MicroRNAs in the Pathogenesis of Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD), or, more accurately, metabolic associated fatty liver disease, accounts for a large proportion of chronic liver disorders worldwide and is closely associated with other conditions such as cardiovascular disease, obesity, and type 2 diabetes mellitus. NAFLD ranges from simple steatosis to nonalcoholic steatohepatitis (NASH) and can progress to cirrhosis and, eventually, also hepatocellular carcinoma. The morbidity and mortality associated with NAFLD are increasing rapidly year on year. Consequently, there is an urgent need to understand the etiology and pathogenesis of NAFLD and identify effective therapeutic targets. MicroRNAs (miRNAs), important epigenetic factors, have recently been proposed to participate in NAFLD pathogenesis. Here, we review the roles of miRNAs in lipid metabolism, inflammation, apoptosis, fibrosis, hepatic stellate cell activation, insulin resistance, and oxidative stress, key factors that contribute to the occurrence and progression of NAFLD. Additionally, we summarize the role of miRNA-enriched extracellular vesicles in NAFLD. These miRNAs may comprise suitable therapeutic targets for the treatment of this condition.     

Circulating Extracellular Vesicles with Specific Proteome and Liver MicroRNAs Are Potential Biomarkers for Liver Injury in Experimental Fatty Liver Disease

Background & Aim

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy.

Design

Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens.

Results

We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r² = 0.64, p<0.05), fibrosis (r² = 0.66, p<0.05) and pathological angiogenesis (r² = 0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192 - two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver.

Conclusions

These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.     

Association between Serum Growth Hormone Levels and Nonalcoholic Fatty Liver Disease: A Cross-Sectional Study

Growth hormone (GH) is an important regulator of metabolism and body composition. GH deficiency is associated with increased visceral body fat and other features of the metabolic syndrome. Here we performed a cross-sectional study to explore the association of GH levels with nonalcoholic fatty liver disease (NAFLD), which is considered to be the hepatic manifestation of the metabolic syndrome. A total of 1,667 subjects were diagnosed as NAFLD according the diagnostic criteria, and 5,479 subjects were defined as the controls. The subjects with NAFLD had significantly lower levels of serum GH than the controls. Those with low GH levels had a higher prevalence of NAFLD and the metabolic syndrome. A stepwise logistic regression analysis showed that GH levels were significantly associated with the risk factor for NAFLD (OR = 0.651, 95%CI = 0.574–0.738, P<0.001). Our results showed a significant association between lower serum GH levels and NAFLD.     

High Serum Uric Acid Increases the Risk for Nonalcoholic Fatty Liver Disease: A Prospective Observational Study

Nonalcoholic fatty liver disease (NAFLD) is a common form of chronic liver disease, and serum uric acid is observed to be significantly elevated in NAFLD patients. However, whether this elevation is causal, a bystander, or a consequence of NAFLD remains unclear. We performed a population-based prospective study among the employees of Zhenhai Refining & Chemical Company Ltd., Ningbo, China to investigate whether the elevation of serum uric acid has a casual role for NAFLD. A total of 6890 initially NAFLD-free subjects were followed up for 3 years. Overall, 11.80% (813/6890) subjects developed NAFLD over 3 years of follow-up. The cumulative incidence of NAFLD increased with progressively higher baseline serum uric acid levels (the cumulative incidence was 7.2%, 9.5%, 11.5%, 13.8%, and 17.2% in quintile 1, quintile 2, 3, 4 and 5, respectively; P value for trend <0.001). Cox proportional hazards regression analyses showed that serum uric acid levels were independently and positively associated with the risk for incident NAFLD; the age-, gender- and metabolic syndrome adjusted hazard ratio (95% CI) for the subjects in quintile 2, 3, 4 and 5 versus quintile 1 was 1.18 (0.91–1.54), 1.32 (1.03–1.70), 1.39 (1.09–1.78) and 1.50 (1.18–1.92), respectively. Taken together, our prospective observational study showed that elevation of serum uric acid levels independently predicts increase risk for incident NAFLD.     

