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。     

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-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。     

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。     

Rheb过表达在前体脂肪细胞株3T3-L1分化中的作用

目的:利用前体脂肪细胞株3T3-L1细胞观察mTOR(mammalian target of rapamycin)信号通路中上游调控因子Rheb(Ras homolog enriched in brain)对其分化的影响。方法:利用高表达Rheb的基因重组质粒转染前体脂肪细胞株,3T3-L1。通过蛋白质免疫印迹实验鉴定质粒成功转染细胞后,诱导该细胞脂肪分化。予以分化第8天的3T3-L1细胞油红染色,并检测细胞内甘油三酯的含量。另外,我们用Western blot方法检测脂肪细胞特异性转录因子PPAR-γ(Peroxisome proliferator-activated receptor-γ)和C/EBP-α(CCAAT-enhancer-binding protein-α)的表达情况来研究Rheb在脂肪细胞分化过程中的作用。结果:我们成功构建了高表达Rheb的3T3-L1细胞株,发现高表达Rheb后可以促进脂滴的生成,油红O染色有显著区别,与对照组相比Rheb高表达组的三酰甘油含量明显升高(P0.05);C/EBP-α和PPAR-γ等脂肪细胞特异性的转录因子蛋白表达量与对照组相比也均有升高(P0.05)。结论:Rheb基因作为mTOR通路上游调控因子,可以促进脂肪细胞的分化。     

槟榔碱对3T3-L1脂肪细胞脂代谢的影响

目的：观察槟榔碱对3T3-L1脂肪细胞脂代谢的影响并探讨其可能机制。方法：采用经典的"鸡尾酒"法诱导3T3-L1前脂肪细胞分化成熟,随后用不同浓度的槟榔碱（0、25、50、100 μmol/L）处理成熟脂肪细胞72 h。72 h后,四甲基偶氮唑盐（MTT）法检测细胞的活性;油红O染色观察胞浆内脂滴情况;Western blot检测脂肪酸合成酶（FAS）、甘油三酯脂肪酶（ATGL）、激素敏感性脂肪酶（HSL）蛋白表达。结果：诱导分化成熟的脂肪细胞胞浆内可见大量脂滴;MTT显示：0~100 μmol/L槟榔碱对脂肪细胞活力无显著影响;油红O染色后脂质含量测定结果表明槟榔碱能减少成熟脂肪细胞中脂质含量;Western blot结果显示：与0 μmol/L组（对照组）相比,槟榔碱可显著降低脂肪细胞内FAS的蛋白表达,增加ATGL和HSL的蛋白表达;其中以50 μmol/L组最为显著。结论：槟榔碱使脂肪细胞脂解增强,可能与降低脂质合成关键酶FAS的表达,增加脂质分解代谢关键酶ATGL和HSL的表达有关。     

绿原酸在小鼠3T3-L1前脂肪细胞分化进程中的作用

目的：探讨绿原酸（CGA）对小鼠3T3-L1前脂肪细胞分化的影响。方法：培养小鼠3T3-L1前脂肪细胞,分别设置空白对照组（CG）、阳性对照组罗格列酮组（RG）、阴性对照组GW9662组 （GG）和绿原酸组（CGA）。油红O染色观察小鼠3T3-L1前脂肪细胞分化后的细胞形态变化以及脂滴形成情况;组织细胞甘油三酯（TAG）酶法测定各组分化后的细胞TAG积累量;实时荧光定量PCR技术（qPCR）检测小鼠3T3-L1前脂肪细胞在分化过程中关键基因PPARγ2 mRNA表达水平。结果：CGA组被油红O染色的区域大于CG组和GG组,但CGA组颜色没有RG组鲜艳,且脂滴形状也与RG组存在差异。CGA组TAG积累量低于CG组和RG组,与CG组比较无显著性差异（P>0.05）,但与RG组比较有显著性差异（P<0.05）。在分化过程中,CGA组和RG组PPARγ2 mRNA的表达量高于CG组和GG组,有显著性差异（P<0.01）,GG组PPARγ2 mRNA的表达量自细胞分化第4d起低于CG组,有显著性差异（P<0.01）。结论：CGA可以促进小鼠3T3-L1前脂肪细胞的分化,同时降低分化后成熟脂肪细胞中TAG的积累量,其机制与分化相关因子PPARγ2的表达有关。     

小鼠3T3-L1前脂肪细胞系的特异性标记(英文)

用增强绿色荧光蛋白特异性标记小鼠 3T3 L1前脂肪细胞系 .构建paP2 promoter EGFP载体 ,电穿孔转染小鼠 3T3 L1前脂肪细胞 ,显微荧光观察和RT PCR确认aP2基因的内源表达 .EGFP基因转入 3T3 L1前脂肪细胞 ,观察到细胞分化过程中EGFP表达和脂肪积累 .RT PCR分析表明 ,EGFP代表了稳定而真实的aP2基因的内源性表达 .建立了由脂肪组织特异表达基因aP2的表达控制的EGFP标记的小鼠 3T3 L1前脂肪细胞系 ,目前尚未见用同样方法对前脂肪细胞进行特异性标记 .该细胞系将为脂肪细胞分化机理研究以及为抗肥胖症和抗糖尿病药物筛选提供有力工具 .     

