BACKGROUND Activation of hepatic stellate cells(HSCs)is a pivotal event in the onset and progression of liver fibrosis.Loss of microRNA-194(miR-194)has been reported in activated HSCs,but the actual role of miR-194 in...BACKGROUND Activation of hepatic stellate cells(HSCs)is a pivotal event in the onset and progression of liver fibrosis.Loss of microRNA-194(miR-194)has been reported in activated HSCs,but the actual role of miR-194 in liver fibrosis remains uncertain.AIM To explore the role and potential mechanism of miR-194-mediated regulation of liver fibrosis in vitro and in vivo.METHODS The expression of miR-194 was examined in human fibrotic liver tissues,activated HSCs,and a carbon tetrachloride(CCl4)mouse model by qPCR.The effects of AKT2 regulation by miR-194 on the activation and proliferation of HSCs were assessed in vitro.For in vivo experiments,we reintroduced miR-194 in mice using a miR-194 agomir to investigate the functions of miR-194 in liver fibrosis.RESULTS MiR-194 expression was notably lacking in activated HSCs from both humans and mice.Overexpression of miR-194(OV-miR-194)inhibitedα-smooth muscle actin(α-SMA)and type I collagen(Col I)expression and suppressed cell proliferation in HSCs by causing cell cycle arrest in G0/G1 phase.AKT2 was predicted to be a target of miR-194.Notably,the effects of miR-194 knockdown in HSCs were almost blocked by AKT2 deletion,indicating that miR-194 plays a role in HSCs via regulation of AKT2.Finally,miR-194 agomir treatment dramatically ameliorated liver fibrosis in CCl4-treated mice.CONCLUSION We revealed that miR-194 plays a protective role by inhibiting the activation and proliferation of HSCs via AKT2 suppression.Our results further propose miR-194 as a potential therapeutic target for liver fibrosis.展开更多
目的:构建针对人microRNA-194过表达及抑制表达的慢病毒表达载体,寻找与探讨感染人骨肉瘤细胞系SO-SP-9607和U2-os的最佳步骤和方法。方法:利用PCR方法调取相应目的基因进行酶切,经电泳回收后与目的基因进行连接,产物转化细菌感受态细胞...目的:构建针对人microRNA-194过表达及抑制表达的慢病毒表达载体,寻找与探讨感染人骨肉瘤细胞系SO-SP-9607和U2-os的最佳步骤和方法。方法:利用PCR方法调取相应目的基因进行酶切,经电泳回收后与目的基因进行连接,产物转化细菌感受态细胞,对克隆进行PCR鉴定和测序对比分析后,构建相应microRNA-194过表达及抑制表达慢病毒表达载体;在人骨肉瘤细胞系SO-SP-9607和U2-os转染及筛选过程中,根据不同阶段及浓度设定相应实验组,并设相应对照组。倒置显微镜观察转染效率,筛选情况,进行比较。结果:PCR及测序结果证实重组慢病毒表达质粒构建正确。过表达及抑制表达重组慢病毒的滴度分别为1.5×108TU/ml及4×108TU/ml。感染复数(multiply of infection,MOI)值测定,实验组及对照组转染效率无明显差异,获得MOI值及感染时间数据。通过新的综合设计,经筛选后获得转染效率满意的目的克隆细胞。结论:成功构建了microRNA-194过表达及抑制表达慢病毒表达载体,并通过新的综合考虑设计,对人骨肉瘤细胞系SO-SP-9607和U2-os进行转染和筛选后,可较快和较高效率获得满意目的细胞。展开更多
