miR-146a抑制酪氨酸酶基因家族在小鼠黑色素细胞中表达

miRNA是在许多生物过程中都起着至关重要作用的一类内源性非编码的小RNA,与癌症、肿瘤的发生有关。现发现很多miRNA在黑色素生成中都有重要的调控作用,但miR-146a是否对黑色素的生成具有影响未见报道。本研究发现miR-146a通过靶向抑制酪氨酸酶相关蛋白1(tyrosinase related protein 1,TYRP1)的表达而使黑色素生成降低。在小鼠黑色素细胞中分别转染miR-146a mimic和miR-146a 抑制剂,通过qRT-PCR与Western印迹分析比较各实验组中TYRP1基因与酪氨酸家族相关基因酪氨酸酶(tyrosinase, TYR)、酪氨酸酶相关蛋白2(tyrosinase related protein 2, TYRP2)的表达差异。双荧光报告实验验证TYRP1与miR-146a的靶向关系,双荧光酶活性结果显示,实验组相比对照组,荧光素酶活性明显降低,说明TYRP1是miR-146a的靶基因之一;qRT-PCR和Western印迹结果显示实验组TYR、TYRP1及TYRP2 在mRNA水平和蛋白质水平表达均显著降低;紫外分光光度法检测黑色素含量,结果显示miR-146a mimic转染组黑色素含量明显下降,而抑制组的黑色素含量呈上升趋势。综上所述,miR-146a通过靶向抑制TYRP1基因的表达,而影响TYR家族成员的表达,调控黑色素的生物合成。     

miR-146a抑制酪氨酸酶基因家族在小鼠黑色素细胞中表达北大核心CSCD

miRNA是在许多生物过程中都起着至关重要作用的一类内源性非编码的小RNA,与癌症、肿瘤的发生有关。现发现很多miRNA在黑色素生成中都有重要的调控作用,但miR-146a是否对黑色素的生成具有影响未见报道。本研究发现miR-146a通过靶向抑制酪氨酸酶相关蛋白1(tyrosinase related protein 1,TYRP1)的表达而使黑色素生成降低。在小鼠黑色素细胞中分别转染miR-146a mimic和miR-146a抑制剂,通过qRT-PCR与Western印迹分析比较各实验组中TYRP1基因与酪氨酸家族相关基因酪氨酸酶(tyrosinase,TYR)、酪氨酸酶相关蛋白2(tyrosinase related protein 2,TYRP2)的表达差异。双荧光报告实验验证TYRP1与miR-146a的靶向关系,双荧光酶活性结果显示,实验组相比对照组,荧光素酶活性明显降低,说明TYRP1是miR-146a的靶基因之一;qRT-PCR和Western印迹结果显示实验组TYR、TYRP1及TYRP2在mRNA水平和蛋白质水平表达均显著降低;紫外分光光度法检测黑色素含量,结果显示miR-146a mimic转染组黑色素含量明显下降,而抑制组的黑色素含量呈上升趋势。综上所述,miR-146a通过靶向抑制TYRP1基因的表达,而影响TYR家族成员的表达,调控黑色素的生物合成。     

miR-30b 的组织特异性研究

目的:研究miR-30b的组织特异性,检测其在心,肝,脑,肾,脾和骨骼肌中的表达情况。方法:选取C57BL/6J雄性小鼠6只,用real-time PCR方法检测小鼠心,肝,脑,肾,脾和骨骼肌中miR-30b的表达量。结果:miR-30b在小鼠肝脏中表达量最低,与肝相比,在心,脑,肾,脾和骨骼肌中的相对表达量分别为13.13±0.899,9.497±0.717,4.478±1.031,6.751±0.596,2.538±0.79。且与肝相比均具有统计学意义(P<0.05)。结论:miR-30b在各组织中的表达存在差异,在心脏中的表达量最高,提示miR-30b可能与心脏的发生发展密切相关,为深入探索miR-30b的功能奠定了基础。     

miR-378促进脂质生成相关靶基因鉴定

旨为验证miR-378在脂肪细胞中的功能,及其脂质相关靶基因的筛选和鉴定。利用miR-378类似物转染3T3-L1细胞,验证miR-378在脂肪细胞中的功能;根据靶标位点的保守性以及促脂功能确定miR-378潜在靶基因;采用microRNA pulldown技术验证miR-378与靶基因的靶标关系;运用双荧光素报告基因实验确定miR-378与靶基因的结合位点。结果发现,miR-378可以通过增加脂质合成和减少脂质分解两条途经来促进脂肪细胞中脂质生成,确定了miR-378与Runx1t1、Galnt3、和RAB10的靶标关系以及结合的靶位点。     

