Liver-specific microRNA Research Articles

Preclinical evaluation of tissue-selective gene therapies for congenital generalised lipodystrophy

Lipodystrophy is a rare disorder which can be life-threatening. Here individuals fail to develop or maintain appropriate adipose tissue stores. This typically causes severe metabolic complications, including hepatic steatosis and lipoatrophic diabetes. There is no cure for lipodystrophy, and treatment options remain very limited. Here we evaluate whether tissue-selective adeno-associated virus (AAV) vectors can provide a targeted form of gene therapy for lipodystrophy, using a preclinical lipodystrophic mouse model of Bscl2 deficiency. We designed AAV vectors containing the mini/aP2 or thyroxine-binding globulin promoter to selectively target adipose or liver respectively. The AAV-aP2 vectors also contained the liver-specific microRNA-122 target sequence, restricting hepatic transgene expression. Systemic delivery of AAV-aP2 vectors overexpressing human BSCL2 restored adipose tissue development and metabolic health in lipodystrophic mice without detectable expression in the liver. High doses (1 × 1012 GCs) of liver-selective vectors led to off target expression and adipose tissue development, whilst low doses (1 × 1010 GCs) expressed selectively and robustly in the liver but did not improve metabolic health. This reveals that adipose tissue-selective, but not liver directed, AAV-mediated gene therapy is sufficient to substantially recover metabolic health in generalised lipodystrophy. This provides an exciting potential new avenue for an effective, targeted, and thereby safer therapeutic intervention.

ESRP2-microRNA-122 axis directs the postnatal onset of liver polyploidization and maturation.

Hepatocyte polyploidy and maturity are critical to acquiring specialized liver functions. Multiple intra- and extracellular factors influence ploidy, but how they cooperate temporally to steer liver polyploidization and maturation or how post-transcriptional mechanisms integrate into these paradigms is unknown. Here, we identified an important regulatory hierarchy in which postnatal activation of Epithelial-Splicing-Regulatory-Protein-2 (ESRP2) stimulates biogenesis of liver-specific microRNA (miR-122), thereby facilitating polyploidization, maturation, and functional competence of hepatocytes. By determining transcriptome-wide protein-RNA interactions in vivo and integrating them with single-cell and bulk hepatocyte RNA-seq datasets, we delineate an ESRP2-driven RNA processing program that drives sequential replacement of fetal-to-adult transcript isoforms. Specifically, ESRP2 binds the primary miR-122 host gene transcript to promote its processing/biogenesis. Combining constitutive and inducible ESRP2 gain- and loss-of-function mice models with miR-122 rescue experiments, we demonstrate that timed activation of ESRP2 augments miR-122-driven program of cytokinesis failure, ensuring proper onset and extent of hepatocyte polyploidization.

Enhanced Virus Translation Enables miR-122-Independent Hepatitis C Virus Propagation.

The 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome forms RNA structures that regulate virus replication and translation. The region contains an internal ribosomal entry site (IRES) and a 5'-terminal region. Binding of the liver-specific microRNA (miRNA) miR-122 to two binding sites in the 5'-terminal region regulates viral replication, translation, and genome stability and is essential for efficient virus replication, but its precise mechanism of action is still unresolved. A current hypothesis is that miR-122 binding stimulates viral translation by facilitating the viral 5' UTR to form the translationally active HCV IRES RNA structure. While miR-122 is essential for detectable replication of wild-type HCV genomes in cell culture, several viral variants with 5' UTR mutations exhibit low-level replication in the absence of miR-122. We show that HCV mutants capable of replicating independently of miR-122 display an enhanced translation phenotype that correlates with their ability to replicate independently of miR-122. Further, we provide evidence that translation regulation is the major role for miR-122 and show that miR-122-independent HCV replication can be rescued to miR-122-dependent levels by the combined impacts of 5' UTR mutations that stimulate translation and by stabilizing the viral genome by knockdown of host exonucleases and phosphatases that degrade the genome. Finally, we show that HCV mutants capable of replicating independently of miR-122 also replicate independently of other microRNAs generated by the canonical miRNA synthesis pathway. Thus, we provide a model suggesting that translation stimulation and genome stabilization are the primary roles for miR-122 in promoting HCV. IMPORTANCE The unusual and essential role of miR-122 in promoting HCV propagation is incompletely understood. To better understand its role, we have analyzed HCV mutants capable of replicating independently of miR-122. Our data show that the ability of viruses to replicate independently of miR-122 correlates with enhanced virus translation but that genome stabilization is required to restore efficient HCV replication. This suggests that viruses must gain both abilities to escape the need for miR-122 and impacts the possibility that HCV can evolve to replicate outside the liver.

