Long Non-Coding RNAs in Liver Cancer and Nonalcoholic Steatohepatitis

This review aims to highlight the recent findings of long non-coding RNAs (lncRNAs) in liver disease. In particular, we focus on the functions of lncRNAs in hepatocellular carcinoma (HCC) and non-alcoholic steatohepatitis (NASH). We summarize the current research trend in lncRNAs and their potential as biomarkers and therapeutic targets for the treatment of HCC and NASH

The Role of Insulin Resistance in Fueling NAFLD Pathogenesis: From Molecular Mechanisms to Clinical Implications

Non-alcoholic fatty liver disease (NAFLD) represents a predominant hepatopathy that is rapidly becoming the most common cause of hepatocellular carcinoma worldwide. The close association with metabolic syndrome's extrahepatic components has suggested the nature of the systemic metabolic-related disorder based on the interplay between genetic, nutritional, and environmental factors, creating a complex network of yet-unclarified pathogenetic mechanisms in which the role of insulin resistance (IR) could be crucial. This review detailed the clinical and pathogenetic evidence involved in the NAFLD-IR relationship, presenting both the classic and more innovative models. In particular, we focused on the reciprocal effects of IR, oxidative stress, and systemic inflammation on insulin-sensitivity disruption in critical regions such as the hepatic and the adipose tissue, while considering the impact of genetics/epigenetics on the regulation of IR mechanisms as well as nutrients on specific insulin-related gene expression (nutrigenetics and nutrigenomics). In addition, we discussed the emerging capability of the gut microbiota to interfere with physiological signaling of the hormonal pathways responsible for maintaining metabolic homeostasis and by inducing an abnormal activation of the immune system. The translation of these novel findings into clinical practice could promote the expansion of accurate diagnostic/prognostic stratification tools and tailored pharmacological approaches

Epitranscriptomic regulation in breast cancer and PCB-induced liver disease.

Post-transcriptional RNA modifications including N6-methyladenosine (m6A) regulate mRNA stability, splicing, and translation. My research examined m6A in two disease models: breast cancer (BCa) and non-alcoholic fatty liver disease (NAFLD). Acquired resistance to endocrine therapies (ET) develops in approximately 20% of BCa patients with estrogen receptor α positive (ER+) tumors following treatment. The mechanisms by which tumor cells evade ET are not completely understood. Using a cell line model, we investigated the role of an m6A reader protein, HNRNPA2B1 (A2B1) that is upregulated in ET-resistant ER+ BCa cells. Stable overexpression of A2B1 in ET-sensitive MCF-7 cells (MCF-7-A2B1), results in ET resistance, whereas knockdown of A2B1 in ET-resistant cells restored ET-sensitivity. microRNAs (miRNAs) downregulated by transient overexpression of A2B1 were identified to target two key enzymes (PSAT1 and PHGDH) in the serine biosynthetic pathway (SSP) which is upregulated in ET-resistant BCa cells and in tumors from patients with ET-resistant disease. Using luciferase assays, PSAT1 and PHGDH were validated as bona fide targets of miRNAs downregulated by A2B1 (miR-145-5p and miR-424-5p targeting PSAT1, miR-34b-5p and miR-876-5p targeting PHGDH). Exogenous overexpression of the validated miRNAs decreased endogenous PSAT1 and PHGDH in ET-resistant BCa cells, resulting in increased sensitivity to ET in vitro. In the second model, alterations in the m6A epitranscriptome were identified in the livers of male C57Bl/6Jmice after a single, oral exposure to polychlorinated biphenyls (PCB), a class of persistent organic pollutants, in combination with 12 weeks on a high fat diet (HFD). Our results demonstrated that exposure to PCBs in combination with a HFD resulted in major changes to the mRNA and miRNA transcriptomes, and m6A epitranscriptome. Pathway analysis of the genes in which m6A peaks were altered identified pathways involved in the progression from steatosis to steatohepatitis in NAFLD. PCB exposures also resulted in changes to alternative splicing (AS) mechanisms and events, suggesting that PCB-induced m6A changes contribute to altered isoforms expression in NAFLD. Taken together, the results in this dissertation demonstrate the significant role of altered m6A in two common human diseases

Molecular Epidemiology towards Deciphering Underlying Mechanisms of Fatty Liver Disease