MicroRNA-449a/b抑制人结肠癌细胞内Fra-1的表达

MicroRNAs (miRNAs) 是一类长度约为22 nt的非编码的调控性小RNA,它们在诸多的生命活动中发挥重要作用,如参与调控细胞的增殖、分化、凋亡以及肿瘤的发生发展. MicroRNA-449a/b (miR 449a/b) 是脊椎动物中进化保守的miRNA,作为抑癌基因,参与了许多癌症的发生过程,但其在结肠癌中的作用尚不清楚. 本文利用实时荧光定量技术研究了miR-449a/b在结肠癌组织中的表达. 利用双荧光素酶报告基因检测系统及Western印迹鉴定miR-449a/b的靶基因. 应用MTS法和Transwell分别检测miR-449a/b对结肠癌细胞增殖和迁移的影响. 检测组蛋白乙酰化酶抑制剂曲古菌素A (trichostatin A, TSA) 对结肠癌细胞中miR-449a/b表达的影响. 研究结果表明：与正常结肠组织相比,miR-449a/b在结肠癌组织中低表达;miR 449a/b能够结合到FRA-1 mRNA 3′-非翻译区 (3′-untranslated region, 3′-UTR),从而抑制结肠癌细胞HCT116内源Fra 1的表达;外源转染miR-449a/b明显抑制结肠癌细胞HCT116的增殖和迁移;并且TSA处理能够诱导结肠癌细胞HCT116中miR-449a/b的表达. 以上结果提示: miR-449a/b可能通过抑制靶基因Fra-1的表达,进而抑制结肠癌细胞的增殖和迁移．     

miR-194a介导饲料维生素D3调节黄颡鱼JAK-STAT免疫通路的研究

为了研究miRNA如何介导饲料维生素D3在黄颡鱼(Pelteobagrus fulvidraco)JAK-STAT通路中调节免疫应答的机制,选择黄颡鱼幼鱼为主要研究对象,设计了3组维生素D₃浓度分别为1120、3950和16600 IU/kg的饲料,开展了为期12周养殖实验,养殖实验结束后进行鮰爱德华氏菌(Edwardsiella ictaluri)攻毒实验。对攻毒后的头肾和脾脏组织进行Illumina高通量测序,筛选差异表达的miRNAs,并对差异表达miRNAs进行靶基因的生物信息学预测和富集分析,发现miR-194a的靶基因富集在JAK-STAT信号通路中;进一步通过双荧光素酶报告基因实验验证了miR-194a对靶基因jak2a和tyk2具有靶向性调控的关系,能够发挥对jak2a和tyk2的抑制作用;通过体外巨噬细胞实验,在细胞中同样检测到miR-194a可以响应培养基中不同浓度的活性维生素D₃,并对靶基因jak2a与tyk2同样发挥抑制作用;通过进一步对靶基因在JAK-STAT通路中的下游基因表达检测,发现随着JAK-STAT信号通路...     

MicroRNA-146a抑制胶质瘤细胞增殖的研究

目的：检测胶质瘤中miR-146a的表达水平,并研究miR-146a对胶质瘤细胞增殖的影响。方法：应用实时定量PCR的方法检测胶质瘤组织和癌旁组织中miR-146a的表达水平,采用脂质体细胞转染miRNA模拟物的方式过表达miR-146a,MTT法检测转染后细胞的增殖率,利用在线软件targetScan预测miRNA可能的靶基因。结果：miR-146a在胶质瘤组织中表达明显降低（P〈0.01）,相对表达水平为癌旁组织的35%,细胞转染miR-146a模拟物后,miR-146a表达明显增加,癌细胞增殖率明显降低（P〈0.01）,仅为原细胞的47%。Notch1基因是miR-146a影响胶质瘤细胞增殖活力的可能靶基因。结论：miR-146a可能通过抑制Notch1基因的表达调控胶质瘤细胞的增殖。     

miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

目的：通过敲低微小RNA （microRNA,miRNA）-449a的方法研究miR-449a对人乳腺癌细胞MCF-7的增殖和迁移能力的影响。方法：采用miRNA芯片在乳腺癌细胞MCF-7和人正常乳腺细胞MCF-10A筛选具有表达差异的miRNA;化学合成法制备miR-449a的抑制剂（inhibitor）,转染后经real-time PCR验证表达的变化;细胞增殖CCK-8实验对转染后细胞增殖能力进行检测;划痕实验检测细胞转移能力,transwell小室实验检测细胞侵袭的改变;蛋白免疫印迹法（Western blot）实验对MCF-7细胞增殖和迁移相关的β-catenin和E-cadherin蛋白进行检测;通过生物信息学软件预测miR-449a潜在靶基因为Notch 1,荧光素酶实验检测Notch 1是miR-449a的靶基因。结果：分别收集MCF-7和MCF-10A细胞,芯片结果显示miR-449a在MCF-7细胞的表达水平显著高于MCF-10A;本研究将细胞分为未处理组（Mock组）,阴性对照组（negative control组,NC组）和处理组,通过收集不同组MCF-7细胞进行试验,CCK-8结果显示miR-449a下调后MCF-7细胞增殖能力显著降低;划痕实验结果显示miR-449a表达降低导致MCF-7细胞转移能力降低;transwell实验结果显示MCF-7细胞侵袭受到抑制;Western blot结果发现miR-449a敲低后β-catenin表达降低,E-cadherin表达增加;荧光素酶试验结果显示,miR-449a能够显著降低Notch 1-3'-UTR质粒的荧光素活性（P<0.01）。结论：在乳腺癌细胞MCF-7中敲低miR-449a能够显著抑制癌细胞增殖和迁移,而这一变化可能通过降低Notch 1蛋白表达实现的。     

MiR-30e调控心肌肥厚的作用机制研究

目的:预测并鉴定miR-30e的靶基因,阐明miR-30e调控心肌肥厚的分子机制.方法:分离新生大鼠心肌细胞,用苯肾上腺素(PE)处理心肌细胞构建心肌细胞肥大模型,48小时后通过定量PCR方法检测miR-30e的表达水平变化.利用生物信息学方法预测miR-30e的靶基因,并通过荧光素酶报告基因实验和蛋白免疫印迹方法验证miR-30e的靶基因.结果:与对照组相比,PE处理48hr后,心肌肥厚标志基因nppa表达明显升高,肥大心肌细胞中miR-30e明显下调.生物信息学预测细胞骨架调控蛋白Twinfilin-1(Twf1)3'UTR有两个miR-30e的结合位点.过表达miR-30e能抑制含有Twf1 3'UTR的荧光素酶报告基因的表达,降低Twf1的蛋白表达水平.结论:Twf1为miR-30e的靶基因,miR-30e通过抑制Twf1的表达调控心肌肥厚.     

豌豆蚜翅分化相关miRNA及其预测靶基因对蜕皮激素的应答及miR-92a-1-p5靶基因的验证

【目的】蜕皮激素对孤雌蚜翅两型性分化具有重要调控作用。在前期研究中我们发现5个微小RNA(microRNA, miRNA)在豌豆蚜Acyrthosiphon pisum翅两型性分化中也发挥关键作用,但蜕皮激素是否与miRNA互作参与蚜虫翅型分化未知。本研究旨在探索蜕皮激素对5个miRNA及其预测靶基因表达的影响,揭示蜕皮激素与miRNA协同调控蚜虫翅型分化的机理。【方法】选择5个与豌豆蚜翅两型性分化相关的miRNA (Let-7, miR-92a, miR-92b, miR-92a-1-p5和miR-277),使用0.1 mol/L蜕皮激素类似物20-羟基蜕皮酮(20E)分别处理孤雌生殖豌豆蚜2龄若蚜10 min和30 min,48 h后取样;qPCR检测这些miRNA及其预测靶基因在20E处理后的表达量;利用双荧光素酶活性检测法对miR-92a-1-p5的预测靶基因flightin进行验证;利用纳米载体/变性剂在孤雌生殖豌豆蚜4龄若蚜中敲低miR-92a-1-p5表达,验证miR-92a-1-p5与其预测靶基因flightin的互作关系。【结果】0.1 mol/L 20E处理30 min可极显著诱导孤雌生殖豌豆蚜2龄若蚜5个miRNA的表达;处理10 min则miR-92a-1-p5和miR-92b表达量较对照极显著下降,而Let-7和miR-277较对照极显著上升。Let-7与预测靶基因abrupt在0.1 mol/L 20E处理后孤雌生殖豌豆蚜豌2龄若蚜中表达趋势相反,miR-92a和miR-277的预测靶基因wingless和Uba1在0.1 mol/L 20E处理10 min时表达量极显著下降,与两个对应的miRNA表达趋势相反;miR-92a-1-p5的预测靶基因flightin在0.1 mol/L 20E处理30 min后表达量极显著下降,与miRNA表达趋势相反。双荧光素酶活性检测结果显示,共转染miR-92a-1-p5模拟物和flightin CDS过表达载体pmirGlO ［flightin］后与转染对照NC mimics相比,荧光素酶活性下降40%,达极显著水平;敲低孤雌生殖豌豆蚜4龄若蚜中miR 92a 1 p5后其表达量下调83%,其预测靶基因flightin表达量上升48%,均达极显著水平,表明flightin是miR-92a-1-p5的真实靶基因。【结论】蜕皮激素能够诱导豌豆蚜体内miRNA的表达;miR-92a-1-p5可能通过调控flightin参与孤雌蚜翅型分化;纳米载体/变性剂可以在蚜虫个体中实现miRNA的有效干涉。该研究为进一步探索蜕皮激素和miRNA互作参与孤雌蚜翅两型性分化机制奠定了基础。     