Serum MicroRNAs as Potential Biomarkers for Early Diagnosis of Hepatitis C Virus-Related Hepatocellular Carcinoma in Egyptian Patients

Circulating microRNAs are deregulated in liver fibrosis and hepatocellular carcinoma (HCC) and are candidate biomarkers. This study investigated the potential of serum microRNAs; miR-19a, miR-296, miR-130a, miR-195, miR-192, miR-34a, and miR-146a as early diagnostic biomarkers for hepatitis C virus (HCV)-related HCC. As how these microRNAs change during liver fibrosis progression is not clear, we explored their serum levels during fibrosis progression in HCV-associated chronic liver disease (CLD) and if they could serve as non-invasive biomarkers for fibrosis progression to HCC. 112 Egyptian HCV-HCC patients, 125 non-malignant HCV-CLD patients, and 42 healthy controls were included. CLD patients were subdivided according to Metavir fibrosis-scoring. Serum microRNAs were measured by qRT-PCR custom array. Serum microRNAs were deregulated in HCC versus controls, and except miR-130a, they were differentially expressed between HCC and CLD or late fibrosis (F3-F4) subgroup. Serum microRNAs were not significantly different between individual fibrosis-stages or between F1-F2 (early/moderate fibrosis) and F3-F4. Only miR-19a was significantly downregulated from liver fibrosis (F1-F3) to cirrhosis (F4) to HCC. Individual microRNAs discriminated HCC from controls, and except miR-130a, they distinguished HCC from CLD or F3-F4 patients by receiver-operating-characteristic analysis. Multivariate logistic analysis revealed a panel of four microRNAs (miR-19a, miR-195, miR-192, and miR-146a) with high diagnostic accuracy for HCC (AUC = 0.946). The microRNA panel also discriminated HCC from controls (AUC = 0.949), CLD (AUC = 0.945), and F3-F4 (AUC = 0.955). Studied microRNAs were positively correlated in HCC group. miR-19a and miR-34a were correlated with portal vein thrombosis and HCC staging scores, respectively. In conclusion, studied microRNAs, but not miR-130a, could serve as potential early biomarkers for HCC in high-risk groups, with miR-19a as a biomarker for liver fibrosis progression to cirrhosis to HCC. We identified a panel of four serum microRNAs with high accuracy in HCC diagnosis. Additional studies are required to confirm this panel and test its prognostic significance.     

Serum Complement C3f and Fibrinopeptide A Are Potential Novel Diagnostic Biomarkers for Non-Alcoholic Fatty Liver Disease: A Study in Qingdao Twins

Aims

To compare the different serum peptidome patterns between twins with and without non-alcoholic fatty liver disease (NAFLD) in order to help understand the pathogenesis of NAFLD and to identify potential diagnostic and therapeutic targets.

Methods

The peptidomics patterns of 63 cases with NAFLD were compared with their twin healthy controls in Qingdao, China. Peptides between 800Da and 3500Da were captured and concentrated using C18 reversed-phase columns, followed by MALDI-TOF mass spectrometry. The sequences of peptides associated with NAFLD were further identified by MALDI-TOF-TOF. Further validation studies were conducted. One hundred additional serum samples were detected by commercially available ELISA kits to calculate the concentrations of complement C3f and fibrinopeptide A, respectively. The differences of these two peptides in the NAFLD and control groups were compared using SPSS 17.0, respectively.

Results

Compared with healthy controls, eleven peaks (861.1, 877.07, 904.5, 1206.57, 1350.64, 1518.7, 1690.9, 1777.94, 2931.29, 3190.4, 3261.4) were up-regulated and 7 peaks (942.44, 1020.47, 1060.06, 1211.7, 1263.63, 1449.76, 2768.3) were down-regulated in the NAFLD group. Two peptides derived from complement C3f and fibrinopeptide A, respectively, had the highest ROC values indistinguishing NAFLD cases from their normal controls. In the validation group, the concentrations of complement C3f and fibrinopeptide A (1466.929±78.306 pg/ml, 4.189±0.326 ng/ml, respevtively) in NAFLD group was higher than in control group (complement C3f 1159.357±99.624 pg/ml, FPA 3.039±0.483 ng/ml; P<0.05).

Conclusions

In this study, we established apeptidomics pattern that could help distinguish NAFLD patients from their twin controls. The differently-regulated peptides identified in our study may be potential diagnostic markers or therapeutic targets for NAFLD.     