MicroRNA-122-functionalized DNA tetrahedron stimulate hepatic differentiation of human mesenchymal stem cells for acute liver failure therapy

As the most abundant liver-specific microRNA, microRNA-122 (miR122) played a crucial role in the differentiation of stem cells into hepatocytes. However, highly efficient miR122 delivery still confronts challenges including poor cellular uptake and easy biodegradation. Herein, we for the first time demonstrated that the tetrahedral DNA (TDN) nanoplatform had great potential in inducing the differentiation of human mesenchymal stem cells (hMSCs) into functional hepatocyte-like cells (HLCs) by transferring the liver-specific miR122 to hMSCs efficiently without any extrinsic factors. As compared with miR122, miR122-functionalized TDN (TDN-miR122) could significantly up-regulate the protein expression levels of mature hepatocyte markers and hepatocyte-specific marker genes in hMSCs, indicating that TDN-miR122 could particularly activate the hepatocyte-specific properties of hMSCs for developing cell-based therapies in vitro. The transcriptomic analysis further indicated the potential mechanism that TDN-miR122 assisted hMSCs differentiated into functional HLCs. The TDN-miR122-hMSCs exhibited hepatic cell morphology phenotype, significantly up-regulated specific hepatocyte genes and hepatic biofunctions in comparison with the undifferentiated MSCs. Preclinical in vivo transplantation appeared that TDN-miR122-hMSCs in combination with or without TDN could efficiently rescue acute liver failure injury through hepatocyte function supplement, anti-apoptosis, cellular proliferation promotion, and anti-inflammatory. Collectively, our findings may provide a new and facile approach for hepatic differentiation of hMSCs for acute liver failure therapy. Further large animal model explorations are needed to study their potential in clinical translation in the future.

MiR-122 dysregulation is associated with type 2 diabetes mellitus-induced dyslipidemia and hyperglycemia independently of its rs17669 variant

miR-122 is a liver specific micro-RNA that participates in the regulation of carbohydrate and lipid metabolism. The rs17669 variant of miR-122 is positioned at the flanking region of miR-122 and may affect its stability and maturation. Therefore, this study was aimed to investigate the association of the rs17669 polymorphism with the miR-122 circulating level, risk of type 2 diabetes mellitus (T2DM) development, and biochemical parameters in T2DM patients and matched healthy controls. This study involved 295 subjects (controls: n = 145 and T2DM: n = 150). The rs17669 variant genotyping was done by ARMS-PCR. Serum biochemical parameters including lipid profile, small-dense low density lipoprotein (sdLDL) and glucose were measured by colorimetric kits. Insulin and Glycated hemoglobin (HbA1c) were assayed using ELISA and capillary electrophoresis methods, respectively. miR-122 expression was measured by real-time PCR. There was no significant difference between study groups in terms of allele and genotype distribution (P > 0.05). The rs17669 variant did not have any significant association with miR-122 gene expression and biochemical parameters (P > 0.05). miR-122 expression level in T2DM patients was significantly higher than that in control subjects (5.7 ± 2.4 vs. 1.4 ± 0.78) (P < 0.001). Furthermore, miR-122 fold change had a positive and significant correlation with low-density lipoprotein cholesterol (LDL-C), sdLDL, fasting blood sugar (FBS), and insulin resistance (P < 0.05). It can be concluded that the rs17669 variant of miR-122 is not associated with the miR-122 expression and T2DM-associated serum parameters. Furthermore, it can be suggested that miR-122 dysregulation is involved in T2DM development through inducing dyslipidemia, hyperglycemia, and resistance to insulin.

Elucidating the distinct contributions of miR-122 in the HCV life cycle reveals insights into virion assembly.

