miR-143通过抑制PTN促进人骨髓间充质干细胞成脂分化

目的：研究miR-143调控人骨髓问充质干细胞（hMSCs）成脂分化的新机理。方法：将NC、miR-143、siPTN、miR-143i转入hMSCs中,诱导成脂分化,检测miR-143对成脂分化的影响。经miRNA靶点分析软件Findtar预测出miR-143在人多效生长因子（hPTN）的3＇-UTR端有靶点。RT-PCR、westernblot研究mjR-143与hPTN的关系。构建hPTN3＇-UTR靶位点荧光检测质粒prltk-PTN及其突变质粒prltk-m,验证miR-143是否在人PTN3＇-UTR上有靶点。结果：miR-143促进hMSCs成脂分化,抑制hPTN的mRNA和蛋白表达水平。荧光报告实验证实miR-143在人PTN的3＇-UTR上有靶点。结论：miR-143通过与hPTN3I-UTR上的靶点相结合而抑制hPTN的表达,从而促进了hMSCs成脂分化进程。     

miR-148a在5-aza诱导间充质干细胞心肌样分化中的调控作用及机制

目的：探讨miR-148a在5-aza诱导人骨髓间充质（hMSCs）成心肌样分化中的表达及miR-148a对hMSCs体外成心肌样分化的生物学作用。方法：免疫荧光检测5-aza诱导hMSCs分化后心肌细胞特异性标志物α-MHC表达水平;qRT-PCR和Western blot分别检测miR-148a和DNMT1在hMSCs成肌样分化中的表达水平。利用Lipofectamine TM 2000将miR-148a mimics和miR-148a inhibitor分别瞬时转染hMSCs,Western blot检测心肌细胞特异性标志物α-MHC的蛋白表达水平。利用生物信息学技术预测miR-148a的靶基因结合位点利用双荧光素酶报告基因系统鉴定其对靶基因3'UTR的结合序列。通过DNMT1 shRNA和miR-148a inhibitors共转到hMSCs中,研究miR-148a在hMSCs成心肌样分化中的调控作用。结果：hMSCs经5-aza诱导分化后,心肌细胞特性标志物α-MHC蛋白水平明显上调。miR-148a在hBMSCs成肌样分化中显著性增加（P<0.01）,DNMT1表达水平显著降低。过表达miR-148a能提高hBMSC中心肌细胞特异性标志物α-MHC表达水平,而抑制miR-148a则能降低其水平（P<0.01）。DNMT1沉默可以阻断miR-148a对hMSCs的诱导成肌样分化作用。结论：miR-148a在hMCCs成肌样分化中表达上调,通过靶定和调控DNMT1基因的表达,并对hMSCs心肌向分化具有正向调控作用。     

miR-143-3p促进C2C12成肌细胞分化

MicroRNAs (miRNAs) 是一类小非编码RNA,近年研究发现其在骨骼肌发育调控中发挥重要作用.为探明miR-143-3p在C2C12成肌细胞分化中的调控作用,采用 real-time PCR 检测了miR-143-3p在小鼠各组织及C2C12成肌细胞分化过程中的表达;使用miR-143-3p 的模拟物和特异性抑制剂分别处理细胞,采用 real-time PCR 和 Western印迹分别检测成肌因子 MyoG和成肌标志基因 MyHC mRNA和蛋白水平的变化;用免疫荧光染色的方法观察肌管的形成.结果显示,miR-143-3p在小鼠各组织中均有表达,并且随着细胞分化表达量逐渐增加;C2C12成肌细胞过表达 miR-143-3p,与对照组相比,成肌调控因子MyoG和成肌标志基因MyHC 的mRNA和蛋白表达均显著升高,肌管数量明显增多;抑制剂处理结果显示,细胞分化被显著抑制.检测miR-143-3p对MyHC各亚型表达的影响发现,miR-143-3p表达的变化并不直接影响MyHC各亚型的表达.以上结果说明, miR-143-3p在骨骼肌和成肌细胞中均有表达,能够促进C2C12成肌细胞分化,但并不直接调控MyHCs的表达.     

