miR-15a靶基因的预测及生物信息学分析

目的:对目前研究较为广泛的miR-15a的靶基因进行预测及相关生物信息学分析,以期为miR-15a靶基因的实验验证提供数据支持,并为深入研究miR-15a的调控机制及生物学功能奠定基础和提供理论指导。方法:选择TargetScan5.1与PicTar两种计算方法预测miR-15a的靶基因的交集作为分析的基因集合,分别进行GO注释描述、GO富集分析和生物通路富集分析。结果与结论:预测靶基因集合分别富集在转录调控、蛋白质修饰、细胞周期等生物学过程和蛋白激酶活性等分子功能上(P0.01);经典miR-15a预测靶基因集合显著富集于KEGG通路数据库中的Wnt信号通路、细胞周期和p53信号通路等5个信号转导通路及前列腺癌、慢性髓细胞性白血病、黑素瘤等7个疾病通路中(P0.05)。     

Hsa-miR-10a-5p靶基因预测及生物信息学分析

利用生物信息学对miR-10a-5p的靶基因进行预测及相关分析,为miR-10a-5p靶基因的实验验证及其调控机制提供理论基础。通过miRBase获取并分析人、大鼠,小鼠等物种的miR-10a-5p的碱基序列特征;使用在线数据库Targetscan7.1,miRDB,mirDIP和DIANA TOOLS等预测miR-10a-5p的靶基因,并用Venny 2.1绘制韦恩图取交集。用在线工具DAVID对交集靶基因进行GO功能注释和KEGG pathway分析。结果表明miR-10a-5p成熟序列在各物种间高度保守。获得的79个靶基因在分子功能上主要涉及染色质结合,转录活性激活等,并显著富集于肝脏发育,脂肪组织发育等生物学过程,涉及cAMP信号通路,TNF信号通路及AMPK信号通路。miR-10a-5p通过调控靶基因参与了生命活动和疾病过程中多个方面,尤其生长发育和癌症过程,为进一步研究提供了生物学基础。     

牦牛miR-383的靶基因预测及生物信息学分析

MicroRNAs (miRNAs)是一类非编码的小分子单链RNA,通过与靶基因的mRNA结合,抑制mRNA的翻译,参与多种生物学过程.本实验室前期通过高通量测序发现90日龄牦牛胚胎的背最长肌中miR-383的表达量显著高于成年牦牛.为探究miR-383在牦牛骨骼肌发育中的分子功能和机制,本研究对miR-383的靶基因进行预测,并进行生物信息学分析.通过TargetScan、miRDB和miRanda 3个软件预测了miR-383的靶基因,然后合并miRTarbase数据库中已被证实的靶基因作为基因集,分别用DAVID和KOBAS3.0在线软件对基因集进行功能注释(GO分析)和Pathway信号通路富集分析.结果 表明,牦牛miR-383序列在各物种间高度保守,靶基因功能富集于CD8阳性T细胞增殖的调控、核糖核蛋白复合物定位和负调控T细胞分化等生物学过程.信号通路分析发现靶基因的信号通路显著富集于PI3K-Ak、AMPK、FoxO和Focal adhesion等与肌肉发育相关的信号通路中.该研究结果将为miR-383功能及调控机制的深入研究提供参考依据,也为解析牦牛肌肉发育的分子机制提供新的研究方向.     

miR-335在肿瘤组织中的表达及其预测靶基因的生物信息学分析

目的:分析miR-335在多种肿瘤组织与癌旁组织中的表达,预测其靶基因并进行相关生物信息学分析,为进一步研究miR-335在肿瘤中的调控机制提供理论基础。方法:分析miR-335的保守性及在多个肿瘤组织中的表达;预测miR-335靶基因,并使用DAIVID对miR-335靶基因进行生物信息学分析。结果:miR-335序列高度保守,在肝癌、肺癌、乳腺癌、肝内胆管癌、脂肪肉瘤中表达下调(P<0.05)。预测miR-335靶基因共34个,靶基因集合功能富集于细胞迁移、凋亡、转录调控,以及蛋白质分子连接、细胞骨架组成等生物学过程和分子功能(P<0.05);主要参与了轴突向导和黏着斑信号通路、黑素瘤疾病信号通路及TGF-β信号通路(P<0.05)。结论:miR-335在多种肿瘤中表达异常,且涉及多个生物学过程和信号转导通路,与肿瘤的发生发展密切相关。     