Efficient hepatitis C virus (HCV) RNA accumulation is dependent upon interactions with the human liver-specific microRNA, miR-122. MiR-122 has at least three roles in the HCV life cycle: it acts as an RNA chaperone, or 'riboswitch', allowing formation of the viral internal ribosomal entry site; it provides genome stability; and promotes viral translation. However, the relative contribution of each role in HCV RNA accumulation remains unclear. Herein, we used point mutations, mutant miRNAs, and HCV luciferase reporter RNAs to isolate each of the roles and evaluate their contribution to the overall impact of miR-122 in the HCV life cycle. Our results suggest that the riboswitch has a minimal contribution in isolation, while genome stability and translational promotion have similar contributions in the establishment phase of infection. However, in the maintenance phase, translational promotion becomes the dominant role. Additionally, we found that an alternative conformation of the 5' untranslated region, termed SLIIalt, is important for efficient virion assembly. Taken together, we have clarified the overall importance of each of the established roles of miR-122 in the HCV life cycle and provided insight into the regulation of the balance between viral RNAs in the translating/replicating pool and those engaged in virion assembly.

PLK1-ELAVL1/HuR-miR-122 signaling facilitates hepatitis C virus proliferation

The liver-specific microRNA, miR-122, plays an essential role in the propagation of hepatitis C virus (HCV) by binding directly to the 5'-end of its genomic RNA. Despite its significance for HCV proliferation, the host factors responsible for regulating miR-122 remain largely unknown. In this study, we identified the cellular RNA-binding protein, ELAVL1/HuR (embryonic lethal-abnormal vision-like 1/human antigen R), as critically contributing to miR-122 biogenesis by strong binding to the 3'-end of miR-122. The availability of ELAVL1/HuR was highly correlated with HCV proliferation in replicon, infectious, and chronically infected patient conditions. Furthermore, by screening a kinase inhibitor library, we identified rigosertib, an anticancer agent under clinical trials, as having both miR-122-modulating and anti-HCV activities that were mediated by its ability to target polo-like kinase 1 (PLK1) and subsequently modulate ELAVL1/HuR-miR-122 signaling. The expression of PLK1 was also highly correlated with HCV proliferation and the HCV positivity of HCC patients. ELAVL1/HuR-miR-122 signaling and its mediation of PLK1-dependent HCV proliferation were demonstrated by performing various rescue experiments and utilizing an HCV mutant with low dependency on miR-122. In addition, the HCV-inhibitory effectiveness of rigosertib was validated in various HCV-relevant conditions, including replicons, infected cells, and replicon-harboring mice. Rigosertib was highly effective in inhibiting the proliferation of not only wild-type HCVs, but also sofosbuvir resistance-associated substitution-bearing HCVs. Our study identifies PLK1-ELAVL1/HuR-miR-122 signaling as a regulatory axis that is critical for HCV proliferation, and suggests that a therapeutic approach targeting this host cell signaling pathway could be useful for treating HCV and HCV-associated diseases.

MicroRNA-122 Regulation of HCV Infections: Insights from Studies of miR-122-Independent Replication.

Despite the advancement in antiviral therapy, Hepatitis C remains a global health challenge and one of the leading causes of hepatitis related deaths worldwide. Hepatitis C virus, the causative agent, is a positive strand RNA virus that requires a liver specific microRNA called miR-122 for its replication. Unconventional to the canonical role of miRNAs in translation suppression by binding to 3′Untranslated Region (UTR) of messenger RNAs, miR-122 binds to two sites on the 5′UTR of viral genome and promotes viral propagation. In this review, we describe the unique relationship between the liver specific microRNA and HCV, the current knowledge on the mechanisms by which the virus uses miR-122 to promote the virus life cycle, and how miR-122 impacts viral tropism and pathogenesis. We will also discuss the use of anti-miR-122 therapy and its impact on viral evolution of miR-122-independent replication. This review further provides insight into how viruses manipulate host factors at the initial stage of infection to establish a successful infection.

MYC Promotes LDHA Expression through MicroRNA-122-5p to Potentiate Glycolysis in Hepatocellular Carcinoma.