共轭亚油酸对前脂肪细胞增殖分化的影响

培养前脂肪细胞3T3-L1,MTT法检测CLA对3T3-L1增殖的影响;以油红O染色检测3T3-L1分化过程胞内脂肪的堆积;同时采用逆转录-聚合酶链式反应(RT-PCR)检测CLA对过氧化物酶体增殖物激活受体γ2(PPARγ2)mRNA表达的影响.结果显示: t10,c12-CLA以及CLA混合物对前脂肪细胞3T3-L1增殖均有显著的抑制作用(P<0.05).油红染色比色结果表明,t10,c12-CLA具有显著的抑制脂肪分化作用(P<0.05).RT-PCR结果显示,在前脂肪细胞3T3-L1分化过程中,经100 μmol/L t10,c12-CLA和50 μmol/L t10,c12-CLA处理后PPARγ2 mRNA表达量分别为对照组的52.1%,83.0%.     

CTSB超表达促进体外培养的猪前体脂肪细胞分化

为研究溶酶体组织蛋白酶B(cathepsin B,CTSB)对脂肪细胞分化的影响,本实验构建了Ctsb重组腺病毒超表达载体,包装并侵染体外培养的猪前体脂肪细胞,采用油红O染色,油红O提取比色法检测猪前体脂肪细胞分化的情况,并通过real-time PCR法检测成脂关键基因mRNA水平的变化.结果显示,重组腺病毒Ctsb载体构建成功,转染猪前体脂肪细胞后,使Ctsb的mRNA和蛋白质表达量分别提高了约16倍和12倍. CTSB超表达能促进脂肪细胞的分化和脂质积累,成脂关键基因过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma, PPARγ)、脂肪酸结合蛋白2(adipocyte protein 2, aP2)的表达量均有显著升高. 研究表明,提高Ctsb的表达能促进猪前体脂肪细胞分化,揭示了Ctsb在猪前体脂肪细胞分化过程中可能发挥关键作用. 研究结果为进一步研究其作用机制奠定了基础.     

猪圆环病毒2型通过外泌体miR-125a-5p靶向Bcl-2诱导淋巴细胞凋亡

为研究miR-125a-5p在猪圆环病毒2型(porcine circovirus type 2,PCV2)诱导淋巴细胞凋亡中的作用及其作用机制,以PCV2感染PK-15细胞外泌体孵育的淋巴细胞为研究对象,采用流式细胞术、蛋白质免疫印迹试验(Western blotting)和实时荧光定量PCR,检测淋巴细胞凋亡率及凋亡相关miRNA表达;合成miR-125a-5p模拟物和抑制物转染PK-15细胞,检测miR-125a-5p过表达或抑制表达后细胞凋亡率;采用生物信息学方法预测miR-125a-5p的靶基因,双荧光素酶报告基因检测miR-125a-5p对靶基因的调控;Western blotting检测外泌体孵育淋巴细胞的线粒体凋亡信号通路相关蛋白Bcl-2、Bax、细胞色素C和caspase-3的表达。结果显示,感染PCV2的PK-15细胞分泌的外泌体极显著提高淋巴细胞凋亡率,在一定浓度范围内呈剂量依赖性;与PCV2诱导细胞凋亡相关的miRNA中,miR-125a-5p表达量极显著升高,miR-125a-5p模拟物转染细胞后极显著提高细胞凋亡率;利用TargetScan预测发现,miR-125a-5p与Bcl-2 3''UTR区有结合位点,miR-125a-5p模拟物极显著抑制pmir-Bcl-2 3''UTR-WT荧光素酶活性,对pmir-Bcl-2 3''UTR-MuT的荧光素酶活性无明显改变;外泌体孵育的淋巴细胞Bcl-2表达量显著降低,Bax、细胞色素C的释放和caspase-3表达量显著升高,Bcl-2/Bax的比值极显著降低。这表明,PCV2通过外泌体诱导淋巴细胞上调miR-125a-5p的表达,进而抑制Bcl-2 mRNA和蛋白表达,激活淋巴细胞线粒体凋亡通路诱导细胞凋亡。     

