microRNA‐143‐3p attenuated development of hepatic fibrosis in autoimmune hepatitis through regulation of TAK1 phosphorylation

Autoimmune hepatitis (AIH) is a chronic liver disease due to autoimmune system attacks hepatocytes and causes inflammation and fibrosis. Intracellular signalling and miRNA may play an important role in regulation of liver injury. This study aimed to investigate the potential roles of microRNA 143 in a murine AIH model and a hepatocyte injury model. Murine AIH model was induced by hepatic antigen S100, and hepatocyte injury model was induced by LPS. Mice and AML12 cells were separated into six groups with or without the treatment of miRNA‐143. Inflammation and fibrosis as well as gene expression were examined by different cellular and molecular techniques. The model was successfully established with the elevation of ALT and AST as well as inflammatory and fibrotic markers. Infection or transfection of mir‐143 in mice or hepatocytes significantly attenuated the development of alleviation of hepatocyte injury. Moreover, the study demonstrated phosphorylation of TAK1‐mediated miRNA‐143 regulation of hepatic inflammation and fibrosis as well as hepatocyte injury. Our studies demonstrated a significant role of miRNA‐143 in attenuation of liver injury in AIH mice and hepatocytes. miRNA‐143 regulates inflammation and fibrosis through its regulation of TAK1 phosphorylation, which warrants TAK1 as a target for the development of new therapeutic strategy of autoimmune hepatitis.     

Induction of hepatocyte‐like cells from human umbilical cord‐derived mesenchymal stem cells by defined microRNAs

Generating functional hepatocyte‐like cells (HLCs) from mesenchymal stem cells (MSCs) is of great urgency for bio‐artificial liver support system (BALSS). Previously, we obtained HLCs from human umbilical cord‐derived MSCs by overexpressing seven microRNAs (HLC‐7) and characterized their liver functions in vitro and in vivo. Here, we aimed to screen out the optimal miRNA candidates for hepatic differentiation. We sequentially removed individual miRNAs from the pool and examined the effect of transfection with remainder using RT‐PCR, periodic acid—Schiff (PAS) staining and low‐density lipoprotein (LDL) uptake assays and by assessing their function in liver injury models. Surprisingly, miR‐30a and miR‐1290 were dispensable for hepatic differentiation. The remaining five miRNAs (miR‐122, miR‐148a, miR‐424, miR‐542‐5p and miR‐1246) are essential for this process, because omitting any one from the five‐miRNA combination prevented hepatic trans‐differentiation. We found that HLCs trans‐differentiated from five microRNAs (HLC‐5) expressed high level of hepatic markers and functioned similar to hepatocytes. Intravenous transplantation of HLC‐5 into nude mice with CCl₄‐induced fulminant liver failure and acute liver injury not only improved serum parameters and their liver histology, but also improved survival rate of mice in severe hepatic failure. These data indicated that HLC‐5 functioned similar to HLC‐7 in vitro and in vivo, which have been shown to resemble hepatocytes. Instead of using seven‐miRNA combination, a simplified five‐miRNA combination can be used to obtain functional HLCs in only 7 days. Our study demonstrated an optimized and efficient method for generating functional MSC‐derived HLCs that may serve as an attractive cell alternative for BALSS.     

The miR‐379/miR‐410 cluster at the imprinted Dlk1‐Dio3 domain controls neonatal metabolic adaptation

In mammals, birth entails complex metabolic adjustments essential for neonatal survival. Using a mouse knockout model, we identify crucial biological roles for the miR‐379/miR‐410 cluster within the imprinted Dlk1‐Dio3 region during this metabolic transition. The miR‐379/miR‐410 locus, also named C14MC in humans, is the largest known placental mammal‐specific miRNA cluster, whose 39 miRNA genes are expressed only from the maternal allele. We found that heterozygote pups with a maternal—but not paternal—deletion of the miRNA cluster display partially penetrant neonatal lethality with defects in the maintenance of energy homeostasis. This maladaptive metabolic response is caused, at least in part, by profound changes in the activation of the neonatal hepatic gene expression program, pointing to as yet unidentified regulatory pathways that govern this crucial metabolic transition in the newborn's liver. Not only does our study highlight the physiological importance of miRNA genes that recently evolved in placental mammal lineages but it also unveils additional layers of RNA‐mediated gene regulation at the Dlk1‐Dio3 domain that impose parent‐of‐origin effects on metabolic control at birth and have likely contributed to mammal evolution.     