miR-29a靶基因预测及其相关生物信息学分析

目的:通过对miR-29a进行靶基因预测及相关生物信息学分析,为miR-29a靶基因的实验验证提供数据支持,以期为深入研究miR-29a的生物学功能和调控机制提供理论指导。方法:利用PubMed检索miR-29a相关文章,通过miRBase在线工具分析miR-29a序列。应用TargetScan及miRNAda两种计算方法预测miR-29a靶基因并取其交集作为分析的基因集合,分别进行基因本体(gene ontology,GO)中的分子功能和生物学过程以及KEGG(Kyoto Encyclopedia of Genes and Genomes)生物通路富集分析。结果:(1)miR-29a序列在多物种间具有高度保守性。(2)两种方法预测miR-29a靶基因交集共191个。(3)miR-29a靶基因GO分子功能集中于转录因子活性、DNA结合和钙离子结合等(P0.05);miR-29a靶基因GO生物学过程集中于调控转录、细胞粘附、细胞增殖与凋亡等(P0.05);KEGG生物通路主要富集于PI3K-AKT信号通路、JAK-STAT信号通路、T细胞受体信号通路和胰岛素信号通路等信号转导通路,以及肺小细胞癌和子宫内膜癌等疾病通路(P0.05)。结论:miR-29a可能通过参与多个靶基因信号通路的调控,在机体的多种生理病理过程中发挥重要作用,是一个颇有研究价值的生物学靶标。     

肝癌细胞HepG2中p53调控miRNA-3661的生物信息分析与功能验证

对已在前期实验中通过Dox诱导肝癌细胞HepG2 DNA损伤发现的受p53调控的hsa-miR-3661进行生物信息学分析,并通过分子生物学实验对其功能进行了验证,为miR-3661在肝肿瘤中的调控机制的研究提供理论基础。获取miR-3661结构与序列信息;预测靶基因,使用DAVID进行miRNA靶基因功能富集分析;分析miR-3661的p53结合位点,通过基因间的相互作用构建调控网络;进行细胞增殖实验验证miR-3661抑制肿瘤功能。结果表明,miR-3661序列保守,启动子区存在p53结合位点,暗示p53与hsa-miR-3661存在直接调控;预测靶基因1 009个,369个显著富集于细胞周期调控、细胞增殖、细胞凋亡等肿瘤相关生物学过程(P0.05),主要参与了癌症信号通路、MAPK信号通路与Erb B信号通路(P0.05);通过268组基因间的相互作用数据构建了p53、hsa-miR-3661和靶基因的调控网络,从系统生物学角度分析了参与多个肿瘤生物进程的关键靶基因;在实验中证实过表达miR-3661可以显著抑制肝癌细胞HepG2的增殖过程(P-value=0.001 46)。miR-3661受p53直接调控,其靶基因显著富集于多种肿瘤相关生物进程与信号通路,过表达miR-3661可显著抑制肝癌细胞增殖。     

Hsa-miR-210-5p靶基因预测及其相关信号通路的生物信息学分析

为深入研究miR-210-5p的调控机制及生物学功能提供理论机制,应用生物信息学方法分析miR-210-5p序列,预测其靶基因,用Veney2.1.0绘制韦恩图得到靶基因集合,并对其靶基因集合进行蛋白质互作分析,GO功能注释分析和KEGG Pathway分析。结果发现,已知的成熟miR-210-5p序列在各物种间高度保守。蛋白质互作分析显示,miR-210-5p预测靶基因所编码蛋白质间相互作用关系较复杂,尤其是靶基因CDK8、MED18、MED13等编码的蛋白质,在互作中起关键作用。GO分析发现其靶基因集合可能参与细胞组分、分子功能、生物调节等生物学过程;KEGG pathway分析发现其靶基因集合主要富集在MAPK、VEGF、癌症、甲状腺激素信号通路等信号通路。miR-210-5p调控靶基因参与多种重要的生物学过程,为后续研究提供了线索。     