miR-30d负性调节HeLa细胞的增殖

有证据显示,microRNA( miRNA)可作为癌基因或抑癌基因发挥功能,参与细胞增殖和凋等 调控;PI3K/Akt通路的持续活化与肿瘤的发生、发展密切相关．在本研究中,我们通过microarray技术在HeLa细胞中检测经ly294002作用24 h阻断PI3K/Akt通路后microRNA的表达变化．Taqman 实时 PCR实验结果显示,has-miR-30d的表达在PI3K/Akt通路被阻断后明显上调．萤光素酶报告基因转染结合Taqman 实时 PCR显示,稳定转染miR- 30d过表达质粒的HeLa细胞系中miR-30d的表达量升高并且有活性．MTT及流式细胞术分析显示,过表达miR-30d的HeLa细胞其增殖受到抑制．这些结果说明,has-miR-30d可抑制HeLa细胞增殖,负性调节细胞生长,是一个潜在的抑癌基因．     

miR-30 family members negatively regulate osteoblast differentiation

miRNAs are endogenously expressed 18- to 25-nucleotide RNAs that regulate gene expression through translational repression by binding to a target mRNA. Recently, it has been indicated that miRNAs are closely related to osteogenesis. Our previous data suggested that miR-30 family members might be important regulators during the biomineralization process. However, whether and how they modulate osteogenic differentiation have not been explored. In this study, we demonstrated that miR-30 family members negatively regulate BMP-2-induced osteoblast differentiation by targeting Smad1 and Runx2. Evidentially, overexpression of miR-30 family members led to a decrease of alkaline phosphatase activity, whereas knockdown of them increased the activity. Then bioinformatic analysis identified potential target sites of the miR-30 family located in the 3' untranslated regions of Smad1 and Runx2. Western blot analysis and quantitative RT-PCR assays demonstrated that miR-30 family members inhibit Smad1 gene expression on the basis of repressing its translation. Furthermore, dual-luciferase reporter assays confirmed that Smad1 is a direct target of miR-30 family members. Rescue experiments that overexpress Smad1 and Runx2 significantly eliminated the inhibitory effect of miR-30 on osteogenic differentiation and provided strong evidence that miR-30 mediates the inhibition of osteogenesis by targeting Smad1 and Runx2. Also, the inhibitory effects of the miR-30 family were validated in mouse bone marrow mesenchymal stem cells. Therefore, our study uncovered that miR-30 family members are key negative regulators of BMP-2-mediated osteogenic differentiation.     

miR-30a suppresses breast cancer cell proliferation and migration by targeting Eya2

Eye absent (Eya) proteins are involved in cell fate determination in a broad spectrum of cells and tissues. Aberrant expression of Eya2 has been documented in a variety of cancers and correlates with clinical outcome. However, whether microRNAs (miRNAs) can regulate Eya2 expression remains unknown. Here, we show that miR-30a represses Eya2 expression by binding to the 3′-untranslated region of Eya2. Overexpression of Eya2 in miR-30a-transfected breast cancer cells effectively rescued the inhibition of cell proliferation and migration caused by miR-30a. Knockdown of Eya2 by small-interfering RNA (siRNA) in breast cancer cells mimicked the effect induced by miR-30a and abolished the ability of miR-30a to regulate breast cancer cell proliferation and migration. The miR-30a/Eya2 axis could regulate G1/S cell cycle progression, accompanied by the modulation of expression of cell cycle-related proteins, including cyclin A, cyclin D1, cyclin E, and c-Myc. Moreover, miR-30a expression was downregulated in breast cancer patients, and negatively correlated with Eya2, which was upregulated in breast cancer patients. These data suggest that the miR-30a/Eya2 axis may play an important role in breast cancer development and progression and that miR-30a activation or Eya2 inhibition may be a useful strategy for cancer treatment.     

