白藜芦醇通过miRNA-122调节神经酰胺水平而治疗非酒精性脂肪肝

目的:探讨白藜芦醇通过微小RNA(miRNA)-122调节神经酰胺水平从而治疗非酒精性脂肪肝的作用机制。方法:高脂高胆固醇饲料喂养C57BL/6J小鼠建立非酒精性脂肪肝模型,实验分为正常对照(control)组、模型(model)组及白藜芦醇80 mg/kg和160 mg/kg给药组,给药4周后检测血清中总胆固醇(TC)和甘油三酯(TG)含量,收集肝脏,行HE染色观察肝脏脂质浸润情况,检测肝组织神经酰胺水平,real-time PCR检测miRNA-122含量,Western blot检测肝组织中丝氨酸棕榈酰转移酶(SPT)水平。人肝癌Hep G2细胞含10%小牛血清的RPMI-1640培养基培养,实验分为正常对照组、模型(油酸诱导)组、模型+白藜芦醇给药组、miRNA-122 siRNA组及白藜芦醇和miRNA-122 siRNA联合给药组,除正常对照组外,各组细胞均同时给予油酸刺激或药物持续孵育24 h,检测细胞匀浆中TC和TG含量、miRNA-122及神经酰胺水平,Western blot法检测各组肝细胞中SPT水平。结果:白藜芦醇低、高剂量组均能显著降低非酒精性脂肪肝小鼠血清TC和TG水平,改善肝组织细胞中脂质浸润情况,减少miRNA-122表达,同时降低肝组织神经酰胺水平及SPT蛋白表达。细胞实验结果显示,与正常对照组相比,油酸诱导脂质沉积模型组及miRNA-122 siRNA组细胞内TC和TG含量均显著增加,神经酰胺及SPT水平亦显著上升;白藜芦醇给药干预能降低油酸诱导模型组细胞中TC和TG含量、神经酰胺水平及SPT蛋白表达;与miRNA-122 siRNA组相比,白藜芦醇与miRNA-122 siRNA共孵育组TC和TG含量、神经酰胺水平及SPT蛋白水平有所降低,但作用有限。结论:白藜芦醇能够显著减少整体及细胞水平非酒精性脂肪肝模型肝细胞中脂质堆积,其机制可能是白藜芦醇通过上调miRNA-122水平、下调SPT蛋白水平而减少肝细胞中神经酰胺水平,从而达到治疗非酒精性脂肪肝的作用。     

高胆红素血症新生儿血清miR-122水平与肝功能各项指标及葡萄糖-6-磷酸脱氢酶缺乏的相关性研究

目的:探讨高胆红素血症(NHB)新生儿血清miR-122表达与肝功能各项指标和葡萄糖-6-磷酸脱氢酶(G-6-PD)缺乏的相关性。方法:将2020年3月至5月于我院新生儿科足月分娩的新生儿217例,根据临床症状和血清总胆红素(TBil)检测结果分为NHB组(n=144)和非NHB组(n=73),比较两组新生儿肝功能各项指标[谷丙转氨酶(ALT)、谷草转氨酶(AST)、TBil、白蛋白(Alb)、碱性磷酸酶(ALP)、谷氨酰转肽酶(GGT)]以及红细胞G-6-PD酶活性。采用实时荧光定量PCR法检测患儿血清miR-122相对表达量,并分析其与各指标之间的关系。结果:与非NHB组相比,NHB组新生儿血清ALT、AST、GGT、Alb、TBil、CRP水平(Z=-11.858~-2.126,均P<0.05)以及血清miR-122相对表达量(t=4.721,P<0.05)显著升高。根据血清TBil水平,将NHB患儿分为轻度、中度、重度3个亚组;与轻度亚组和中度亚组相比,重度亚组NHB新生儿血清ALT、AST、GGT、TBil、CRP、PCT水平以及血清miR-122相对表达量均升高(H=6.045~63.896,均P<0.05)。NHB患儿血清miR-122相对表达量与ALT、AST、GGT、TBil、PCT水平呈正相关性(r=0.173~0.550,均P<0.05)。22例新生儿G-6-PD缺乏,其血清miR-122相对表达量显著高于G-6-PD正常新生儿(Z=36.831,P<0.05)。结论:在NHB新生儿中,血清miR-122相对表达量普遍升高,这与AST、ALT、AST、GGT、TBil、PCT水平升高、G-6-PD酶缺乏有关,因此加强血清miR-122水平检测有助于更准确地评估NHB患儿肝损伤情况。     