MYC is a notorious oncogene in a vast network of malignancies, whereas liver-specific microRNA- (miR-) 122-5p is downregulated in hepatocellular cancer (HCC). Here, we studied the possible correlation between these two and their involvement in glycolysis in HCC. MYC was overexpressed in HCC tissues and cells compared to normal liver tissues and normal hepatocytes NHC, which predicted a poor survival of HCC sufferers. Functional assays demonstrated that silencing of MYC inhibited the glycolysis in HCC cells, as evidenced by significantly weaker glucose consumption, lactate production, adenosine triphosphate (ATP) levels, and downregulated HK1 and HK2 protein expression. Moreover, MYC bound to the miR-122-5p promoter and repressed the miR-122-5p expression. Rescue experiments showed that miR-122-5p inhibitor rescued the diminished glycolysis after MYC silencing. In addition, lactate dehydrogenase (LDHA) was identified as a downstream target of miR-122-5p. The overexpression of LDHA mitigated the effects of si-MYC and miR-122-5p mimic on glycolysis of HCC cells, respectively. In conclusion, the MYC/miR-122-5p/LDHA axis modulates glycolysis in HCC cells and possibly affects HCC progression.

Sevoflurane attenuates hepatic ischemia reperfusion injury by the miR-122/Nrf2 pathway

BackgroundSevoflurane can protect organs from ischemia-reperfusion (IR) injury, but the mechanism is still unclear. MicroRNA-122 (miR-122) is a liver-specific microRNA (miRNA) and regulates liver function. Therefore, this study aims to elucidate the relationship between the protective effect of sevoflurane and miR-122 in liver IR injury.MethodsWistar rats were divided into the following groups: sham, IR, IR + sevoflurane, IR + miR-122 antagomir, and IR + miR-122 antagomir + sevoflurane. Hematoxylin and eosin (H&E) staining and Suzuki score were used to evaluate the pathological damage of the liver. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 in the serum and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO) in the liver homogenate supernatant were detected by using the corresponding kit. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and flow cytometry was applied to evaluate the apoptosis of liver tissues. The expression of nuclear factor E2-related factor 2 (Nrf2), miR-122, p53, and HO-1 in liver tissue was evaluated by using immunohistochemistry, qRT-PCR, and western blot as needed.ResultsCompared to the IR group, the sevoflurane post-treatment or miR-122 antagomir groups showed improved liver injury, decreased Suzuki score, inhibited the levels of AST, ALT, LDH, MDA, NO, TNF-α, IL-1β, and IL-6, increased levels of SOD, IL-10, and inhibited hepatocyte apoptosis. Regarding the molecular mechanism, sevoflurane post-treatment fostered the expression of HO-1, promoted the transport of Nrf2 from cytoplasm to the nucleus, and decreased the expression of miR-122 and p53. The combined use of miR-122 antagomir and sevoflurane enhanced the protective effect of miR-122 antagomir in liver injury in IR rats.ConclusionsSevoflurane protected the liver from IR damage by regulating the miR-122/Nrf2/HO-1 pathway.

MicroRNA 122 Affects both the Initiation and the Maintenance of Hepatitis C Virus Infections.

A liver-specific microRNA, miR-122, anneals to the hepatitis C virus (HCV) genomic 5' terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full-length RNAs with specific mutations in the 5' untranslated region (UTR) can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having encephalomyocarditis virus internal ribosomal entry site (EMCV IRES)-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by the amount of viral RNA delivered. HCV RNAs replicating independently of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of an miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication, suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection and also supports new replication complex formation during ongoing infection and after infected cell division. IMPORTANCE The mechanism by which miR-122 promotes the HCV life cycle is not well understood, and a role in directly promoting genome amplification is still debated. In this study, we have shown that miR-122 increases the rate of viral RNA accumulation and promotes the establishment of an HCV infection in a greater number of cells than in the absence of miR-122. However, we also confirm a minor role in promoting ongoing virus replication and propose a role in the initiation of new replication complexes throughout a virus infection. This study has implications for the use of anti-miR-122 as a potential HCV therapy.