家蚕miR-301对化学感受蛋白基因csp9表达的调控

【目的】探索家蚕Bombyx mori miRNAs对化学感受蛋白基因表达的调控作用,以进一步研究miRNAs及其靶基因在昆虫化学识别中的作用。【方法】利用生物信息学方法预测和筛选可能作用于家蚕化学感受蛋白CSPs基因家族成员的miRNAs;实时荧光定量PCR分析预测获得的候选miR-301和其作用的靶基因在家蚕成虫不同组织中的表达变化;构建miR-301预测靶基因3′-UTR的双荧光素酶报告载体,与合成的miR-301 mimics或阴性对照转染人胚肾细胞HEK293,通过双荧光素酶报告基因检测系统中荧光素酶活性变化检测miR-301对其靶基因表达的调控作用。【结果】生物信息学分析结果发现,家蚕化学感受蛋白基因csp9是miR-301的预测靶基因,二者的结合位点位于csp9的3′-UTR区。实时荧光定量PCR检测结果表明,miR-301在交配后家蚕雌雄成虫触角和雄成虫头部都显著上调表达,靶基因csp9在对应组织中表达则显著下调。二者共转染HEK293细胞后,双荧光素酶检测结果表明,miR-301可以通过与csp9 3′-UTR的互作,显著抑制上游荧光素酶报告基因的表达。【结论】家蚕化学感受蛋白基因csp9是miR-301的靶基因,miR-301通过与靶基因3′-UTR的结合,在翻译水平上抑制csp9的表达。     

过表达miR-103促进猪前体脂肪细胞分化

为阐明miR-103在猪前体脂肪细胞分化过程中的调控作用,采用Real-time PCR检测猪前体脂肪细胞成脂分化过程中的miR-103表达谱,明确了其在分化过程中的表达趋势;使用miR-103的腺病毒超表达载体感染猪原代脂肪细胞,随后采用Real-time PCR和Western blotting分别检测成脂标记基因PPARγ、aP2的mRNA和蛋白表达量变化;油红O染色观察腺病毒miR-103侵染的前体脂肪细胞诱导分化第8天的成脂情况。结果显示,miR-103的表达量随着脂肪细胞分化而增加,在miR-103超表达的猪原代脂肪细胞的诱导分化过程中,成脂标记基因PPARγ、aP2的表达量与对照相比显著升高,分化第8天观察到明显的脂滴。说明miR-103能够促进猪前体脂肪细胞分化。     

MiR-186-5p对3T3-L1前脂肪细胞增殖分化的影响研究

目的:探究miR-186-5p对小鼠3T3-L1前脂肪细胞增殖、分化的影响及其潜在的分子机制。方法:qRT-PCR检测miR-186-5p在不同周龄小鼠白色脂肪组织及3T3-L1前脂肪细胞增殖分化过程中的表达变化;通过脂质体将miR-186-5p mimics、inhibitors转染入增殖液或分化液培养的3T3-L1细胞后,利用CCK-8、EdU和qRT-PCR检测3T3-L1前脂肪细胞增殖变化,油红O染色观察其脂滴形态;通过生物信息软件TargetScan和双荧光报告系统分别对miR-186-5p靶基因进行预测和确认。结果:(1) miR-186-5p在1～6周龄小鼠的白色脂肪组织及3T3-L1前脂肪细胞自然分化过程中表达量均逐渐上调。(2)与阴性对照相比,mimics或inhibitors转染分别显著地促进或抑制了miR-186-5p的表达。(3)过表达miR-186-5p后,3T3-L1前脂肪细胞的增殖速率减慢,脂滴增大增多;而抑制miR-186-5p后,3T3-L1前脂肪细胞增殖速率增快,脂滴数量减少,且粒径变小。其中过表达miR-186-5p显著地降低了野生型Wnt5a和Mapk1 3'-UTR活性,而突变相应的绑定位点可解除该抑制作用。结论:miR-186-5p可抑制3T3-L1前脂肪细胞增殖,且通过直接靶向Wnt5a和Mapk1以促进其分化为成熟脂肪细胞。     

miR-143-3p通过靶向TAK1抑制肺癌增殖和侵袭

目的:分析miR-143-3p在肺癌细胞中的表达情况以及对肺癌进展的作用。方法:通过starBase数据库和肺癌病例组织检测分析miR-143-3p在肺癌组织和正常对照组织中的表达差异;分析miR-143-3p在肺癌细胞HCC27、H1975、A549和肺上皮细胞BEAS-2B中的mRNA水平差异;采用CCK-8法检测miR-143-3p对肺癌细胞增殖活性的影响;采用Transwell实验检测miR-143-3p对肺癌细胞迁移和侵袭的影响;通过qRT-PCR检测miR-143-3p对整合素α6(ITGA6)、锚蛋白重复及PH结构域3(ASAP3)、黑色素瘤相关抗原A9(MAGE-A9)和转化生长因子β激活激酶1(TAK1)表达的影响;通过Western印迹检测miR-143-3p对TAK1蛋白表达的影响。结果:starBase分析和肺癌病例组织检测结果显示miR-143-3p在肺癌组织中低表达,同样地,miR-143-3p在肺癌细胞中的表达也显著低于正常肺上皮细胞;过表达miR-143-3p抑制了肺癌细胞的增殖活性、迁移和侵袭能力;过表达miR-143-3p显著抑制TAK1的表达。结论:miR-143-3p在肺癌中通过靶向TAK1抑制肺癌的增殖和侵袭,miR-143-3p在肺癌进展中详细的分子作用机制和信号通路仍须进一步探讨。     