Signature of Circulating MicroRNAs as Potential Biomarkers in Vulnerable Coronary Artery Disease

Aims

MicroRNAs (miRNAs) play important roles in the pathogenesis of cardiovascular diseases. Circulating miRNAs were recently identified as biomarkers for various physiological and pathological conditions. In this study, we aimed to identify the circulating miRNA fingerprint of vulnerable coronary artery disease (CAD) and explore its potential as a novel biomarker for this disease.

Methods and Results

The Taqman low-density miRNA array and coexpression network analyses were used to identify distinct miRNA expression profiles in the plasma of patients with typical unstable angina (UA) and angiographically documented CAD (UA group, n = 13) compared to individuals with non-cardiac chest pain (control group, n = 13). Significantly elevated expression levels of miR-106b/25 cluster, miR-17/92a cluster, miR-21/590-5p family, miR-126*, and miR-451 were observed in UA patients compared to controls. These findings were validated by real-time PCR in another 45 UA patients, 31 stable angina patients, and 37 controls. In addition, miR-106b, miR-25, miR-92a, miR-21, miR-590-5p, miR-126* and miR-451 were upregulated in microparticles (MPs) isolated from the plasma of UA patients (n = 5) compared to controls (n = 5). Using flow cytometry and immunolabeling, we further found that Annexin V⁺ MPs were increased in the plasma samples of UA patients compared to controls, and the majority of the increased MPs in plasma were shown to be Annexin V⁺ CD31⁺ MPs. The findings suggest that Annexin V⁺ CD31⁺ MPs may contribute to the elevated expression of the selected miRNAs in the circulation of patients with vulnerable CAD.

Conclusion

The circulating miRNA signature, consisting of the miR-106b/25 cluster, miR-17/92a cluster, miR-21/590-5p family, miR-126* and miR-451, may be used as a novel biomarker for vulnerable CAD.

Trial Registration

Chinese Clinical Trial Register, ChiCTR-OCH-12002349.     

Plasma MicroRNAs as Potential Noninvasive Biomarkers for In-Stent Restenosis

Objective

To investigate whether microRNAs (miRs) can serve as novel biomarkers for in-stent restenosis (ISR).

Methods

This retrospective, observational single-centre study was conducted at the cardiovascular department of a tertiary hospital centre in the north of China. Follow-up coronary angiography at 6 to 12 months was performed in 181 consecutive patients implanted with drug-eluting stents. Fifty-two healthy volunteers served as the control group. The plasma miRs levels were analyzed by quantitative real-time PCR. Receiver-operating characteristic curve (ROC) analysis was performed to investigate the characters of these miRs as potential biomarkers of ISR.

Results

MiR-21 levels in ISR patients were significantly higher than those in non-ISR patients and healthy controls (P<0.05), while miR-100 (P<0.05), miR-143 (P<0.001) and miR-145 (P<0.0001) levels were significantly decreased in ISR patients. Further analysis showed that miR-21 levels were remarkably increased (P = 0.045), while miR-100 (P = 0.041), miR-143 (P = 0.029) and miR-145 (P<0.01) levels were dramatically decreased in patients with diffuse ISR compared to those with focal ISR. ROC analysis demonstrated that the area under curve of miR-145, miR-143, miR-100 and miR-21 were 0.880 (95% confidence interval; CI = 0.791–0.987, P<0.001), 0.818 (95% confidence interval; CI = 0.755–0.963, P<0.001), 0.608 (95% confidence interval; CI = 0.372–0.757, P<0.05) and 0.568 (95% confidence interval; CI = 0.372–0.757, P<0.05), with specificity of 83.1%, 80.1%, 68.9% and 68.6%, and sensitivity of 88.7%, 82.1%, 60.2% and 50.1%, respectively.

Conclusions

Circulating miR-143 and miR-145 levels are associated with the occurrence of ISR and can serve as novel noninvasive biomarkers for ISR.     