Abstract 8879: Circulating Levels of MicroRNA-122 and Hepatic Fat Changes in Response to Lifestyle Intervention: Central Magnetic Resonance Imaging Randomized Controlled Trial

Introduction: Compelling evidence has linked liver-specific microRNA-122 (miR-122) expression with hepatic fat distribution; however, little is known about the relations between changes in circulating miR-122 and liver fat in response to weight-loss interventions. We aimed to prospectively investigate the association between serum miR-122 and changes of hepatic fat content during 18-month diet and physical activity interventions. Methods: The CENTRAL trial is an 18-month randomized controlled trial among adults with abdominal obesity or dyslipidemia. Subjects were randomly assigned to a low-fat (LF) diet or a Mediterranean/low-carbohydrate (MED/LC) diet. After six months of dietary intervention, each diet group was further randomized into added physical activity groups (LFPA+, MED/LCPA+) or no added physical activity groups (LFPA-, MED/LCPA-) for the following 12 months of intervention. The current study included 220 participants with repeated measurements on serum miR-122 and hepatic fat content (percentage) over 18 months. Results: In the study participants, serum concentration of miR-122 significantly increased from baseline to 18 months, while no difference was observed across the four intervention groups. We found a significant association between miR-122 and hepatic fat percentage at baseline, as per unit increment in log-transformed miR-122 was associated with 3.79 higher hepatic fat content ( P trend&lt;0.001). In addition, we found that a higher elevation in log-transformed miR-122 was associated with a greater reduction in hepatic fat percentage during 18-month interventions, independent of weight loss (β=1.56, P trend=0.002). We also found a significant interaction between changes in miR-122 and baseline fasting plasma glucose with hepatic fat content changes in 18 months ( P interaction=0.02). The association between changes in miR-122 and hepatic fat content changes appeared to be stronger among participants with higher baseline fasting glucose levels than those with lower levels. Conclusion: Our data indicate that participants with higher serum miR-122 levels may benefit more in reduction of hepatic fat content in response to Mediterranean/low-carbohydrate diet and physical activity interventions.

Correlation of serum microRNA-122 level with the levels of Alanine aminotransferase and HBV-DNA in Chronic HBV-infected patients.

Background: The microRNA‐122 (miR‐122) is a liver‐specific microRNA that can be used as a potential molecular marker for predicting liver injury. There is a positive correlation between miR‐122 level in serum and hepatitis B virus (HBV) replication in patients infected with this virus. The present study was conducted to study the clinical importance and expression of miR-122 in asymptomatic and symptomatic patients with HBV infection in comparison to the healthy group. Methods: This study was performed on 60 samples to examine the presence of HBsAg and total HBc antibody (IgM-IgG) using an enzyme-linked immunosorbent assay. HBV-DNA extraction and real time polymerase chain reaction (PCR) assay were performed on all samples via the Real ART HBV LC PCR kit on a LightCycler instrument. RNA was extracted from the serum of all participants. Next, miRNA expression was assessed using quantitative real time reverse-transcription PCR. Also, ALT levels were measured as a surrogate parameter for liver injury using Pars Azmoon Biochemical assay Kit on Hitachi autoanalyzer. The Levene, Kruskal Wallis, Mann-Whitney and Spearman’s correlation tests were used for assessing the differences between the studied groups. Results: Based on the obtained results, miR-122 expression in patients with HBV without clinical symptoms was 1.6 times, while in patients with clinical symptoms was 2.7 times more than the control group (p=0.001). A significant increase was observed in the ALT enzyme of symptomatic patients (p=0.001). HBV DNA in the people with clinical symptoms was higher than 105 copies/mL and in the asymptomatic group was less than 103 copies/mL, suggesting a statistically significant increase in a group with clinical symptoms (p=0.001). Finally, it was found that the miR‐122 serum concentration correlated with HBV DNA and serum ALT (p=0.001). Conclusion: Based on the obtained results, measuring the miR-122 levels can serve as a biomarker and an indicator of hepatitis B replication, especially in cases where ALT levels are unchanged; however, more research and more samples are needed.

Functional Recellularization of Acellular Rat Liver Scaffold by Induced Pluripotent Stem Cells: Molecular Evidence for Wnt/B-Catenin Upregulation.