miR-138-5p靶向缺氧诱导因子1α对胰腺癌PANC-1细胞生长和转移的调节作用

目的探索miR-138-5p对胰腺癌细胞PANC-1生长、转移的影响及其相关机制。方法应用荧光实时定量PCR (real-time quantitative PCR, RT-PCR)检测miR-138-5p及其缺氧诱导因子1α(hypoxia inducible factor 1 alpha, HIF-1α)在PANC-1细胞中的表达。应用荧光素酶报告检测验证miR-138-5p与HIF-1α之间的生物学关系。通过体外试验研究miR-138-5p、HIF-1α在PANC-1细胞中的生物学功能,Western blot检测蛋白表达情况;CCK-8检测PANC-1细胞增殖能力;Transwell试验检测PANC-1细胞侵袭能力;划痕试验检测PANC-1细胞迁移能力。结果 miR-138-5p表达明显下调HIF-1α表达水平(P<0.01),生物信息学预测和荧光素酶报告试验证明miR-138-5p通过直接结合HIF-1α 3′-未翻译区域(3′-UTR)抑制HIF-1α。在PANC-1细胞中,miR-138-5p过表达可抑制HIF-1α表达及细胞增殖、侵袭、迁移,且差异有统计学意义(P<0.01)。结论 miR-138-5p结合HIF-1α 3′-UTR的沉默HIF-1α;miR-138-5p通过打靶HIF-1α而抑制胰腺癌细胞PANC-1增殖和转移。HIF-1α可能是胰腺癌的治疗靶点。     

MiR-186-5p对3T3-L1前脂肪细胞增殖分化的影响研究 *

目的 探究miR-186-5p对小鼠3T3-L1前脂肪细胞增殖,分化的影响及其潜在的分子机制.方法: qRT-PCR检测miR-186-5p在不同周龄小鼠白色脂肪组织及3T3-L1前脂肪细胞增殖分化过程中的表达变化;通过脂质体将miR-186-5p mimics,inhibitors转染入增殖液或分化液培养的3T3-L1细胞后,利用CCK-8,EdU和qRT-PCR检测3T3-L1前脂肪细胞增殖变化,油红O染色观察其脂滴形态;通过生物信息软件TargetScan和双荧光报告系统分别对miR-186-5p靶基因进行预测和确认.结果: (1)miR-186-5p在1~6周龄小鼠的白色脂肪组织及3T3-L1前脂肪细胞自然分化过程中表达量均逐渐上调.(2)与阴性对照相比,mimics或inhibitors转染分别显著地促进或抑制了miR-186-5p的表达.(3)过表达miR-186-5p后,3T3-L1前脂肪细胞的增殖速率减慢,脂滴增大增多;而抑制miR-186-5p后,3T3-L1前脂肪细胞增殖速率增快,脂滴数量减少,且粒径变小.其中过表达miR-186-5p显著地降低了野生型Wnt5a和Mapk1 3'-UTR活性,而突变相应的绑定位点可解除该抑制作用.结论: miR-186-5p可抑制3T3-L1前脂肪细胞增殖,且通过直接靶向Wnt5a和Mapk1以促进其分化为成熟脂肪细胞.     