A substantial burden of illness and mortality worldwide is caused by fatty liver disease (FLD) and its complications, including type 2 diabetes (T2D), obesity, hypertension, NBHJY98and dyslipidemia. Among the FLD patients, the majority of fatty liver patients develop non-alcoholic fatty liver disease (NAFLD), which is the most common cause of chronic liver disease worldwide. Although there are no or few symptoms, a subpopulation can progress to end-stage liver disease or even liver cancer. Recently, a consensus by an international panel of experts recommended a change in name for NAFLD to metabolic dysfunction associated fatty liver disease (MAFLD). It does not only eliminate alcohol from both the name and definition. However, the underlying mechanism of FLD and how to early diagnose FLD are still not clear. Understanding the pathophysiology of FLD can help to facilitate the prevention and treatment strategies. In this thesis, I aim to provide novel insights into the molecular mechanisms of FLD through several omics approaches and integration of multi-omics, centering on genomics, transcriptomics, metabolomics, and gut microbiota.In Chapter 2, I found that in Rotterdam Study, lower sex hormone-binding globulin (SHBG) was associated with NAFLD both in men and women. Lower testosterone was associated with NAFLD among women. Similarly, the meta-analysis of 16 studies also indicated that there is no sex-specific association between SHBG and NAFLD. Moreover, men with NAFLD had lower testosterone levels than those without NAFLD, while women with NAFLD had higher testosterone levels than those without. In addition, men with NAFLD had lower estradiol levels than those without NAFLD.Chapter 3 focused on investigating the impact of klotho (KL) rs495392 C>A polymorphism on the histological severity of NAFLD. In Chapter 3.1, I found that carriage of the klotho rs495392 A allele, a variant commonly reported to be associated with liver diseases, had a protective effect on steatosis severity in Chinese patients. The effect on NAFLD was confirmed in the Rotterdam Study cohort. The findings also showed the association between serum vitamin D levels and NAFLD specifically in rs495392 A allele carriers, but not in non-carriers. Moreover, I found that the rs495392 A allele attenuated the detrimental impact of the PNPLA3 rs738409 G allele on the risk of severe NAFLD in the Chinese cohort.In Chapter 4, I mainly focused on exploring the epigenetic regulation of FLD and metabolic disorders. In Chapter 4.1, I found two recent epigenome-wide association studies conducted among large population-based cohorts that have reported the association between cg06690548 (SLC7A11) and FLD. Moreover, several studies have demonstrated the association between microRNAs (miRNAs) and FLD, in which miR-122, miR-34a, and miR-192 were recognized as the most relevant miRNAs as biomarkers for FLD. In Chapter 4.2, I found few circulating miRNAs correlated with various non-invasive markers of fatty liver, due to either non-alcoholic or alcohol FLD. The response to the editorial that commented on our paper can be found in Chapter 4.3. In Chapter 4.4, I found the relationship between plasma levels of miRNAs with obesity, body fat distribution, and fat mass using data from the Rotterdam Study. Interestingly, miR-193a-5p, the top obesity-associated miRNA was among the miRNAs I found to be significantly associated with FLD in Chapter 4.1.Finally, in Chapter 5 I explored the potential role of the gut microbiome in liver health and disease. Chapter 5.1, I found that lower microbial alpha diversity was associated with a higher prevalence of MAFLD. Three genera were significantly associated with MAFLD after the false discovery rate was corrected (q-valu

Diabetes and associated cardiovascular complications: The role of microRNAs

Diabetes mellitus (DM) refers to a complex cluster of metabolic disorders characterized by hyperglycemia caused by inadequate insulin secretion, insulin resistance, or excessive glucagon secretion. If not correctly treated, the prolonged effects of DM-associated metabolic perturbations lead to systemic vascular complications and cardiovascular disease (CVD), the principal cause of mortality among patients with DM. Given the increase in the global prevalence of diabetes, novel diagnostic and therapeutic procedures are necessary for its effective identification and treatment. Recent findings point to an important role of microRNA (miRNAs) in DM initiation and progression, as well as the occurrence of associated cardiovascular complications. miRNAs are short, highly conserved, single-stranded, non-coding RNAs that contribute to the maintenance of physiological homeostasis through the regulation of crucial processes such as metabolism, cell proliferation, and apoptosis. The increased availability of high-throughput methodologies for identifying and characterizing non-coding RNAs has led to considerable interest in miRNAs as potential biomarkers and therapeutic agents for DM. In this review, we first comprehensively detail the regulatory miRNAs involved in the pathophysiology of DM and diabetic cardiomyopathy (DCMP). Subsequently, we summarize findings regarding the utility of several of these miRNAs as potential prognostic and diagnostic biomarkers for DM and DM-associated CVD. Finally, we evaluate the potential of miRNA-based therapeutic approaches for treating DM and DCMP in the clinical setting

Prevention and Treatment of Atherosclerosis

This open access book is supported by the European Atherosclerosis Society Association (EAS). This follow-up edition of the well-received Handbook volume 'Atherosclerosis: Diet and Drugs' reflects the state-of-the-art and most recent developments in atherosclerosis research. Outstanding international experts give a comprehensive overview of the field covering topics, such as improving the treatment focusing on established targets, novel drug developments addressing pre-defined targets, hypothesis-based and hypothesis-free approaches to unravel novel targets