Nanoparticle delivery of stable miR-199a-5p agomir improves the osteogenesis of human mesenchymal stem cells via the HIF1a pathway

Elucidating the regulatory mechanisms of osteogenesis of human mesenchymal stem cell (hMSC) is important for the development of cell therapies for bone loss and regeneration. Here we showed that hsa-miR-199a-5p modulated osteogenic differentiation of hMSCs at both early and late stages through HIF1a pathway. hsa-miR-199a expression was up-regulated during osteogenesis for both of two mature forms, miR-199a-5p and -3p. Over-expression of miR-199a-5p but not -3p enhanced differentiation of hMSCs in vitro, whereas inhibition of miR-199a-5p reduced the expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and mineralization. Furthermore, over-expression of miR-199a enhanced ectopic bone formation in vivo. Chitosan nanoparticles were used for delivery of stable modified hsa-miR-199a-5p (agomir) both in vitro and in vivo, as a proof-of-concept for stable agomir delivery on bone regeneration. The hsa-mir199a-5p agomir were mixed with Chitosan nanoparticles to form nanoparticle/hsa-mir199a-5p agomir plasmid (nanoparticle/agomir) complexes, and nanoparticle/agomir complexes could improve the in vivo regeneration of bone. Further mechanism studies revealed that hypoxia enhanced osteogenesis at early stage and inhibited osteogenesis maturation at late stage through HIF1a-Twist1 pathway. At early stage of differentiation, hypoxia induced HIF1a-Twist1 pathway to enhance osteogenesis by up-regulating miR-199a-5p, while at late stage of differentiation, miR-199a-5p enhanced osteogenesis maturation by inhibiting HIF1α-Twist1 pathway.     

Dysregulated Serum MicroRNA Expression Profile and Potential Biomarkers in Hepatitis C Virus-infected Patients

Objectives: Circulating microRNAs (miRNAs) play critical roles in pathogen-host interactions. Aberrant miRNA expression profiles might have specific characteristics for virus strains, and could serve as noninvasive biomarkers for screening and diagnosing infectious diseases. In this study, we aimed to find new potential miRNA biomarkers of hepatitis C virus (HCV) infection.Methods: Expression levels of broad-spectrum miRNAs in serum samples from 10 patients with HCV viremia and 10 healthy volunteers were analyzed using miRNA PCR arrays. Subsequently, the differential expression of four selected miRNAs (miR-122, miR-134, miR-424-3p, and miR-629-5p) was verified by qRT-PCR in the serum of 39 patients compared with that in 29 healthy controls. Receiver operating characteristic (ROC) curve analysis was performed to evaluate their potential for the diagnosis of HCV infection.Results: miRNA PCR array assays revealed differential expression of 106 miRNAs in sera of HCV patients compared with that in healthy controls. Serum hsa-miR-122, miR-134, miR-424-3p, and miR-629-5p were well identified. The ROC curves showed that miR-122, miR-134, miR-424-3p, and miR-629-5p could distinguish HCV patients with preferable sensitivity and specificity. In addition, Correlation analysis indicated serum miR-122 expression was positive correlation with ALT/AST levels. Functional analysis of target proteins of these miRNAs indicated the involvement of viral replication, inflammation, and cell proliferation.Conclusion: HCV patients have a broad ''fingerprint'' profile with dysregulated serum miRNAs compared with that in healthy controls. Among these, serum hsa-miR-122, miR-134, miR-424-3p, and miR-629-5p are identified as promising indication factors of the serum miRNA profile of HCV infection. Particularly, miR-122 could be one of serum biomarkers for early pathological process of HCV. However, more miRNA biomarkers and biological functions of these miRNAs require further investigation.     