基于数据挖掘分析microRNAs在冠状动脉支架内再狭窄中的调控机制

目的:通过寻找支架内再狭窄相关的循环microRNAs(miRNAs)及其作用靶基因,阐释miRNAs在经皮冠状动脉介入治疗的同时带来的支架内再狭窄(ISR)中的网络调控作用及机制。方法:由美国NCBI的GEO公共数据平台下载GSE60959中的miRNAs芯片数据,通过GeneSpringGX芯片分析软件分析得到ISR患者和对照组之间差异表达的循环miRNAs;采用miRNAs靶基因预测算法TargetScan和miRanda预测差异表达miRNAs的靶基因,通过DAVID平台和KEGG数据库分析得到靶基因所参与的信号通路;用GSE46560的mRNA芯片数据分析得到ISR患者较非ISR患者循环mRNAs差异表达谱,用来验证miRNAs调控ISR所作用的靶基因;采用Cytoscape软件构建基于miRNAs-信号通路、miRNAs-靶基因的共表达网络。结果:与对照组相比,ISR组存在131个差异表达循环miRNAs,其中上调41个。前5个上调miRNAs分别是miR-1183、miR-512-5p、miR-187-5p、miR-144-3p和miR-1225-5p。5个miRNAs的预测靶基因共6587个,信号通路富集分析显示这些基因主要参与的信号通路包括MAPK signaling pathway、ErbB signaling pathway、Wnt signaling pathway、Focal adhesion、Neurotrophin signaling pathway和Regulation of actin cytoskeleton。进一步对mRNAs芯片进行差异分析显示,与对照组相比,ISR患者差异循环mRNAs共171个,其中最可能受上述5个miRNAs调控的基因共43个。构建的共表达网络显示,miR-512-5p是作用靶基因和参与信号通路最多的miRNAs。结论:ISR患者循环miRNAs和mRNAs表达谱均存在改变,多个差异表达miRNAs通过作用于多个靶mRNAs,进而影响多个信号通路的活性,最终对ISR的发生、发展形成网络调控作用。     

hsa-miR-381-3p靶基因预测及其相关信号通路的生物信息学分析

应用生物信息学方法分析miR-381-3p序列,预测其靶基因,并对靶基因进行蛋白质互作分析、功能富集分析及信号转导通路富集分析。结果发现,已知的成熟miR-381-3p序列在不同物种间高度保守。蛋白质互作分析显示,mi R-381-3p预测靶基因所编码蛋白质间存在复杂的相互作用,尤其是靶基因BTBD1、NUP160、STX16等编码的蛋白质,在互作中起关键作用。GO分析发现其靶基因集合可能参与细胞过程、生物调节、细胞大分子代谢等生物学过程;KEGG通路分析发现其靶基因集合显著富集于mTOR、Wnt、p53、MAPK等信号通路中。分析结果初步提示miR-381-3p通过调控靶基因广泛参与多种重要的生物学过程,为后续的实验性研究提供了线索。     