Exosomes derived from miR‐181‐5p‐modified adipose‐derived mesenchymal stem cells prevent liver fibrosis via autophagy activation

Proliferating hepatic stellate cells (HSCs) respond to liver damage by secreting collagens that form fibrous scar tissue, which can lead to cirrhosis if in appropriately regulated. Advancement of microRNA (miRNA) hepatic therapies has been hampered by difficulties in delivering miRNA to damaged tissue. However, exosomes secreted by adipose‐derived mesenchymal stem cells (ADSCs) can be exploited to deliver miRNAs to HSCs. ADSCs were engineered to overexpress miRNA‐181‐5p (miR‐181‐5p‐ADSCs) to selectively home exosomes to mouse hepatic stellate (HST‐T6) cells or a CCl4‐induced liver fibrosis murine model and compared with non‐targeting control Caenorhabditis elegans miR‐67 (cel‐miR‐67)‐ADSCs. In vitro analysis confirmed that the transfer of miR‐181‐5p from miR‐181‐5p‐ADSCs occurred via secreted exosomal uptake. Exosomes were visualized in HST‐T6 cells using cyc3‐labelled pre‐miRNA‐transfected ADSCs with/without the exosomal inhibitor, GW4869. The effects of miRNA‐181‐5p overexpression on the fibrosis associated STAT3/Bcl‐2/Beclin 1 pathway and components of the extracellular matrix were assessed. Exosomes from miR181‐5p‐ADSCs down‐regulated Stat3 and Bcl‐2 and activated autophagy in the HST‐T6 cells. Furthermore, the up‐regulated expression of fibrotic genes in HST‐T6 cells induced by TGF‐β1 was repressed following the addition of isolated miR181‐5p‐ADSC exosomes compared with miR‐67‐ADSCexosomes. Exosome therapy attenuated liver injury and significantly down‐regulated collagen I, vimentin, α‐SMA and fibronectin in liver, compared with controls. Taken together, the effective anti‐fibrotic function of engineered ADSCs is able to selectively transfer miR‐181‐5p to damaged liver cells and will pave the way for the use of exosome‐ADSCs for therapeutic delivery of miRNA targeting liver disease.     

miR‐19b controls cardiac fibroblast proliferation and migration

Cardiac fibrosis is a fundamental constituent of a variety of cardiac dysfunction, making it a leading cause of death worldwide. However, no effective treatment for cardiac fibrosis is available. Therefore, novel therapeutics for cardiac fibrosis are highly needed. Recently, miR‐19b has been found to be able to protect hydrogen peroxide (H₂O₂)‐induced apoptosis and improve cell survival in H9C2 cardiomyocytes, while down‐regulation of miR‐19b had opposite effects, indicating that increasing miR‐19b may be a new therapeutic strategy for attenuating cellular apoptosis during myocardial ischaemia–reperfusion injury. However, considering the fact that microRNAs might exert a cell‐specific role, it is highly interesting to determine the role of miR‐19b in cardiac fibroblasts. Here, we found that miR‐19b was able to promote cardiac fibroblast proliferation and migration. However, miR‐19b mimics and inhibitors did not modulate the expression level of collagen I. Pten was identified as a target gene of miR‐19b, which was responsible for the effect of miR‐19b in controlling cardiac fibroblast proliferation and migration. Our data suggest that the role of miR‐19b is cell specific, and systemic miR‐19b targeting in cardiac remodelling might be problematic. Therefore, it is highly needed and also urgent to investigate the role of miR‐19b in cardiac remodelling in vivo.     

Analysing the relationship between lncRNA and protein‐coding gene and the role of lncRNA as ceRNA in pulmonary fibrosis

Long non‐coding RNAs (lncRNAs) are involved in various pathophysiologic processes and human diseases. However, their dynamics and corresponding functions in pulmonary fibrosis remain poorly understood. In this study, portions of lncRNAs adjacent or homologous to protein‐coding genes were determined by searching the UCSC genome bioinformatics database. This was found to be potentially useful for exploring lncRNA functions in disease progression. Previous studies showed that competing endogenous RNA (ceRNA) hypothesis is another method to predict lncRNA function. However, little is known about the function of ceRNA in pulmonary fibrosis. In this study, we selected two differentially expressed lncRNAs MRAK088388 and MRAK081523 to explore their regulatory mechanisms. MRAK088388 and MRAK081523 were analysed as long‐intergenic non‐coding RNAs (lincRNAs), and identified as orthologues of mouse lncRNAs AK088388 and AK081523, respectively. qRT‐PCR and in situ hybridization (ISH) showed that they were significantly up‐regulated, and located in the cytoplasm of interstitial lung cells. We also showed that MRAK088388 and N4bp2 had the same miRNA response elements (MREs) for miR‐200, miR‐429, miR‐29, and miR‐30, whereas MRAK081523 and Plxna4 had the same MREs for miR‐218, miR‐141, miR‐98, and let‐7. Moreover, the expression levels of N4bp2 and Plxna4 significantly increased in fibrotic rats, and were highly correlated with those of MRAK088388 and MRAK081523, respectively. Among their shared miRNAs, miR‐29b‐3p and let‐7i‐5p decreased in the model group, and were negatively correlated with the expression of MRAK088388 and MRAK081523, respectively. MRAK088388 and MRAK081523 could regulate N4bp2 and Plxna4 expression by sponging miR‐29b‐3p and let‐7i‐5p, respectively, and possessed regulatory functions as ceRNAs. Thus, our study may provide insights into the functional interactions of lncRNA, miRNA and mRNA, and lead to new theories for the pathogenesis and treatment of pulmonary fibrosis.     