Background. Liver transplantation remains the only viable therapy for liver failure but has a severely restricted utility. Here, we aimed to decellularize rat livers to form acellular 3D bio-scaffolds suitable for seeding with induced pluripotent cells (iPSCs) as a tool to investigate the role of Wnt/β-catenin signaling in liver development and generation. Methods. Dissected rat livers were randomly divided into three groups: I (control); II (decellularized scaffolds) and III (recellularized scaffolds). Liver decellularization was established via an adapted perfusion procedure and assessed through the measurement of extracellular matrix (ECM) proteins and DNA content. Liver recellularization was assessed through histological examination and measurement of transcript levels of Wnt/β-catenin pathway, hepatogenesis, liver-specific microRNAs and growth factors essential for liver development. Adult rat liver decellularization was confirmed by the maintenance of ECM proteins and persistence of growth factors essential for liver regeneration. Results. iPSCs seeded rat decellularized livers displayed upregulated transcript expression of Wnt/β-catenin pathway-related, growth factors, and liver specification genes. Further, recellularized livers displayed restored liver-specific functions including albumin secretion and urea synthesis. Conclusion. This establishes proof-of-principle for the generation of three-dimensional liver organ scaffolds as grafts and functional re-establishment.

Transduction Properties of an Adenovirus Vector Containing Sequences Complementary to a Liver-Specific microRNA, miR-122a, in the 3'-Untranslated Region of the E4 Gene in Human Hepatocytes from Chimeric Mice with Humanized Liver.

Replication-incompetent adenovirus (Ad) vectors are promising gene delivery vehicles, especially for hepatocytes, due to their superior hepatic tropism; however, in vivo application of an Ad vector often results in hepatotoxicity, mainly due to the leaky expression of Ad genes from the Ad vector genome. In order to reduce the Ad vector-induced hepatotoxicity, we previously developed an Ad vector containing the sequences perfectly complementary to a liver-specific microRNA (miRNA), miR-122a, in the 3'-untranslated region (UTR) of the E4 gene. This improved Ad vector showed a significant reduction in the leaky expression of Ad genes and hepatotoxicity in the mouse liver and primary mouse hepatocytes; however, the safety profiles and transduction properties of this improved Ad vector in human hepatocytes remained to be elucidated. In this study, we examined the transgene expression and safety profiles of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in human hepatocytes from chimeric mice with humanized liver. The transgene expression levels of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene were significantly higher than those of the conventional Ad vectors. The leaky expression levels of Ad genes of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in the primary human hepatocytes were largely reduced, compared with the conventional Ad vectors, resulting in an improvement in Ad vector-induced cytotoxicity. These data indicated that this improved Ad vector was a superior gene delivery vehicle without severe cytotoxicity for not only mouse hepatocytes but also human hepatocytes.

Oleanolic acid inhibits the migration and invasion of hepatocellular carcinoma cells by promoting microRNA-122 expression.

MicroRNAs are emerging as important endogenous regulators of gene function and they are playing an important role in the occurrence and development of cancer. They are also regarded as robust biomarkers of cancer diagnosis and prognosis. Hepatocellular carcinoma (HCC) is a common and complex human malignancy with high mortality and morbidity in the world. MicroRNA-122 (miR-122) is a liver-specific microRNA and is closely associated with HCC metastasis, which makes miR-122 a promising target for drug design and development. In this study, we performed a cell-based screening method for discovering miR-122 activators and found that oleanolic acid (OA), a natural pentacyclic triterpene, specifically increased miR-122 expression in a concentration-dependent manner. Two HCC cell lines (HepG2 and Sk-hep-1 cells) were used to evaluate the effect of OA on cell migration and invasion abilities. The results indicated that OA attenuated the migration and invasion abilities of HCC cells by upregulating miR-122 expression. In addition, OA increased the expression of E-cadherin and decreased the expression of β-catenin, N-cadherin and vimentin. After knocking down miR-122 with miR-122 inhibitor, we found that the effect of OA on these epithelial-to-mesenchymal transition (EMT) related molecules was significantly weakened, indicating OA exhibited anti-EMT effect by increasing the expression of miR-122. These finding may help to better understand the molecular mechanism of OA's anti-metastasis activity.