Nonalcoholic Fatty Liver Disease and Risk of Diabetes: A Prospective Study in China

Objective: We aimed to investigate whether liver steatosis severity affects the risk of developing diabetes in a large cohort study.Methods: We prospectively examined the association in 41,650 Chinese adults with negative hepatitis-B surface antigen who were free of alcohol consumption, diabetes, and liver cirrhosis at baseline. Cox proportional models were used to estimate the risk of diabetes after a mean of 3.6 years of follow-up. Nonalcoholic fatty liver disease (NAFLD) was assessed with hepatic ultrasonography. Elevated alanine transaminase (ALT) was defined as ALT concentrations >19 and >30 U/L in females and males, respectively. Diabetes was defined as a fasting glucose ³7.0 mmol/L or treatment with hypoglycemic medication.Results: Liver steatosis severity was significantly associated with higher risks of developing diabetes (adjusted hazard ratio [HR] for severe vs. without NAFLD = 2.66, 95% confidence interval [CI]: 2.17–3.25, P-trend<.001) and impaired fasting glucose (fasting glucose between 5.6 and 6.9 mmol/L, adjusted HR = 1.36, 95% CI: 1.16–1.59, P-trend<.001), as well as a faster increase rate of fasting glucose concentrations (P-trend<.001), during 3.6 years of follow-up. Elevated ALT was also associated with incident diabetes (HR = 1.12, 95% CI: 1.02–1.22), adjusting for NAFLD and other covariates.Conclusion: We observed a dose-response relationship between liver steatosis severity and increased diabetes risk, and ALT may predict incident diabetes independently of NAFLD.Abbreviations: ALT = alanine transaminase; BP = blood pressure; CI = confidence interval; HCV = hepatitis C virus; HR = hazard ratio; IFG = impaired fasting glucose; NAFLD = nonalcoholic fatty liver disease; ULN = upper limit of normal     

MicroRNAs as Potential Diagnostic New Biomarkers in Diagnosis of Sepsis in Pediatric Patients

Background:Sepsis is one of the most common causes of multiorgan failure. Sepsis requires the presence of infection with a resultant systemic inflammatory state. Organ dysfunction occurs from the combination of the two processes. Sepsis is the main cause of mortality at intensive care units, with 30-50% mortality rate for all septic episodes. MicroRNA (miRNA) profile data could be beneficial as a specific diagnostic biomarker for sepsis and systemic inflammatory response syndrome (SIRS).Methods:Expression of miRNAs -122, -181b, -223 and -146a levels were assayed by quantitative real time polymerase chain reaction (qRT-PCR) in a prospective case control study, where forty septic cases were compared to 40 healthy controls of matched age and gender. Results:miRNAs -122 and -181b were significantly upregulated during early septic conditions, indicating that they could be sensitive and specific biomarkers for diagnosing sepsis. miRNA-223 and miRNA-146a could also represent highly specific and sensitive diagnostic biomarkers, as they were found to be significantly down-regulated. Serum levels of miRNA-223 could be used to predict poor prognosis with 70% sensitivity and 75% specificity, whereas the other three miRNAs could not predict prognosis.Conclusion:Our study shows that all tested miRNAs can be used for early detection of sepsis, with miRNA-223 being predictive of mortality, hence preventing multi-organ failure and reducing mortality, and predicting poor outcomes, thereby assisting in early categorization of ICU patients for rapid appropriate treatment and medico legal aspects.Key Words: Micrna-223, Systemic inflammatory response syndrome (SIRS), Sepsis, Biomarker     

非酒精性脂肪性肝病蛋白质组学研究进展

非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)是一种常见慢性肝脏疾病,其发病率呈逐年上升趋势,但发病机制尚未明确,诊疗手段仍不完善.蛋白质组学(proteomics)的出现使NAFLD研究有了进一步的发展,相关研究已达21个.目前,蛋白质组学技术可以研究疾病相关的分子改变,从而寻找新的生物标志物和治疗靶标.在此,对蛋白质组学在NAFLD诊断及分期、发病机制和其他相关领域研究进展作一个较为全面的综述.首先,对研究中遇到的研究对象、样本种类、实验方法和标志物特征选择进行经验性总结.其次,除了介绍如何运用蛋白质组学研究病因、危险因素和重要分子在NAFLD发病机制中的作用,还介绍NAFLD发病机制的亚细胞蛋白质组学、修饰蛋白质组学以及蛋白质组学与转录组学相结合的研究实例.此外,对差异蛋白质的分析策略和价值作了重点阐述,收集到一些有望成为NAFLD治疗靶标的候选分子.最后,结合新技术展望研究新空间,以期能够有助于推动蛋白质组学在寻找新的疾病标志物、探索疾病分子机制和治疗靶标中开辟新的途径.     