Overexpression of miR-493-3p suppresses ovarian cancer cell proliferation,migration and invasion through downregulating DPY30

Accumulating evidence has verified that the aberrant expression level of miR-493?3p is often associated with the occurrence of numerous cancers. Nevertheless, the expression level and effect of this microRNA in ovarian cancer (OC) remain largely unclear. Therefore, the molecular function of miR-493?3p in OC progression was systematically investigated in this study.The expression of miR-493?3p and DPY30 was assessed by qRT-PCR. The protein expression level of DPY30 in cell lines was further assessed by western blot. Cell viability was respectively examined in vitro functional experiments including CCK-8 assay, EdU assay, wound healing assay, colony formation and apoptosis assays as well as the scratch test and transwell assay. Bioinformatics analysis and luciferase reporter assays were performed to predict and clarity of the correlation between miR-493?3p and DPY30.The expression of miR-493?3p was significantly reduced in OC tissues and cells. Functional experimental results showed that miR-493?3p suppressed cellular proliferation, migration, invasion, but promoted apoptosis in OC cells. Mechanistically, we also confirmed that DPY30 could be directly targeted by miR-493?3p based on bioinformatics and dual-luciferase reporter analysis. Rescue experiments results indicated that the inhibitory effect of miR-493?3p on cellular proliferation, migration and invasion and the promotive effect of miR-493?3p on apoptosis was abolished by DPY30 overexpression.Our findings demonstrated the antitumor effect of miR-493?3p through targeting DPY30 in ovarian cancer, indicating that miR-493?3p might represent a promising target for ovarian cancer diagnosis and treatment.     

ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR-573 binding

Rho GTPase activating protein 26 (ARHGAP26) is a negative regulator of the Rho family that converts the small G proteins RhoA and Cdc42 to their inactive GDP-bound forms. It is essential for the CLIC/GEEC endocytic pathway, cell spreading, and muscle development. The present study shows that ARHGAP26 mRNA undergoes extensive A-to-I RNA editing in the 3′ UTR that is specifically catalyzed by ADAR1. Furthermore, the mRNA and protein levels of ARHGAP26 were decreased in cells in which ADAR1 was knocked down. Conversely, ADAR1 overexpression increased the abundance of ARHGAP26 mRNA and protein. In addition, we found that both miR-30b-3p and miR-573 target the ARHGAP26 gene and that RNA editing of ARHGAP26 mediated by ADAR1 abolished the repression of its expression by miR-30b-3p or miR-573. When ADAR1 was overexpressed, the reduced abundance of ARHGAP26 protein mediated by miR-30b-3p or miR-573 was rescued. Importantly, we also found that knocking down ADAR1 elevated RhoA activity, which was consistent with the reduced level of ARHGAP26. Conversely, when ADAR1 was overexpressed, the amount of RhoA-GTP decreased. The similar expression patterns of ARHGAP26 and ADAR1 in human tissue samples further confirmed our findings. Taken together, our results suggest that ADAR1 regulates the expression of ARHGAP26 through A-to-I RNA editing by disrupting the binding of miR-30b-3p and miR-573 within the 3′ UTR of ARHGAP26. This study provides a novel insight into the mechanism by which ADAR1 and its RNA editing function regulate microRNA-mediated modulation of target genes.     

食道癌组织miR-21、miR-182表达与临床病理特征及预后的关系

目的:探讨食道癌组织微小RNA-21(miR-21)、微小RNA-182(miR-182)表达与临床病理特征及预后的关系。方法:选取2011年4月到2013年7月期间在我院接受手术治疗的食道癌患者84例,取患者的癌组织和癌旁正常组织作为检验标本,比较癌组织和癌旁正常组织中miR-21、mi R-182的表达水平,并分析食道癌组织中mi R-182、mi R-21的表达与临床病理特征及预后的关系。结果:癌组织中mi R-21、mi R-182的相对表达量明显高于癌旁正常组织,差异有统计学意义(P<0.05)。食道癌组织中mi R-21的表达与淋巴结转移、临床分期有关(P<0.05),与性别、年龄、分化程度、肿瘤大小无关(P>0.05);食道癌患者癌组织中miR-182的表达与年龄、性别、肿瘤大小无关(P>0.05),与分化程度、临床分期、淋巴结转移有关(P<0.05)。食道癌癌组织中miR-21、miR-182高表达患者的中位生存时间均低于低表达患者,差异有统计学意义(P<0.05)。结论:食道癌组织mi R-21、mi R-182表达与患者的部分临床病理特征及预后有关,两者有望成为食道癌新的治疗靶点。