Overexpression of miR‐483‐5p/3p cooperate to inhibit mouse liver fibrosis by suppressing the TGF‐β stimulated HSCs in transgenic mice

The transition from liver fibrosis to hepatocellular carcinoma (HCC) has been suggested to be a continuous and developmental pathological process. MicroRNAs (miRNAs) are recently discovered molecules that regulate the expression of genes involved in liver disease. Many reports demonstrate that miR‐483‐5p and miR‐483‐3p, which originate from miR‐483, are up‐regulated in HCC, and their oncogenic targets have been identified. However, recent studies have suggested that miR‐483‐5p/3p is partially down‐regulated in HCC samples and is down‐regulated in rat liver fibrosis. Therefore, the aberrant expression and function of miR‐483 in liver fibrosis remains elusive. In this study, we demonstrate that overexpression of miR‐483 in vivo inhibits mouse liver fibrosis induced by CCl₄. We demonstrate that miR‐483‐5p/3p acts together to target two pro‐fibrosis factors, platelet‐derived growth factor‐β and tissue inhibitor of metalloproteinase 2, which suppress the activation of hepatic stellate cells (HSC) LX‐2. Our work identifies the pathway that regulates liver fibrosis by inhibiting the activation of HSCs.     

Aldehyde dehydrogenase 2 activation ameliorates CCl4‐induced chronic liver fibrosis in mice by up‐regulating Nrf2/HO‐1 antioxidant pathway

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is critical in the pathogenesis of alcoholic liver cirrhosis. However, the effect of ALHD2 on liver fibrosis remains to be further elucidated. This study aimed to demonstrate whether ALDH2 regulates carbon tetrachloride (CCl₄)‐induced liver fibrosis and to investigate the efficacy of Alda‐1, a specific activator of ALDH2, on attenuating liver fibrosis. ALDH2 expression was increased after chronic CCl₄ exposure. ALDH2 deficiency accentuated CCl₄‐induced liver fibrosis in mice, accompanied by increased expression of collagen 1α1, α‐SMA and TIMP‐1. Moreover, ALDH2 knockout triggered more ROS generation, hepatocyte apoptosis and impaired mitophagy after CCl₄ treatment. In cultured HSC‐T6 cells, ALDH2 knockdown by transfecting with lentivirus vector increased ROS generation and α‐SMA expression in an in vitro hepatocyte fibrosis model using TGF‐β1. ALDH2 overexpression by lentivirus or activation by Alda‐1 administration partly reversed the effect of TGF‐β1, whereas ALDH2 knockdown totally blocked the protective effect of Alda‐1. Furthermore, Alda‐1 administration protected against liver fibrosis in vivo, which might be mediated through up‐regulation of Nrf2/HO‐1 cascade and activation of Parkin‐related mitophagy. These findings indicate that ALDH2 deficiency aggravated CCl₄‐induced hepatic fibrosis through ROS overproduction, increased apoptosis and mitochondrial damage, whereas ALDH2 activation through Alda‐1 administration alleviated hepatic fibrosis partly through activation of the Nrf2/HO‐1 antioxidant pathway and Parkin‐related mitophagy, which indicate ALDH2 as a promising anti‐fibrotic target and Alda‐1 as a potential therapeutic agent in treating CCl₄‐induced liver fibrosis.     