hsa-miR-342-3p生物信息学分析

通过生物信息学方法预测hsa-miR-342-3p靶基因及其功能机制。检索Pub Med有关hsa-miR-342-3p的研究报道并进行功能分析;检索miRBase获取hsa-miR-342-3p序列;通过Target Scan,Pictar和PITA数据库预测靶基因并取交集,对其进行组织和疾病特异性表达谱分析、功能富集分析(GO enrichment analysis)、信号转导通路富集分析(Pathway enrichment analysis)和蛋白质相互作用网络分析(PPI analysis)。结果发现:hsa-miR-342-3p序列在多物种间具有高度保守性;hsa-miR-342-3p在肾脏组织和急性淋巴细胞性白血病、乳腺癌疾病中表达水平较高(RPM≥1 000);预测得到14个hsa-miR-342-3p靶基因;靶基因分子功能分别富集于转化生长因子活性、DNA结合和蛋白激酶激活等(P0.05);hsa-miR-342-3p靶基因GO生物学过程主要集中于大分子代谢抑制,肺部组织发育、呼吸系统发育及管状组织发育建成(P0.05);细胞信号通路主要富集于TGF-Beta信号通路、细胞因子、受体作用信号通路及前列腺疾病信号通路(P0.01)。hsa-miR-342-3p在体内分布广泛,预测的靶向TGF-Beta信号通路可能在疾病发生中发挥重要调控作用,是具有潜在研究价值的生物学靶标。     

MiR-21 is involved in cervical squamous cell tumorigenesis and regulates CCL20

MicroRNA 21 (miR-21) has been implicated in various aspects of carcinogenesis. However, its function and molecular mechanism in cervical squamous carcinoma have not been studied. Using TaqMan quantitative real-time PCR and Northern blot, we confirmed that miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines. Remarkably, we showed that the level of miR-21 correlates with the tumor differentiation and nodal status by ISH. Furthermore, we demonstrated that miR-21 regulates proliferation, apoptosis, and migration of HPV16-positive cervical squamous cells. In order to identify candidate target genes for miR-21, we used gene expression profiling. By luciferase reporter assays, we confirmed that CCL20 is one of its target genes, which is related to the HPV16 E6 and E7 oncogenes. Our results suggest that miR-21 may be involved in cervical squamous cell tumorigenesis.     

鸡miR-21的组织表达谱及其功能预测分析

已知miR-21在多种生物学过程中发挥重要的调控作用,然而有关鸡miR-21功能的研究尚未见报道。为了解鸡miR 21的潜在生物学功能,采用qRT-PCR检测了固始鸡5个发育阶段、15种组织中miR-21的表达情况。同时,利用Pictar和TargetScan算法预测了miR-21的靶基因并对预测的靶基因进行了Gene Ontology分析和通路分析。结果显示,miR-21的表达具有明显的时序特征,除14胚龄鸡的小脑和腺胃外,胚胎期各组织中miR-21的表达水平均显著低于出壳后对应组织的表达水平;出壳后鸡的下丘脑、大脑、小脑、小肠、肝脏、胸肌和肾脏等组织中miR-21的表达水平随发育进程均显著上调。生物信息学分析显示,预测的miR-21的靶基因在基因表达调控、大分子代谢调控、转录调控、细胞代谢调控、细胞衰老和增殖、呼吸系统发育、骨骼发育、心脏发育和神经系统发育等广泛的生物学过程中显著富集。总之,鸡miR-21为广泛性时序表达miRNA,可能参与鸡出壳后诸多器官发育相关的生物学过程的调控。     

Disease-linked microRNA-21 exhibits drastically reduced mRNA binding and silencing activity in healthy mouse liver

MicroRNAs (miRNAs) bind to mRNAs and fine-tune protein output by affecting mRNA stability and/or translation. miR-21 is a ubiquitous, highly abundant, and stress-responsive miRNA linked to several diseases, including cancer, fibrosis, and inflammation. Although the RNA silencing activity of miR-21 in diseased cells has been well documented, the roles of miR-21 under healthy cellular conditions are not well understood. Here, we show that pharmacological inhibition or genetic deletion of miR-21 in healthy mouse liver has little impact on regulation of canonical seed-matched mRNAs and only a limited number of genes enriched in stress response pathways. These surprisingly weak and selective regulatory effects on known and predicted target mRNAs contrast with those of other abundant liver miRNAs such as miR-122 and let-7. Moreover, miR-21 shows greatly reduced binding to polysome-associated target mRNAs compared to miR-122 and let-7. Bioinformatic analysis suggests that reduced thermodynamic stability of seed pairing and target binding may contribute to this deficiency of miR-21. Significantly, these trends are reversed in human cervical carcinoma (HeLa) cells, where miRNAs including miR-21 show enhanced target binding within polysomes and where miR-21 triggers strong degradative activity toward target mRNAs. Taken together, our results suggest that, under normal cellular conditions in liver, miR-21 activity is maintained below a threshold required for binding and silencing most of its targets. Consequently, enhanced association with polysome-associated mRNA is likely to explain in part the gain of miR-21 function often found in diseased or stressed cells.     