目的研究microRNA-194(miR-194)缓解高脂环境下肝细胞脂质代谢及炎症反应的分子机制。方法以低脂饲料(low-fat diet,LFD)及高脂饲料(high-fat diet,HFD)喂养构建对照组及脂肪肝小鼠模型,HE染色、油红染色观察建模情况。qPCR检测二组小...目的研究microRNA-194(miR-194)缓解高脂环境下肝细胞脂质代谢及炎症反应的分子机制。方法以低脂饲料(low-fat diet,LFD)及高脂饲料(high-fat diet,HFD)喂养构建对照组及脂肪肝小鼠模型,HE染色、油红染色观察建模情况。qPCR检测二组小鼠肝组织中miR-194、促脂质合成因子SCD1、ACC、促炎因子TNF-α、IL-6表达水平。棕榈酸(palmitic acid,PA)刺激肝细胞株LO2,过表达miR-194后qPCR检测人肝细胞系LO2细胞内SCD1、ACC、TNF-α、IL-6表达水平,油红染色检测LO2细胞内脂质沉积情况,免疫荧光检测LO2细胞内TNF-α变化。结果脂肪肝小鼠模型构建成功。与LFD组小鼠相比,HFD组小鼠miR-194表达明显降低[LFD:1.000±0.147 vs HFD:0.634±0.116,(t=3.478,P=0.025)]。SCD1、ACC1表达明显升高[SCD1 LFD:1.000±0.287 vs HFD:1.658±0.216,(t=4.802,P=0.009);ACC LFD:1.000±0.252 vs HFD:1.851±0.245,(t=4.194,P=0.015)],TNF-α、IL-6表达明显升高[TNF-αLFD:1.000±0.172 vs HFD:1.952±0.147,(t=7.288,P=0.002);IL-6 LFD:1.000±0.207 vs HFD:1.452±0.108,(t=3.242,P=0.029)]。在高脂环境下,过表达miR-194后,qPCR结果显示LO2细胞内SCD1、ACC表达明显下降[SCD1 miR-NC:1.000±0.149 vs miR-194:0.625±0.112,(t=3.340,P=0.029);ACC miR-NC:1.000±0.204 vs miR-194:0.572±0.124,(t=3.105,P=0.038)],TNF-α、IL-6表达明显减少[TNF-αmiR-NC:1.000±0.149 vs miR-194:0.563±0.059,(t=4.723,P=0.009);IL-6 miR-NC:1.000±0.156 vs miR-194:0.685±0.112,(t=2.853,P=0.048)]。油红染色显示LO2细胞内脂质沉积明显减少,免疫荧光结果显示细胞内TNF-α表达明显下[miR-NC:1.000±0.124 vs miR-194:0.655±0.152,(t=3.020,P=0.039)],炎症反应明显减轻。结论miR-194可以延缓肝细胞内脂质沉积,减轻高脂处理诱导的炎症反应,延缓脂肪肝的进展,这可能成为治疗非酒精性脂肪性肝病的新靶点。展开更多
基金the National Natural Science Foundation of China,No.81600480,No.81570547,and No.81770597the Development Program of China during the 13th Five-year Plan Period,No.2017ZX10203202003005
文摘BACKGROUND Activation of hepatic stellate cells(HSCs)is a pivotal event in the onset and progression of liver fibrosis.Loss of microRNA-194(miR-194)has been reported in activated HSCs,but the actual role of miR-194 in liver fibrosis remains uncertain.AIM To explore the role and potential mechanism of miR-194-mediated regulation of liver fibrosis in vitro and in vivo.METHODS The expression of miR-194 was examined in human fibrotic liver tissues,activated HSCs,and a carbon tetrachloride(CCl4)mouse model by qPCR.The effects of AKT2 regulation by miR-194 on the activation and proliferation of HSCs were assessed in vitro.For in vivo experiments,we reintroduced miR-194 in mice using a miR-194 agomir to investigate the functions of miR-194 in liver fibrosis.RESULTS MiR-194 expression was notably lacking in activated HSCs from both humans and mice.Overexpression of miR-194(OV-miR-194)inhibitedα-smooth muscle actin(α-SMA)and type I collagen(Col I)expression and suppressed cell proliferation in HSCs by causing cell cycle arrest in G0/G1 phase.AKT2 was predicted to be a target of miR-194.Notably,the effects of miR-194 knockdown in HSCs were almost blocked by AKT2 deletion,indicating that miR-194 plays a role in HSCs via regulation of AKT2.Finally,miR-194 agomir treatment dramatically ameliorated liver fibrosis in CCl4-treated mice.CONCLUSION We revealed that miR-194 plays a protective role by inhibiting the activation and proliferation of HSCs via AKT2 suppression.Our results further propose miR-194 as a potential therapeutic target for liver fibrosis.