MiR‐122 modification enhances the therapeutic efficacy of adipose tissue‐derived mesenchymal stem cells against liver fibrosis

Mesenchymal stem cell (MSC) transplantation alone may be insufficient for treatment of liver fibrosis because of complicated histopathological changes in the liver. Given that miR‐122 plays an essential role in liver fibrosis by negatively regulating the proliferation and transactivation of hepatic stellate cells (HSCs), this study investigated whether miR‐122 modification can improve the therapeutic efficacy of adipose tissue‐derived MSCs in treating liver fibrosis. MiR‐122‐modified AMSCs (AMSC‐122) were constructed through lentivirus‐mediated transfer of pre‐miR‐122. MiR‐122‐modified AMSCs expressed high level of miR‐122, while they retained their phenotype and differentiation potential as naïve AMSCs. AMSC‐122 more effectively suppressed the proliferation of and collagen maturation in HSCs than scramble miRNA‐modified AMSCs. In addition, AMSC‐derived exosomes mediated the miR‐122 communication between AMSCs and HSCs, further affecting the expression levels of miR‐122 target genes, such as insulin‐like growth factor receptor 1 (IGF1R), Cyclin G(1) (CCNG1) and prolyl‐4‐hydroxylase α1 (P4HA1), which are involved in proliferation of and collagen maturation in HSCs. Moreover, miR‐122 modification enhanced the therapeutic efficacy of AMSCs in the treatment of carbon tetrachloride (CCl₄)‐induced liver fibrosis by suppressing the activation of HSCs and alleviating collagen deposition. Results demonstrate that miR‐122 modification improves the therapeutic efficacy of AMSCs through exosome‐mediated miR‐122 communication; thus, miR‐122 modification is a new potential strategy for treatment of liver fibrosis.     

MicroRNA‐379 suppresses osteosarcoma progression by targeting PDK1

Osteosarcoma is the most common primary bone tumour. Increasing evidence has demonstrated the pathogenic role of microRNA (miRNAs) dysregulation in tumour development. miR‐379 was previously reported to function as an oncogenic or tumour‐suppressing miRNA in a tissue‐dependent manner. However, its function in osteosarcoma remains unknown. In this study, we found that the expression of miR‐379 was downregulated in osteosarcoma tissues and cell lines. Further functional characterization revealed that miR‐379 suppressed osteosarcoma cell proliferation and invasion in vitro and retarded the growth of osteosarcoma xenografts in vivo. Mechanistically, PDK1 was identified as the direct target of miR‐379 in osteosarcoma, in which PDK1 expression was up‐regulated and showed inverse correlation with miR‐379. Enforced expression of PDK1 promoted osteosarcoma cell proliferation and rescued the anti‐proliferative effect of miR‐379. These data suggest that miR‐379 could function as a tumour‐suppressing miRNA via targeting PDK1 in osteosarcoma.     

MicroRNA‐30a ameliorates hepatic fibrosis by inhibiting Beclin1‐mediated autophagy

We explored the role of microRNA‐30a (miR‐30a) and the mechanism involved in hepatic fibrosis. MiR‐30a overexpression was achieved by miR‐30a mimics transfection in hepatic stellate cells (HSCs) (HSC‐T6, LX‐2), and miR‐30a agomir (ago‐miR‐30a) treatment in mice. MiR‐30a levels were measured using TaqMan miRNA assay system, and the localization of miR‐30a was detected by fluorescence in situ hybridization (FISH). The interaction of miR‐30a and Beclin1 was confirmed by dual‐luciferase reporter assay. Autophagic flux was analysed using tandem mRFP‐GFP‐LC3 fluorescence microscopy, electron microscopy and Western blot of LC3‐II/I ratio. MiR‐30a was notably down‐regulated in activated HSCs and LX‐2‐exosomes induced by TGF‐β1; overexpression of miR‐30a down‐regulated extracellular matrix (ECM), such as α‐SMA, TIMP‐1, and Collagen I expression, and suppressed cell viability in HSCs. MiR‐30a was significantly down‐regulated in hepatic fibrosis mice and overexpression of miR‐30a prevented BDL‐induced fibrogenesis, concomitant with the down‐regulation of ECM. MiR‐30a inhibited HSCs autophagy and increased lipid accumulation in HSCs and in mice fibrotic hepatic tissues. MiR‐30a inhibited its downstream effector of Beclin1 by direct targeting its 3′‐UTR region. Moreover, Knock‐down of Beclin1 by small interfering RNA (siRNA) inhibited HSC autophagy and activation in LX‐2 cells. In conclusion, miR‐30a is down‐regulated in hepatic fibrosis models and its overexpression prevents liver fibrogenesis by directly suppressing Beclin1‐mediated autophagy; therefore, miR‐30a may be a new potential therapeutic target for controlling hepatic fibrosis.     