miR-122在D-氨基半乳糖联合脂多糖诱导急性肝衰竭小鼠体内的表达及其意义

目的 通过监测急性肝功能衰竭小鼠体内miR-122的表达,探讨miR-122与小鼠急性肝衰竭疾病程度和进展之间的关系,为肝功能衰竭的早期诊断提供新的生物学标志物. 方法将BALB/C小鼠随机分为4组,实验组用D-氨基半乳糖(D-GalN,900 mg/kg)联合脂多糖(LPS,10 μg/kg)腹腔注射建立肝衰竭模型,对照组3组,分别予以D-GalN(900 mg/kg),LPS(10 μg/kg)和等渗盐水腹腔注射,在不同时间点观察小鼠病死率、肝脏组织学变化,给药后0、1、3、5、7、9 h分别留取血清、肝脏组织标本,实时定量逆转录多聚酶链反应检测小鼠体内miRNA-122和炎症因子的表达,LNA(锁核酸)-Northern blot验证miRNA-122的表达,生化分析仪检测血清中ALT、AST水平,酶联免疫吸附法检测血清中炎症因子水平.组间均数比较用two-WayANOVA方差分析,相关性分析采用Pearson和Spearman相关分析.结果 D-GalN/LPS给药24 h,小鼠病死率率达80%以上,而3个对照组则无一只小鼠死亡;肝脏特异性miR-122在正常小鼠肝脏内含量丰富(ct≈14),D-GalN/LPS诱导后1 h,miR-122即发生了明显的变化(P=0.013),表现为上调,之后随疾病的进展,miR-122表达进行性下降,9 h下调最为明显(ct≈15,P=0.002);ALT/AST于给药1 h无明显变化,3 h后呈明显上升趋势,7 h达高峰,之后ALT/AST急剧下降;对miR-122和ALT的表达对比,发现在该模型中miR-122比ALT变化快,且持久;肝衰竭相关炎症因子肿瘤坏死因子(TNF)α和白细胞介素(IL)-6在肝组织和血清中的变化一致,均上调(P<0.05); miR-122和ALT、TNFα和IL-6的相关性分析显示miR-122与以上三项指标均呈良好的相关性(相关系数分别为-0.505、0.493和0.674、).结论 肝衰竭小鼠体内肝脏特异性miR-122和ALT呈负相关关系,但又较ALT更敏感,更持久地反映肝细胞损伤程度,且miR-122表达变化与肝脏炎症损伤相关因素TNF α、IL-6均具良好的相关性,推测miR-122有望成为判断急性肝衰竭肝细胞损伤程度的一个新的分子生物学标志物.     

Targeting microRNA-122 to Treat Hepatitis C Virus Infection

An important host factor for hepatitis C virus (HCV) is microRNA-122 (miR-122). miR-122 is a liver-specific member of a family of small, non-coding RNA molecules known as microRNAs that play major roles in the regulation of gene expression by direct interaction with RNA targets. miR-122 binds directly to two sites in the 5' untranslated region (UTR) of HCV RNA and positively regulates the viral life cycle. The mechanism by which this regulation occurs is still not fully understood. There has been a great deal of interest in potential therapeutics based on small RNAs, and targeting miR-122 to combat HCV is one of the furthest advanced. Chemical inhibitors of miR-122 can be introduced into mammals intravenously and result in potent and specific knockdown of the microRNA, with no detectable adverse effects on liver physiology. This strategy was recently applied to chimpanzees chronically infected with HCV and resulted in a sustained reduction in viral load in the animals. Inhibition of miR-122 therefore presents a very attractive novel approach to treating HCV, a virus for which improved therapeutics are urgently needed.     

MiR-122稳定转染细胞系的建立及其脂代谢特征

目的:利用稳定表达人miR-122前体的表达载体,转染低表达miR-122的人肝癌细胞系HepG2细胞,获得过量表达miR-122的稳定转染细胞系,探讨miR-122对肝脏细胞脂代谢的影响及其机制。方法:对已构建完成的表达质粒pEZX-hsa-mir-122和对照质粒pEZX-mir-control进行酶切鉴定,对酶切正确的克隆进行DNA测序,测序正确的克隆应用脂质体试剂转染,绿色荧光蛋白表达监测转染效率,应用嘌呤霉素筛选稳定转染细胞系,实时定量PCR在RNA水平鉴定miR-122的表达,蛋白质印迹法从靶分子水平验证miR-122的抑制功能,尼罗红染色观察细胞内脂质。结果:pEZX-hsa-mir-122质粒成功转染HepG2细胞,质粒携带的miR-122前体序列整合到细胞基因组中,获得过量表达miR-122的HepG2细胞株,细胞内成熟体miR-122可抑制靶基因的翻译,细胞内脂质明显增加。结论:人miR-122前体序列可整合在HepG2细胞基因组中,过量表达miR-122的HepG2细胞出现脂质代谢异常。     

Charcot-Marie-Tooth neuropathy type 1A mutation: Apparent crossovers with D17S122 are due to a duplication