MicroRNA-129-5p inhibits 3T3-L1 preadipocyte proliferation by targeting G3BP1

MicroRNAs have been regarded to play a crucial role in the proliferation of different cell types including preadipocytes. In our study, we observed that miR-129-5p was down-regulated during 3T3-L1 preadipocyte proliferation, while the expression of G3BP1 showed a contrary tendency. 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay and flow cytometry showed that overexpression of miR-129-5p could bring about a reduction in S-phase cells and G2-phase arrest. Additional study indicated that miR-129-5p impaired cell cycle-related genes in 3T3-L1 preadipocytes. Importantly, it showed that miR-129-5p directly targeted the 3′UTR of G3BP1 and the expression of G3BP1 was inhibited by miR-129-5p mimic. Moreover, miR-129-5p mimic activated the p38 signaling pathway through up-regulating p38 and the phosphorylation level of p38. In a word, results in our study revealed that miR-129-5p suppressed preadipocyte proliferation via targeting G3BP1 and activating the p38 signaling pathway.     

Targeting of EIF4EBP1 by miR-99a-3p affects the functions of B lymphocytes via autophagy and aggravates SLE disease progression

Excessive activation of immune cells plays a key role in the pathogenesis of systemic lupus erythematosus (SLE). The regulation of immune cells by miRNAs is a research hotspot. In this study, second-generation high-throughput sequencing revealed a reduction in miR-99a-3p expression in patients with SLE; however, the specific mechanism underlying this phenomenon remains unclear. After transfection with an miR-99a-3p agomir, the proliferation of Ball-1 cells decreased and the levels of their apoptosis increased. The opposite effects were observed in cells transfected with the miR-99a-3p antagomir. Luciferase reporter assay indicated that miR-99a-3p directly targeted EIF4EBP1. Rescue experiments confirmed the proposed interaction between miR-99a-3p and EIF4EBP1. In vitro, in vivo and clinical investigations further confirmed that the miR-99a-3p agomir reduced the expression of EIF4EBP1, LC3B and LAMP-2A. In the in vivo experiments, serum levels of anti-nuclear antibodies, double-stranded DNA, IgE, IgM, IL-6, IL-10 and B lymphocyte stimulator were higher in mice from the antagomir group than those in mice from the MRL/lpr group. Furthermore, the protein and mRNA levels of EIF4EBP1, LC3B and LAMP-2A, the intensity of immunohistochemical staining of EIF4EBP1, LC3B and LAMP-2A, the urinary protein levels, and the C3 immunofluorescence deposition increased in mice from the antagomir group. The upregulation of miR-99a-3p expression protected B cells from EIF4EBP1-mediated autophagy, whilst the downregulation of miR-99a-3p expression induced autophagy via the EIF4EBP1-mediated regulation of the autophagy signalling pathway in B cells isolated from individuals with SLE. Based on these results, miR-99a-3p and EIF4EBP1 may be considered potential targets for SLE treatment.     

Interfering histone deacetylase 4 inhibits the proliferation of vascular smooth muscle cells via regulating MEG3/miR-125a-5p/IRF1

In this study, we investigated the role ofhistone deacetylase 4 (HDAC4) and MEG3/miR-125a-5p/interferonregulatoryfactor 1 (IRF1) on vascular smooth muscle cell (VSMCs)proliferation. Platelet derived growth factor (PDGF)-BB was used toinduce the proliferation and migration of VSMCs. The expressionsof MEG3, miR-125a-5p, HDAC4 and IRF1in VSMCs were detectedby qRT-PCR and western blot, respectively. ChIP assay was usedto determine the relationship between MEG3 and HDAC4. Doubleluciferase reporter assay was used to test the regulation betweenmiR-125-5p and IRF1. Results showed that PDGF-BB decreasedthe expression of MEG3 and IRF1, while increased the expressionof miR-125a-5p and HDAC4. In addition, HDAC4 knockdowninhibited the proliferation and migration of VSMCs via upregulatingMEG3 and downregulating miR-125a-5p. MiR-125a-5p inhibitorcould repress the proliferation and migration of VSMCs andalleviate intimal hyperplasia (IH) by directly upregulating IRF1expression. These results suggested that HDAC4 interferenceinhibited PDGF-BB-induced VSMCs proliferation via regulatingMEG3/miR-125a-5p/IRF1 axis, and then alleviated IH.     