MiR-122–based therapies select for three distinct resistance mechanisms based on alterations in RNA structure

Hepatitis C virus (HCV) is a positive-sense RNA virus that interacts with a liver-specific microRNA called miR-122. miR-122 binds to two sites in the 5' untranslated region of the viral genome and promotes HCV RNA accumulation. This interaction is important for viral RNA accumulation in cell culture, and miR-122 inhibitors have been shown to be effective at reducing viral titers in chronic HCV-infected patients. Herein, we analyzed resistance-associated variants that were isolated in cell culture or from patients who underwent miR-122 inhibitor-based therapy and discovered three distinct resistance mechanisms all based on changes to the structure of the viral RNA. Specifically, resistance-associated variants promoted riboswitch activity, genome stability, or positive-strand viral RNA synthesis, all in the absence of miR-122. Taken together, these findings provide insight into the mechanism(s) of miR-122-mediated viral RNA accumulation and provide mechanisms of antiviral resistance mediated by changes in RNA structure.

High HBV Load Weakens Predictive Effect of Serum miR-122 on Response to Sorafenib in Hepatocellular Carcinoma Patients.

Background MiR-122 is a liver-specific microRNA. The aim of the study was to explore the association of serum miR-122 with response to sorafenib in hepatitis B virus- (HBV-) related hepatocellular carcinoma (HCC) patients and to further reveal the effect of the virus load on such potential relationship. Methods A total of 588 patients with HCC were retrospectively included. All of them were diagnosed with HBV-related locally advanced HCC and were treated with sorafenib. Therapeutic and prognostic information and other information were collected from medical records. Stored blood specimens that were obtained before sorafenib treatment were adopted to detect miR-122. Results The patients were divided into high-level group and low-level group according to the median of serum miR-122 level, and each group contained 294 patients. During the first 24 weeks after sorafenib treatment, the patients in the high-level group had more opportunities to experience progression-free survival (PFS) and overall survival (OS) than those in the low-level group (HR: 2.47, 95%CI: 1.24∼4.88; HR: 1.20, 95%CI: 1.09∼1.32). In the subgroup analysis, the relationship between serum miR-122 level and overall survival still existed in the patients with relatively lower HBV load (HR: 1.22, 95%CI: 1.09∼1.36), but not in the patients with higher HBV load (HR: 1.12, 95%CI: 0.93∼1.35). Conclusion Higher serum level of miR-122 at baseline was associated with a better response to sorafenib in HBV-related locally advanced HCC patients, and relatively high HBV load weakened such predictive effect mentioned above.

Pathogenesis, MicroRNA-122 Gene-Regulation, and Protective Immune Responses After Acute Equine Hepacivirus Infection.

Background and AimsEquine hepacivirus (EqHV) is phylogenetically the closest relative of HCV and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses.Approach and ResultsSeven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same and, subsequently, a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred nonsterilizing immunity, resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV‐specific T cells were identified. Additionally, an interferon‐stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver‐specific microRNA (miR), miR‐122. Interestingly, we found that EqHV infection sequesters enough miR‐122 to functionally affect gene regulation in the liver. This RNA‐based mechanism thus could have consequences for pathology.ConclusionsEqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.

Preferentially released miR-122 from cyclodextrin-based star copolymer nanoparticle enhances hepatoma chemotherapy by apoptosis induction and cytotoxics efflux inhibition

Chemotherapy, as one of the most commonly used treatment modalities for cancer therapy, provides limited benefits to hepatoma patients, owing to its inefficient delivery as well as the intrinsic chemo-resistance of hepatoma. Bioinformatic analysis identified the therapeutic role of a liver-specific microRNA — miR-122 for enhancing chemo-therapeutic efficacy in hepatoma. Herein, a cyclodextrin-cored star copolymer nanoparticle system (sCDP/DOX/miR-122) is constructed to co-deliver miR-122 with doxorubicin (DOX) for hepatoma therapy. In this nanosystem, miR-122 is condensed by the outer cationic poly (2-(dimethylamino) ethyl methacrylate) chains of sCDP while DOX is accommodated in the inner hydrophobic cyclodextrin cavities, endowing a sequential release manner of miR-122 and DOX. The preferentially released miR-122 not only directly induces cell apoptosis by down regulation of Bcl-w and enhanced p53 activity, but also increases DOX accumulation through inhibiting cytotoxic efflux transporter expression, which realizes synergistic performance on cell inhibition. Moreover, sCDP/DOX/miR-122 displays remarkably increased anti-tumor efficacy in vivo compared to free DOX and sCDP/DOX alone, indicating its great promising in hepatoma therapy.