文摘目的:构建针对人microRNA-194过表达及抑制表达的慢病毒表达载体,寻找与探讨感染人骨肉瘤细胞系SO-SP-9607和U2-os的最佳步骤和方法。方法:利用PCR方法调取相应目的基因进行酶切,经电泳回收后与目的基因进行连接,产物转化细菌感受态细胞,对克隆进行PCR鉴定和测序对比分析后,构建相应microRNA-194过表达及抑制表达慢病毒表达载体;在人骨肉瘤细胞系SO-SP-9607和U2-os转染及筛选过程中,根据不同阶段及浓度设定相应实验组,并设相应对照组。倒置显微镜观察转染效率,筛选情况,进行比较。结果:PCR及测序结果证实重组慢病毒表达质粒构建正确。过表达及抑制表达重组慢病毒的滴度分别为1.5×108TU/ml及4×108TU/ml。感染复数(multiply of infection,MOI)值测定,实验组及对照组转染效率无明显差异,获得MOI值及感染时间数据。通过新的综合设计,经筛选后获得转染效率满意的目的克隆细胞。结论:成功构建了microRNA-194过表达及抑制表达慢病毒表达载体,并通过新的综合考虑设计,对人骨肉瘤细胞系SO-SP-9607和U2-os进行转染和筛选后,可较快和较高效率获得满意目的细胞。
文摘目的研究microRNA-194(miR-194)缓解高脂环境下肝细胞脂质代谢及炎症反应的分子机制。方法以低脂饲料(low-fat diet,LFD)及高脂饲料(high-fat diet,HFD)喂养构建对照组及脂肪肝小鼠模型,HE染色、油红染色观察建模情况。qPCR检测二组小鼠肝组织中miR-194、促脂质合成因子SCD1、ACC、促炎因子TNF-α、IL-6表达水平。棕榈酸(palmitic acid,PA)刺激肝细胞株LO2,过表达miR-194后qPCR检测人肝细胞系LO2细胞内SCD1、ACC、TNF-α、IL-6表达水平,油红染色检测LO2细胞内脂质沉积情况,免疫荧光检测LO2细胞内TNF-α变化。结果脂肪肝小鼠模型构建成功。与LFD组小鼠相比,HFD组小鼠miR-194表达明显降低[LFD:1.000±0.147 vs HFD:0.634±0.116,(t=3.478,P=0.025)]。SCD1、ACC1表达明显升高[SCD1 LFD:1.000±0.287 vs HFD:1.658±0.216,(t=4.802,P=0.009);ACC LFD:1.000±0.252 vs HFD:1.851±0.245,(t=4.194,P=0.015)],TNF-α、IL-6表达明显升高[TNF-αLFD:1.000±0.172 vs HFD:1.952±0.147,(t=7.288,P=0.002);IL-6 LFD:1.000±0.207 vs HFD:1.452±0.108,(t=3.242,P=0.029)]。在高脂环境下,过表达miR-194后,qPCR结果显示LO2细胞内SCD1、ACC表达明显下降[SCD1 miR-NC:1.000±0.149 vs miR-194:0.625±0.112,(t=3.340,P=0.029);ACC miR-NC:1.000±0.204 vs miR-194:0.572±0.124,(t=3.105,P=0.038)],TNF-α、IL-6表达明显减少[TNF-αmiR-NC:1.000±0.149 vs miR-194:0.563±0.059,(t=4.723,P=0.009);IL-6 miR-NC:1.000±0.156 vs miR-194:0.685±0.112,(t=2.853,P=0.048)]。油红染色显示LO2细胞内脂质沉积明显减少,免疫荧光结果显示细胞内TNF-α表达明显下[miR-NC:1.000±0.124 vs miR-194:0.655±0.152,(t=3.020,P=0.039)],炎症反应明显减轻。结论miR-194可以延缓肝细胞内脂质沉积,减轻高脂处理诱导的炎症反应,延缓脂肪肝的进展,这可能成为治疗非酒精性脂肪性肝病的新靶点。