MicroRNA 21 Is a Homeostatic Regulator of Macrophage Polarization and Prevents Prostaglandin E2-Mediated M2 Generation

Macrophages dictate both initiation and resolution of inflammation. During acute inflammation classically activated macrophages (M1) predominate, and during the resolution phase alternative macrophages (M2) are dominant. The molecular mechanisms involved in macrophage polarization are understudied. MicroRNAs are differentially expressed in M1 and M2 macrophages that influence macrophage polarization. We identified a role of miR-21 in macrophage polarization, and found that cross-talk between miR-21 and the lipid mediator prostaglandin E₂ (PGE₂) is a determining factor in macrophage polarization. miR-21 inhibition impairs expression of M2 signature genes but not M1 genes. PGE₂ and its downstream effectors PKA and Epac inhibit miR-21 expression and enhance expression of M2 genes, and this effect is more pronounced in miR-21-/- cells. Among potential targets involved in macrophage polarization, we found that STAT3 and SOCS1 were enhanced in miR-21-/- cells and further enhanced by PGE₂. We found that STAT3 was a direct target of miR-21 in macrophages. Silencing the STAT3 gene abolished PGE₂-mediated expression of M2 genes in miR-21-/- macrophages. These data shed light on the molecular brakes involved in homeostatic macrophage polarization and suggest new therapeutic strategies to prevent inflammatory responses.     

The Oncogenic MicroRNA-21 Inhibits the Tumor Suppressive Activity of FBXO11 to Promote Tumorigenesis

The microRNA miR-21 is overexpressed in most human cancers and accumulating evidence indicates that it functions as an oncogene. Since miRNAs suppress the expression of their target genes, we hypothesized that some miR-21 targets may act as tumor suppressors, and thus their expression would be anticipated to be reduced by the high miR-21 levels observed in various human cancers. By microarray analysis and quantitative PCR we identified and validated FBXO11 (a member of the F-box subfamily lacking a distinct unifying domain) as a miR-21 target gene. FBXO11 is a component of the SKP1-CUL1-F-box ubiquitin ligase complex that targets proteins for ubiquitination and proteosomal degradation. By loss of function and gain of function studies, we show that FBXO11 acts as a tumor suppressor, promotes apoptosis and mediates the degradation of the oncogenic protein BCL6. The critical role that FBXO11 plays in miR-21-mediated tumorigenesis was demonstrated by a rescue experiment, in which silencing FBXO11 in miR-21KD cancer cells restored their high tumorigenicity. Expression of miR-21 and FBXO11 are inversely correlated in tumor tissue, and their expression correlates with patient survival and tumor grade. High FBXO11 expression correlates with better patient survival and lower tumor grade consistent with its tumor suppressor activity. In contrast high miR-21 expression, which correlates with poor patient survival and higher tumor grade, is consistent with its oncogenic activity. Our results identify FBXO11 as a novel miR-21 target gene, and demonstrate that the oncogenic miRNA miR-21 decreases the expression of FBXO11, which normally acts as a tumor suppressor, and thereby promotes tumorigenesis.     