Targeted Therapeutics and Novel Signaling Pathways in Non-Alcohol-Associated Fatty Liver/Steatohepatitis (NAFL/NASH)

Non-alcohol-associated fatty liver/steatohepatitis (NAFL/NASH) has become the leading cause of liver disease worldwide. NASH, an advanced form of NAFL, can be progressive and more susceptible to developing cirrhosis and hepatocellular carcinoma. Currently, lifestyle interventions are the most essential and effective strategies for preventing and controlling NAFL without the development of fibrosis. While there are still limited appropriate drugs specifically to treat NAFL/NASH, growing progress is being seen in elucidating the pathogenesis and identifying therapeutic targets. In this review, we discussed recent developments in etiology and prospective therapeutic targets, as well as pharmacological candidates in pre/clinical trials and patents, with a focus on diabetes, hepatic lipid metabolism, inflammation, and fibrosis. Importantly, growing evidence elucidates that the disruption of the gut-liver axis and microbe-derived metabolites drive the pathogenesis of NAFL/NASH. Extracellular vesicles (EVs) act as a signaling mediator, resulting in lipid accumulation, macrophage and hepatic stellate cell activation, further promoting inflammation and liver fibrosis progression during the development of NAFL/NASH. Targeting gut microbiota or EVs may serve as new strategies for the treatment of NAFL/NASH. Finally, other mechanisms, such as cell therapy and genetic approaches, also have enormous therapeutic potential. Incorporating drugs with different mechanisms and personalized medicine may improve the efficacy to better benefit patients with NAFL/NASH

Mastiha has efficacy in immune-mediated inflammatory diseases through a microRNA-155 Th17 dependent action

Mastiha is a natural nutritional supplement with known anti-inflammatory properties. Non-alcoholic fatty liver disease (NAFLD) and Inflammatory bowel disease (IBD) are immune mediated inflammatory diseases that share common pathophysiological features. Mastiha has shown beneficial effects in both diseases. MicroRNAs have emerged as key regulators of inflammation and their modulation by phytochemicals have been extensively studied over the last years. Therefore, the aim of this study was to investigate whether a common route exists in the anti-inflammatory activity of Mastiha, specifically through the regulation of miRNA levels. Plasma miR-16, miR-21 and miR-155 were measured by Real-Time PCR before and after two double blinded and placebo-controlled randomized clinical trials with Mastiha. In IBD and particularly in ulcerative colitis patients in relapse, miR-155 increased in the placebo group (p = 0.054) whereas this increase was prevented by Mastiha. The mean changes were different in the two groups even after adjusting for age, sex and BMI (p = 0.024 for IBD and p = 0.042). Although the results were not so prominent in NAFLD, miR-155 displayed a downward trend in the placebo group (p = 0.054) whereas the levels did not changed significantly in the Mastiha group in patients with less advanced fibrosis. Our results propose a regulatory role for Mastiha in circulating levels of miR-155, a critical player in T helper-17 (Th17) differentiation and function

Molecular Epidemiology towards Deciphering Underlying Mechanisms of Fatty Liver Disease