miR-196a-5p对3T3-L1前脂肪细胞增殖和分化的影响效应

目的:探究miR-196a-5p对小鼠前体脂肪细胞增殖、分化的影响及其潜在的分子机制。方法:(1)构建小鼠肥胖模型,RT-PCR检测脂肪组织中miR-196a-5p表达量;(2)鸡尾酒法诱导3T3-L1前脂肪细胞分化,RT-PCR检测分化过程中miR-196a-5p的表达变化;(3)合成miR-196a-5p mimics和inhibitors转染3T3-L1细胞,以CCK8、EdU试剂盒检测miR-196a-5p对3T3-L1前脂肪细胞增殖的影响作用;(4)运用油红O染色、甘油三酯测定评估miR-196a-5p对3T3-L1细胞分化的影响;(5) RT-PCR检测miR-196a-5p对前脂肪细胞增殖、分化相关基因的影响;(6)结合前人文献,运用生物信息软件、萤光素酶报告系统对miR-196a-5p调控脂肪细胞分化的靶基因进行筛选和验证。结果:(1) miR-196a-5p在肥胖小鼠脂肪组织中高表达,在3T3-L1前脂肪细胞分化过程中先升高后下降;(2)与阴性对照组相比,mimics转染抑制了3T3-L1细胞增殖,inhibitors转染促进了3T3-L1细胞增殖;(3)与阴性对照组相比,mimics组积累了大量油红着色的脂滴,甘油三酯含量增多,而inhibitors组的脂滴少而小,甘油三酯含量相对降低;(4)与阴性对照组相比,mimics转染抑制了增殖标志基因Cyclin D1、Cyclin E、CDK2和CDK4表达,促进了分化标志基因PPARγ、C/EBPα、LPL、aP2等的表达,inhibitors转染则表现出与mimics转染相反的作用;(5) miR-196a-5p可显著抑制野生型MAP4K3和MAPK1 3'UTR萤光素酶活性,而突变绑定位点可废除该抑制效应。结论:miR-196a-5p不仅可抑制3T3-L1前脂肪细胞增殖,还可促进其诱导分化、沉积脂滴;miR-196a-5p可能通过靶向调节MAP4K3和MAPK1来介导3T3-L1前脂肪细胞分化。     

MicroRNA-153 Physiologically Inhibits Expression of Amyloid-β Precursor Protein in Cultured Human Fetal Brain Cells and Is Dysregulated in a Subset of Alzheimer Disease Patients

Regulation of amyloid-β (Aβ) precursor protein (APP) expression is complex. MicroRNAs (miRNAs) are expected to participate in the molecular network that controls this process. The composition of this network is, however, still undefined. Elucidating the complement of miRNAs that regulate APP expression should reveal novel drug targets capable of modulating Aβ production in AD. Here, we investigated the contribution of miR-153 to this regulatory network. A miR-153 target site within the APP 3'-untranslated region (3'-UTR) was predicted by several bioinformatic algorithms. We found that miR-153 significantly reduced reporter expression when co-transfected with an APP 3'-UTR reporter construct. Mutation of the predicted miR-153 target site eliminated this reporter response. miR-153 delivery in both HeLa cells and primary human fetal brain cultures significantly reduced APP expression. Delivery of a miR-153 antisense inhibitor to human fetal brain cultures significantly elevated APP expression. miR-153 delivery also reduced expression of the APP paralog APLP2. High functional redundancy between APP and APLP2 suggests that miR-153 may target biological pathways in which they both function. Interestingly, in a subset of human AD brain specimens with moderate AD pathology, miR-153 levels were reduced. This same subset also exhibited elevated APP levels relative to control specimens. Therefore, endogenous miR-153 inhibits expression of APP in human neurons by specifically interacting with the APP 3'-UTR. This regulatory interaction may have relevance to AD etiology, where low miR-153 levels may drive increased APP expression in a subset of AD patients.     

miR-21 downregulates the tumor suppressor P12 CDK2AP1 and stimulates cell proliferation and invasion

The present study was undertaken to investigate the regulation of P12(CDK2AP1) by miRNAs. A conserved target site for miR-21 within the CDK2AP1-3'-UTR at nt 349-370 was predicted by bioinformatics software and an inverse correlation of miR-21 and CDK2AP1 protein was observed. Highly specific amplification and quantification of miR-21 was achieved using real-time RT-PCR. Transfection of HaCaT cells with pre-miR-21 significantly suppressed a luciferase reporter including the CDK2AP1-3'-UTR, whereas transfection of Tca8113 with anti-miR-21 increased activity of this reporter. This was abolished when a construct mutated at the miR-21/nt 349-370 target site was used instead. Anti-miR-21-transfected Tca8113 cells showed an increase of CDK2AP1 protein and reduced proliferation and invasion. Resected primary tumors and tumor-free surgical margins of 18 patients with head and neck squamous cell carcinomas demonstrated an inverse correlation between miR-21 and P12(CDK2AP1). This study shows that P12(CDK2AP1) is downregulated by miR-21 and that miR-21 promotes proliferation and invasion in cultured cells.     

miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells

In spite of the extensive potential of human mesenchymal stem cells (hMSCs) in cell therapy, little is known about the molecular mechanisms that regulate their therapeutic properties. We aimed to identify microRNAs (miRNAs) involved in controlling the transition between the resting and reparative phenotypes of hMSCs, hypothesizing that these miRNAs must be present in the undifferentiated cells and downregulated to allow initiation of distinct activation/differentiation programs. Differential miRNA expression analyses revealed that miR-335 is significantly downregulated upon hMSC differentiation. In addition, hMSCs derived from a variety of tissues express miR-335 at a higher level than human skin fibroblasts, and overexpression of miR-335 in hMSCs inhibited their proliferation and migration, as well as their osteogenic and adipogenic potential. Expression of miR-335 in hMSCs was upregulated by the canonical Wnt signaling pathway, a positive regulator of MSC self-renewal, and downregulated by interferon-γ (IFN-γ), a pro-inflammatory cytokine that has an important role in activating the immunomodulatory properties of hMSCs. Differential gene expression analyses, in combination with computational searches, defined a cluster of 62 putative target genes for miR-335 in hMSCs. Western blot and 3'UTR reporter assays confirmed RUNX2 as a direct target of miR-335 in hMSCs. These results strongly suggest that miR-335 downregulation is critical for the acquisition of reparative MSC phenotypes.     

Expression of CD44 3'-untranslated region regulates endogenous microRNA functions in tumorigenesis and angiogenesis

The non-coding 3'-untranslated region (UTR) plays an important role in the regulation of microRNA (miRNA) functions, since it can bind and inactivate multiple miRNAs. Here, we show the 3'-UTR of CD44 is able to antagonize cytoplasmic miRNAs, and result in the increased translation of CD44 and downstream target mRNA, CDC42. A series of cell function assays in the human breast cancer cell line, MT-1, have shown that the CD44 3'-UTR inhibits proliferation, colony formation and tumor growth. Furthermore, it modulated endothelial cell activities, favored angiogenesis, induced tumor cell apoptosis and increased sensitivity to Docetaxel. These results are due to the interaction of the CD44 3'-UTR with multiple miRNAs. Computational algorithms have predicted three miRNAs, miR-216a, miR-330 and miR-608, can bind to both the CD44 and CDC42 3'-UTRs. This was confirmed with luciferase assays, western blotting and immunohistochemical staining and correlated with a series of siRNA assays. Thus, the non-coding CD44 3'-UTR serves as a competitor for miRNA binding and subsequently inactivates miRNA functions, by freeing the target mRNAs from being repressed.     

MiR-218对滋养层细胞迁移侵袭的影响及其机制研究

目的：研究miR-218是否通过下调SOX4影响滋养层细胞系HTR-8细胞的迁移和侵袭。方法：妊娠期高血压疾病（HDCP）患者46例,平均年龄（31 ±4.6）岁,收缩期血压≥ 140 mmHg和/或舒张期血压> 90 mmHg;以血压正常孕妇50例为对照,实时荧光定量PCR（RT-PCR）检测两组患者静脉血中miR-218的表达情况。转染miR-218mimic和miR-NC至离体培养的HTR-8细胞中,将细胞分为对照组（加入DMEM）、空质粒组（加入miR-NC）和过表达miR-218组（加入miR-218 mimic）3组,检测细胞的迁移侵袭情况以及细胞中MMP-2和MMP-9的表达,,生物信息学预测miR-218潜在靶基因为SOX4,利用荧光素酶素试验验证SOX4是miR-218的靶基因;再通过转染过表达SOX4的质粒至HTR-8细胞,HTR-8细胞分为过表达miR-218组、过表达miR-218+空质粒组、过表达miR-218+SOX4组,以上方法检测HTR-8细胞的迁移侵袭情况。结果：相比于正常孕妇组,HDCP组患者血清中miR-218表达减少（P <0.01）。相比于空质粒组,转染miR-218mimic后,HTR-8细胞中MMP-2、MMP-9、SOX4的表达减少（P < 0.01）,细胞迁移和侵袭能力下降（P < 0.01）;荧光素酶试验结果显示,miR-218能够显著降低SOX4-3'-UTR质粒的荧光素活性（P< 0.01）;相比于miR-218+空质粒组,转染过表达SOX4质粒后,HTR-8细胞迁移和侵袭能力增加（P < 0.01）。结论：HDCP患者血清中miR-218表达减少,miR-218可以通过下调SOX4从而抑制HTR-8细胞的迁移和侵袭。