Induction of Specific MicroRNAs Inhibits Cutaneous Wound Healing

Chronic nonhealing wounds, such as venous ulcers (VUs), are a widespread and serious medical problem with high morbidity and mortality. The molecular pathology of VUs remains poorly understood, impeding the development of effective treatment strategies. Using mRNA expression profiling of VUs biopsies and computational analysis, we identified a candidate set of microRNAs with lowered target gene expression. Among these candidates, miR-16, -20a, -21, -106a -130a, and -203 were confirmed to be aberrantly overexpressed in a cohort study of 10 VU patients by quantitative PCR and in situ hybridizations. These microRNAs were predicted to target multiple genes important for wound healing, including early growth response factor 3, vinculin, and leptin receptor (LepR). Overexpression of the top up-regulated miRNAs, miR-21 and miR-130a, in primary human keratinocytes down-regulated expression of the endogenous LepR and early growth response factor 3. The luciferase reporter assay verified LepR as a direct target for miR-21 and miR-130a. Both miR-21 and miR-130a delayed epithelialization in an acute human skin wound model. Furthermore, in vivo overexpression of miR-21 inhibited epithelialization and granulation tissue formation in a rat wound model. Our results identify a novel mechanism in which overexpression of specific set of microRNAs inhibits wound healing, resulting in new potential molecular markers and targets for therapeutic intervention.     

The Curcumin Analog EF24 Targets NF-κB and miRNA-21, and Has Potent Anticancer Activity In Vitro and In Vivo

EF24 is a curcumin analog that has improved anticancer activity over curcumin, but its therapeutic potential and mechanism of action is unknown, which is important to address as curcumin targets multiple signaling pathways. EF24 inhibits the NF-κB but not the JAK-STAT signaling pathway in DU145 human prostate cancer cells and B16 murine melanoma cells. EF24 induces apoptosis in these cells apparently by inhibiting miR-21 expression, and also enhances the expression of several miR-21 target genes, PTEN and PDCD4. EF24 treatment significantly suppressed the growth of DU145 prostate cancer xenografts in immunocompromised mice and resulted in tumor regression. EF24 enhanced the expression of the miR-21 target PTEN in DU145 tumor tissue, but suppressed the expression of markers of proliferating cells (cyclin D1 and Ki67). In syngeneic mice injected with B16 cells, EF24 treatment inhibited the formation of lung metastasis, prolonged animal survival, inhibited miR-21 expression and increased the expression of miR-21 target genes. Expression profiling of miRNAs regulated by EF24 in vitro and in vivo showed that the antitumor activity of EF24 reflected the enhanced expression of potential tumor suppressor miRNAs as well as the suppressed expression of oncogenic miRNAs, including miR-21. Taken together, our data suggest that EF24 is a potent anticancer agent and selectively targets NF-κB signaling and miRNA expression, indicating that EF24 has significant potential as a therapeutic agent in various cancers.     

miR-15a-5p and miR-21-5p contribute to chemoresistance in cytogenetically normal acute myeloid leukaemia by targeting PDCD4, ARL2 and BTG2

Cytarabine and daunorubicin are old drugs commonly used in the treatment of acute myeloid leukaemia (AML). Refractory or relapsed disease because of chemotherapy resistance is a major issue. microRNAs (miRNAs) were incriminated in resistance. This study aimed to identify miRNAs involved in chemoresistance in AML patients and to define their target genes. We focused on cytogenetically normal AML patients with wild-type NPM1 without FLT3-ITD as the treatment of this subset of patients with intermediate-risk cytogenetics is not well established. We analysed baseline AML samples by small RNA sequencing and compared the profile of chemoresistant to chemosensitive AML patients. Among the miRNAs significantly overexpressed in chemoresistant patients, we revealed miR-15a-5p and miR-21-5p as miRNAs with a major role in chemoresistance in AML. We showed that miR-15a-5p and miR-21-5p overexpression decreased apoptosis induced by cytarabine and/or daunorubicin. PDCD4, ARL2 and BTG2 genes were found to be targeted by miR-15a-5p, as well as PDCD4 and BTG2 by miR-21-5p. Inhibition experiments of the three target genes reproduced the functional effect of both miRNAs on chemosensitivity. Our study demonstrates that miR-15a-5p and miR-21-5p are overexpressed in a subgroup of chemoresistant AML patients. Both miRNAs induce chemoresistance by targeting three pro-apoptotic genes PDCD4, ARL2 and BTG2.