A locus for the slow conducting form of Charcot-Marie-Tooth neuropathy (CMT1A) was localised to the proximal short arm of chromosome 17, in band p11.2, distal to D17S58. Linkage studies of CMT1A in 3 large Australian families with the marker loci D17S58, D17S71, and D17S57 suggested the order, pter–CMT1A–D17S71–D17S58–centromere–D17S57. However, the estimate of the recombination fraction between CMT1A and D17S122, also assigned to p11.2, was incompatible with known map distances. The impasse was resolved when the D17S122 genotypes were revised to take into account a dosage effect due to a duplication. After correction of the genotypes, the maximum lod score between CMT1A and D17S122 increased from 0.53 at a recombination fraction of 0.3 to 34.28 at zero recombination. This result emphasizes that genotypes for markers in the p12–p11.2 region should be examined very carefully as ignoring the duplication changes the linkage results dramatically. The fact that no crossovers were found between CMT1A and D17S122 suggests that the duplication may cause the disease phenotype. © Wiley-Liss, Inc.     

miR-122对脑缺血再灌注损伤小鼠脑梗死体积及胰岛素样生长因子-1受体通路的影响

目的：探讨miR-122 对脑缺血再灌注损伤小鼠脑梗死体积及胰岛素样生长因子-1 受体（IGF-1R）通路的 影响。方法：将SPF 级雄性 C57BL 小鼠,运用线栓法建立脑缺血再灌注损伤（I/R）模型,分对照组（ 假手术 组）、模型组（I/R 模型组）、模拟物组（I/R 模型+miR-122 模拟物）、抑制物组（I/R 模型+miR-122 抑制物）,比 较小鼠神经功能和认知功能,测定小鼠脑梗死体积,H-E 染色检测脑组织结构改变,RT-PCR 测定miR-122、IGF- 1R mRNA水平,免疫印迹测定IGF-1R 蛋白表达,流式细胞术检测神经元细胞凋亡情况。结果：与对照组相比, 模型组和模拟物组小鼠旋转圈数明显增多,悬挂实验评分降低,且模拟物组小鼠旋转圈数多于模型组,悬挂实验 评分低于模型组;与模型组相比,抑制物组小鼠旋转圈数明显减少,悬挂实验评分明显升高。与对照组相比,模 型组和模拟物组小鼠PT%、T% 值均降低,且模拟物组较模型组降低的更为显著;与模型组相比,抑制物组小鼠 PT%、T% 值显著升高。与对照组相比,模型组和模拟物组大鼠脑梗死体积明显增加,且模拟物脑梗死体积大于 模型组;与模型组相比,抑制剂组大鼠脑梗死体积显著减小。对照组脑组织结构完整,神经元排列紧密,无染色 不匀现象,模型组脑组织神经元排列呈疏松状态,着色不匀,有大量空泡样病理结构改变,模拟物组脑组织结构 改变较模型组严重,抑制物组脑组织损伤病理变化逐渐减弱,神经元排列较密,空泡样病理改变减少。与对照组 对比,模型组miR-122 水平呈显著升高状态,其余3 组miR-122 水平从高到低依次为模拟物组、模型组、抑制物组。 与对照组比较,模型组、模拟物组IGF-1R mRNA 水平均呈下降趋势;与模型组比较,模拟物组 IGF-1R mRNA 水平有所升高。与对照组比较,模型组IGF-1R 蛋白表达明显下降,模型组IGF-1R 蛋白高于模拟物组,抑制物组 IGF-1R 蛋白高于模型组、模拟物组。神经元细胞凋亡从高到低依次为模拟物组、模型组、抑制物组、对照组,4 组神经元细胞凋亡率比较差异有统计学意义。结论：miR-122 低表达可减少脑缺血再灌注损伤小鼠脑梗死体积, 增加IGF-1R 通路活性,对脑缺血再灌注损伤小鼠脑组织有一定的预防作用。     

miR-122在PM2.5诱导的肺损伤中的作用机制

目的探索PM2.5引起的肺损伤的分子机制,以期为肺损伤的诊疗提供新的靶点。 方法对PM2.5诱导下肺损伤的多组学数据进行差异基因筛选及分子功能分析,确定肺损伤相关的分子靶标,构建PM2.5诱导的SD大鼠肺损伤模型并进行初步验证。 结果HE染色结果显示,暴露组的大鼠肺泡间隔增宽,肺泡腔增大,部分肺泡断裂、融合;Masson染色结果发现暴露组大鼠肺部呈现出少量纤维化。本次实验结果发现,在PM2.5诱导条件下,miR-122表达显著上调,其靶基因胰岛素样生长因子1受体(insulin-like growth factor 1, IGF1R)表达显著下调。 结论PM2.5会导致大鼠的肺损伤,推测miR-122通过调控其靶基因IGF1R进而影响PI3K-AKT信号通路并引起细胞凋亡,加剧肺损伤。