved in the development of IgAN.     

miR-146a-5p improves the decidual cytokine microenvironment by regulating the toll-like receptor signaling pathway in unexplained spontaneous abortion

Spontaneous abortion (SA) is a common pregnancy failure, but the cause of numerous cases remains unexplained. Decidual immune cells (DICs)-mediated cytokine microenvironment is involved in pregnancy and regulated by many microRNAs, but whether microRNA-146a-5p (miR-146a) regulate the decidual cytokine microenvironment and the potential mechanisms in unexplained SA pathogenesis have rarely been reported. In this study, the levels of cytokines and miR-146a in healthy and unexplained SA deciduae were first investigated, and the correlation between them was analyzed. Then, the effect of miR-146a inhibitor on cytokines was assessed in healthy deciduae-derived DICs. Third, the downstream targets and related molecular mechanisms of miR-146a were analyzed by bioinformatics, and the levels of the predicted targets in deciduae were assessed, followed by the correlation analysis between the levels of miR-146a and the targets. Finally, the effect of miR-146a on the predicted targets and inflammatory cytokines was validated in unexplained SA deciduae-derived DICs. As a result, decreased miR-146a correlated with the cytokine disorder in unexplained SA deciduae, and inhibition of miR-146a promoted pro-inflammatory response in healthy deciduae-derived DICs. One hundred four target genes and related molecular mechanisms of miR-146a were predicted, among which the toll-like receptor (TLR) pathway might be associated with the decidual cytokine regulation. Upregulation of miR-146a inhibited the expression of the predicted molecules enriched in the TLR pathway and improved the cytokine disorder in unexplained SA deciduae-derived DICs. Collectively, miR-146a improves the decidual cytokine microenvironment by regulating the TLR pathway in unexplained SA, providing novel potential targets for further therapeutic research.     

Altered expression of microRNAs may predict therapeutic response in rheumatoid arthritis patients

BackgroundEpigenetic alternations of microRNAs (miRNAs) can contribute to the pathogenesis and progression of rheumatoid arthritis (RA). This study aimed to measure the expression level of peripheral blood miRNAs, as well as their target mRNAs, in RA patients and healthy controls (HCs), and to evaluate the potential of miRNAs as promising non-invasive biomarkers of treatment response.MethodsThe peripheral expression of miRNAs, including miR-146a, miR-146b, miR-150, miR-155, miR-125a-5p, miR-223, miR-26a, and miR-21, as well as their target mRNAs, was analyzed in 90 RA patients and 30 HCs via quantitative real-time polymerase chain reaction (RT-PCR) assay. We compared differences between the patients in terms of good response (GR; n = 55) and poor response (PR; n = 35) to the conventional therapeutic approach.ResultsAll miRNAs were significantly overexpressed in RA patients. The expression of miR-155, miR-150, miR-146a, miR-146b, miR-125a-5p, and miR-223 increased in both groups of RA patients, compared to HCs, and miR-26a and miR-21 were the only upregulated miRNAs in the GR group versus HCs. Among the upregulated miRNAs, miR-125a-5p expression significantly changed in GR and PR patients (P = 0.047). The ROC curve analysis indicated the potential involvement of miR-125a-5p in the pathogenesis of RA. We also observed the downregulated expression of GATA3, RORC, FOXP3, TBX21, STAT1, and TRAF6 in RA patients versus HCs.ConclusionOur findings indicated that different expression levels of miR-125a-5p in the GR and PR groups of patients may serve as a therapeutic response biomarker, which can be also used as a target for therapeutic interventions.     

MiR-22-3p in exosomes increases the risk of heart failure after down-regulation of FURIN

Heart failure (HF) is often the inevitable manifestation of myocardial ischemia. Hypoxia can induce cardiomyocytes to express many microRNAs (miRNAs), which are highly expressed in exosomes. In addition, miR-22-3p is a marker in heart failure. Therefore, miR-22-3p was taken as the research object to explore its role and mechanism in HF. HF differentially expressed miRNAs were screened by bioinformatic analysis. The HF rats model was constructed and identified by detecting serum brain natriuretic peptide (BNP) and ultrasound analysis [left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS)]. The extracted exosomes were identified by transmission electron microscopy, and Western blot was used to detect the expressions of Tsg101 and CD63. Quantitative real-time polymerase chain reaction detected miR-22-3p expression in serum, exosomes, and serum without exosomes, while the cardiomyocytes cytotoxicity was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and PKH26 staining. After overexpressing/silencing miR-22-3p in cells, cell viability, apoptosis, and apoptosis-associated markers were detected. Bioinformatic analysis screened the target gene of miR-22-3p, which was verified by dual-luciferase assay. Regulation of miR-22-3p on FURIN was measured by rescue tests. In vivo experiments were verified the above results. MiR-22-3p was identified as the research object. BNP was increased in the model group, while LVEF and LVFS were decreased. MiR-22-3p was overexpressed in HF-treated serum and exosomes. Normal exosomes did not affect cardiomyocyte function, while high concentrations of HF-treated exosomes were cytotoxic. By regulating apoptosis-related genes, overexpressed miR-22-3p inhibited cell activity and promoted cell apoptosis. Silenced miR-22-3p with opposite effects counteracted effects of HF-treated exosomes. FURIN, target gene of miR-22-3p, was negatively regulated by miR-22-3p, while overexpressed FURIN promoted cell activity and inhibited apoptosis. In vivo research was consistent with the results of cell experiments. By regulating FURIN, miR-22-3p in exosomes increases the risk of HF damage.