miR-125a-3p suppresses the growth and progression of papillary thyroid carcinoma cell by targeting MMP11

The dysregulation of miR-125a-3p has been observed in multiple tumor types. Nevertheless, the function of miR-125a-3p in papillary thyroid carcinoma (PTC) is yet to be explored. Herein, we find that miR-125a-3p is markedly downregulated in PTC tissues, and its level is inversely related to the histological grade of PTC. Upregulation of miR-125a-3p suppresses the pulmonary metastatic ability as well as the tumor growth of PTC cell in vivo. Consistently, the colony formation ability and other metastasis-related traits of PTC cell are inhibited by miR-125a-3p transfection in vitro. In addition, we identify that matrix metalloprotease 11 (MMP11) is the direct target gene of miR-125a-3p, and that miR-125a-3p inhibits cell viability, migration, and invasiveness of PTC cell by reducing MMP11 expression in vitro. Together, these data testify that the miR-125a-3p/MMP11 axis plays vital roles in the growth and progression of human PTC cells.     

miR-324-5p promotes adipocyte differentiation and lipid droplet accumulation by targeting Krueppel-like factor 3 (KLF3)

miRNAs, a kind of noncoding small RNA, play a significant role in adipose differentiation. In this study, we explored the effect of miR-324-5p in adipose differentiation, and found that miR-324-5p could promote adipocytes differentiation and increase body weight in mice. We overexpressed miR-324-5p during adipocytes differentiation, by oil red O and bodipy staining found that lipid accumulation was increased, and the expression level of adipogenic related genes were significantly increased. And the opposite experimental results were obtained after inhibiting miR-324-5p. In vivo, we injected miR-324-5p agomiR in obese mice and found that body weight, adipocyte area, and adipogenic-related gene expression level were significantly increased but lipolytic genes were decreased. To further explore the mechanism of miR-324-5p regulation in lipid accumulation, we constructed Krueppel-like factor 3 (KLF3) 3′-untranslated region luciferase reporter vector and KLF3 pcDNA 3.1 overexpression vector, and found that miR-324-5p was able to directly target KLF3. Overall, in this study we found that miR-324-5p could promote mice preadipoytes differentiation and increase mice fat accumulation by targeting KLF3.     

miR-125a inhibits porcine preadipocytes differentiation by targeting ERRα

MicroRNAs are a family of small, non-coding RNAs that regulate gene expression in a sequence-specific manner. Estrogen-related receptor α (ERRα) is an orphan nuclear receptor which plays an important role in adipocyte differentiation. Our previous Solexa sequencing results indicated a high expression of miR-125a in adult pig backfat. In this study, we predicated and experimentally validated ERRα as a target of miR-125a. To explore the role of miR-125a in porcine preadipocytes differentiation, miRNA agomir and antagomir were used to perform miR-125a overexpression or knockdown, respectively. Our results showed that overexpression of miR-125a could dramatically reduce the mRNA expression of adipogenic markers PPARγ, LPL, and aP2, as well as its target gene ERRα. Western blotting showed the protein level of aP2 and ERRα was also significantly down-regulated. The overexpression of miR-125a also led to a notable reduction in lipid accumulation which was detected by Oil Red O staining. In contrast, we observed promoted differentiation of porcine preadipocytes upon miR-125a inhibition. In conclusion, we verified miR-125a inhibits porcine preadipocytes differentiation through targeting ERRα for the first time, which may provide new insights in pork quality improvement and obesity control.     

miR-125a-3p调控人结肠癌细胞浸润转移的作用及机制研究

目的:探讨miR-125a-3p在结肠癌细胞浸润与转移中的作用及其可能机制。方法:通过qRT-PCR方法检测miR-125a-3p在结肠癌细胞及组织样本中的表达;在结肠癌细胞过表达或沉默miR-125a-3p后,通过平板克隆实验、MTT实验、划痕实验、Transwell实验检测结肠癌细胞增殖、迁移及侵袭能力的变化;采用Western blot方法检测miR-125a-3p过表达后相关标志分子的表达水平变化情况。结果:miR-125a-3p在结肠癌细胞及组织呈现异常低表达;过表达miR-125a-3p抑制结肠癌细胞HCT116及SW480的增殖能力;过表达或沉默miR-125a-3p分别抑制或增强结肠癌细胞的迁移与侵袭能力;过表达miR-125a-3p在mRNA及蛋白水平均能够显著抑制Snail、N-cadherin及Vimentin的表达,而增加E-cadherin的表达。结论:miR-125a-3p参与调节结肠癌细胞浸润与转移,其机制可能是通过调控上皮间质转化途径介导的。