A substantial burden of illness and mortality worldwide is caused by fatty liver disease (FLD) and its complications, including type 2 diabetes (T2D), obesity, hypertension, NBHJY98and dyslipidemia. Among the FLD patients, the majority of fatty liver patients develop non-alcoholic fatty liver disease (NAFLD), which is the most common cause of chronic liver disease worldwide. Although there are no or few symptoms, a subpopulation can progress to end-stage liver disease or even liver cancer. Recently, a consensus by an international panel of experts recommended a change in name for NAFLD to metabolic dysfunction associated fatty liver disease (MAFLD). It does not only eliminate alcohol from both the name and definition. However, the underlying mechanism of FLD and how to early diagnose FLD are still not clear. Understanding the pathophysiology of FLD can help to facilitate the prevention and treatment strategies. In this thesis, I aim to provide novel insights into the molecular mechanisms of FLD through several omics approaches and integration of multi-omics, centering on genomics, transcriptomics, metabolomics, and gut microbiota.In Chapter 2, I found that in Rotterdam Study, lower sex hormone-binding globulin (SHBG) was associated with NAFLD both in men and women. Lower testosterone was associated with NAFLD among women. Similarly, the meta-analysis of 16 studies also indicated that there is no sex-specific association between SHBG and NAFLD. Moreover, men with NAFLD had lower testosterone levels than those without NAFLD, while women with NAFLD had higher testosterone levels than those without. In addition, men with NAFLD had lower estradiol levels than those without NAFLD.Chapter 3 focused on investigating the impact of klotho (KL) rs495392 C>A polymorphism on the histological severity of NAFLD. In Chapter 3.1, I found that carriage of the klotho rs495392 A allele, a variant commonly reported to be associated with liver diseases, had a protective effect on steatosis severity in Chinese patients. The effect on NAFLD was confirmed in the Rotterdam Study cohort. The findings also showed the association between serum vitamin D levels and NAFLD specifically in rs495392 A allele carriers, but not in non-carriers. Moreover, I found that the rs495392 A allele attenuated the detrimental impact of the PNPLA3 rs738409 G allele on the risk of severe NAFLD in the Chinese cohort.In Chapter 4, I mainly focused on exploring the epigenetic regulation of FLD and metabolic disorders. In Chapter 4.1, I found two recent epigenome-wide association studies conducted among large population-based cohorts that have reported the association between cg06690548 (SLC7A11) and FLD. Moreover, several studies have demonstrated the association between microRNAs (miRNAs) and FLD, in which miR-122, miR-34a, and miR-192 were recognized as the most relevant miRNAs as biomarkers for FLD. In Chapter 4.2, I found few circulating miRNAs correlated with various non-invasive markers of fatty liver, due to either non-alcoholic or alcohol FLD. The response to the editorial that commented on our paper can be found in Chapter 4.3. In Chapter 4.4, I found the relationship between plasma levels of miRNAs with obesity, body fat distribution, and fat mass using data from the Rotterdam Study. Interestingly, miR-193a-5p, the top obesity-associated miRNA was among the miRNAs I found to be significantly associated with FLD in Chapter 4.1.Finally, in Chapter 5 I explored the potential role of the gut microbiome in liver health and disease. Chapter 5.1, I found that lower microbial alpha diversity was associated with a higher prevalence of MAFLD. Three genera were significantly associated with MAFLD after the false discovery rate was corrected (q-valu

Molecular Mechanisms Underlying SSRI-induced Non-alcoholic Fatty Liver Disease

This thesis aims to investigate fluoxetine, a widely prescribed SSRI antidepressant, for its role in the pathogenesis of NAFLD and uncover novel mechanisms by which it may contribute to drug-induced steatosis. We demonstrated that increased hepatic lipid accumulation was mediated, in part, via elevated serotonin production. The inhibition of hepatic serotonin synthesis prevented lipid accumulation in fluoxetine-treated hepatocytes demonstrating a causal role for serotonin in fluoxetine-induced hepatic steatosis. Interestingly, in several studies, serotonin signaling has been shown to impact prostaglandin biosynthesis. As prostaglandins have been implicated in the development of NAFLD, and fluoxetine has previously been shown to alter the production of prostaglandins I assessed the role of prostaglandins in fluoxetine-induced hepatic lipid accumulation. Fluoxetine treatment increased mRNA expression of prostaglandin biosynthetic enzymes, increased production of prostaglandin 15-deoxy-Δ12,14PGJ2 (PPARG agonist), and elevated PPARG targets involved in fatty acid uptake. Fluoxetine-induced lipid accumulation, 15-deoxy-Δ12,14PGJ2 production, and the expression of PPARG lipogenic genes were attenuated with a PTGS1 specific inhibitor. Taken together these findings suggested that fluoxetine-induced lipid accumulation was mediated via PTGS1 and its downstream product 15-deoxy-Δ12,14PGJ2. Given that Pparg was elevated following fluoxetine treatment, and PPARG regulates microRNA involved in hepatic lipid accumulation, my final project focused on PPARG’s role in altered miRNA expression. Indeed, fluoxetine treatment increased the miRNA expression of miR-122, an effect that was attenuated when fluoxetine treatment was combined with the PPARG antagonist GW9662, suggesting a fluoxetine-PPARG-miR122 axis contributing to hepatic steatosis. While these studies have only been performed in vitro, an understanding of the molecular changes associated with SSRI treatment may lead to the development of strategies to prevent the increased risk of adverse metabolic outcomes associated with the use of SSRI antidepressants.DissertationDoctor of Philosophy (Medical Science)In adults, major depressive disorder (depression) is one of the most common psychiatric illnesses. Recent data suggests that there are more than 4.1 million Canadians who currently suffer from depression. Depression is commonly treated using selective serotonin reuptake inhibitor (SSRI) antidepressants. While these antidepressants do help manage depressive symptoms, they can also cause unwanted side effects including a build-up of fat in the liver, leading to fatty liver disease. The goal of my research is to understand the link between SSRI use and the development of fatty liver disease. This thesis investigates the effects of fluoxetine (Prozac®), a commonly used SSRI antidepressant, on molecular pathways that can lead to the development of fatty liver disease. An understanding of the molecular changes with SSRI treatment may lead to the development of strategies to prevent the harmful effects of SSRI antidepressants on the liver