非酒精性脂肪性肝病的药物治疗进展

非酒精性脂肪性肝病已日渐成为目前慢性肝病的主要病因,其与肥胖、2型糖尿病和代谢综合征等疾病密切相关,疾病谱主要包括非酒精性单纯性脂肪肝、非酒精性脂肪性肝炎和肝硬化。早期诊断和及时治疗可望减轻NAFLD患者肝炎的严重程度并延缓肝纤维化的进展,减少并发症的出现。目前认为其发病与胰岛素抵抗、氧化应激及脂质过氧化和肠道菌群失调等因素有关。通过饮食调整和适当运动而减轻体重被认为是最基础的治疗措施,但单纯依靠减肥治疗脂肪性肝病(FLD)的效果并不理想,药物在脂肪性肝炎防治中的作用同样不可忽视。目前没有根治这一疾病的特效药物,单纯针对某一发病机制的药物亦难以治愈NAFLD这种复杂的疾病,本文主要从改善胰岛素抵抗、降脂、保肝抗炎及改善肠道菌群等四方面介绍一下本病的药物治疗进展。提倡改变生活方式的非药物治疗与药物干预治疗紧密结合,以取得最理想的治疗效果。     

Genetics of Nonalcoholic Fatty Liver Disease:An Overview

Nonalcoholic fatty liver disease(NAFLD) is the most common liver disease in the world today.Its incidence in adults and children is rising rapidly due to the ongoing epidemics of obesity and type 2 diabetes.Hence,it has become a global public health issue.Environmental factors have been found to play a major role in the etiology of NAFLD,especially for genetically susceptible populations. Among these,one of the most important factors is junk food,especially the typical "Western-style" diet rich in simple carbohydrates, saturated fat,and highly processed food materials.Genetic predisposition to NAFLD does occur;however,a precise definition of genetic factors responsible for NAFLD is still lacking.Specific variants of different genes have been shown to present a risk for NAFLD.Genetic studies might be helpful in the management of the disease by developing novel treatment strategies based on individual’s genotype.     

A Meta-analysis on Associated Risk of Mortality in Nonalcoholic Fatty Liver Disease

ObjectiveNonalcoholic fatty liver disease (NAFLD) affects much of the worldwide population and poses a significant burden to the global healthcare. The rising numbers of individuals with NAFLD and instances of mortality point toward the importance of understanding the association causes of mortality in NAFLD. This meta-analysis aimed to seek the associations of NAFLD with all-cause, cardiovascular disease (CVD)-related, liver-related, and cancer-related mortality.MethodsMEDLINE and Embase were searched for articles relating to causes of mortality between NAFLD and non-NAFLD. The DerSimonian and Laird random-effects model was used to analyze adjusted hazard ratios (HR), and a sensitivity analysis was conducted to reduce heterogeneity through a graphical display of study heterogeneity.ResultsFifteen studies involving 10 286 490 patients were included. Individuals with NAFLD exhibited an increased risk of all-cause mortality (HR, 1.32; 95% CI, 1.09-1.59; P < .01; I² = 96.00%), CVD-related mortality (HR, 1.22; 95% CI, 1.06-1.41; P < .01; I² = 81.00%), and cancer-related mortality (HR, 1.67; 95% CI, 1.15-2.41; P < .01; I² = 95.00%). However, no significant association was found between liver-related mortality and NAFLD (HR, 3.58; 95% CI, 0.69-18.46; P =.13; I² = 96.00%). The sensitivity analysis conducted with graphic display of heterogeneity and only population-based studies found similar results.ConclusionNAFLD was associated with an increased risk of all-cause, CVD-related, and cancer-related mortality but not liver-related mortality. The finding is likely because of low fibrosis prevalence in the community. However, the significant burden in other causes of mortality beyond the liver points to a need for multidisciplinary efforts to reduce the mortality risks.