Serum miRNAs are potential biomarkers for the detection of disc degeneration,among which miR‐26a‐5p suppresses Smad1 to regulate disc homeostasis

Disc degeneration is a common clinical condition in which damaged discs cause chronic pain; however, a laboratory diagnosis method for its detection is not available. As circulating miRNAs have potential as biomarkers, their application in disc degeneration has not been explored. Here, we prepared serum miRNAs from a mouse disc degeneration model and performed miRNA‐Seq and quantitative PCR to characterize disc degeneration–associated miRNAs. We identified three miRNAs, including miR‐26a‐5p, miR‐122‐5p and miR‐215‐5p, undergoing perturbation during the pathogenesis of disc degeneration. Specifically, the levels of miR‐26a‐5p in the serum demonstrated steady increases in the model of disc degeneration, compared with those in the pre‐injury samples of younger age or compared with normal controls of the same age but without disc degeneration, whereas the miRNAs miR‐122‐5p and miR‐215‐5p exhibited lower expression in post‐injury samples than in their counterparts without the surgery. Moreover, we found that miR‐26a‐5p targets Smad1 expression, and Smad1 negatively regulates Vegfa expression in disc cells, and thus, miR‐26a‐5p promotes disc degeneration. In summary, we established a method that consistently profiles circulating miRNAs and identified multiple miRNAs as promising biomarkers for disc degeneration, among which miR‐26a‐5p enhances VEGF expression during disc degeneration through targeting Smad1 signalling.     

miRNA‐532‐5p functions as an oncogenic microRNA in human gastric cancer by directly targeting RUNX3

Accumulating data reveal that microRNAs are involved in gastric carcinogenesis. To date, no information was reported about the function and regulatory mechanism of miR‐532‐5p in human gastric cancer (GC). Thus, our study aims to determine the role and regulation of miR‐532‐5p in GC. Here, we found that transient and stable overexpression of miR‐532‐5p dramatically increased the potential of colony formation and migration of GC cells, decreased the percentage of cells in G1 phase and cell apoptosis in vitro, and increased the weight of mice lungs and number of lung xenografts in vivo. Gain‐of‐function, loss‐of‐function and luciferase activity assays demonstrated that miR‐532‐5p negatively regulated the expression of RUNX3 and its targets directly. We also found that miR‐532‐5p level was negatively correlated with RUNX3 gene expression in various GC cell lines. Our results indicate that miR‐532‐5p functions as an oncogenic miRNA by promoting cell growth, migration and invasion in human GC cells.     

MicroRNA‐30e‐5p promotes cell growth by targeting PTPN13 and indicates poor survival and recurrence in lung adenocarcinoma

Aberrant microRNA expression is involved in the regulation of various cellular processes, such as proliferation and metastasis in multiple diseases including cancers. MicroRNA‐30e‐5p (miR‐30e) was previously reported as an oncogenic or tumour suppressing miRNA in some malignancies, but its function in lung adenocarcinoma (LAC) remains largely undefined. In this study, we found that the expression of miR‐30e was increased in LAC tissues and cell lines, associated with tumour size and represented an independent prognostic factor for overall survival and recurrence of LAC patients. Further functional experiments showed that knockdown of miR‐30e suppressed cell growth while its overexpression promoted growth of LAC cells and xenografts in vitro and in vivo. Mechanistically, PTPN13 was identified as the direct target of miR‐30e in LAC, in which PTPN13 expression was down‐regulated in LAC tissues and showed the inverse correlation with miR‐30e expression. Overexpression of PTPN13 inhibited cell growth and rescued the proliferation‐promoting effect of miR‐30e through inhibition of the EGFR signalling. Altogether, our findings suggest that miR‐30e could function as an oncogene in LAC via targeting PTPN13 and act as a potential therapeutic target for treating LAC.     

Alcohol‐mediated miR‐34a modulates hepatocyte growth and apoptosis

MicroRNAs (miRs) have been recently shown to be heavily involved in the development of alcoholic liver disease (ALD) and suggested as a potential therapeutic target in ALD. The miR‐34a was consistently reported to be significantly elevated in several ALD rodent models, but it remains unclear how miR‐34a modulates the cellular behaviours of hepatocytes in ALD development and progression. This study aims to characterize alcohol‐induced miR‐34a impact on hepatocytes growth and apoptosis. The miRNA array was performed to assess changes in miRNA after chronic alcohol feeding. Liver and blood samples were used to examine ALD progression. The miR‐34a was overexpressed in human hepatocytes to evaluate its impact on cell growth and apoptosis. Real‐time quantitative PCR and Western blot were used to determine the growth and apoptosis molecular signalling pathways associated with miR‐34a. Alcohol feeding significantly promoted fatty liver progression, serum ALT levels, apoptosis and miR‐34a expression in rat liver. Overexpression of miR‐34a in human hepatocytes suppressed cell growth signallings, including c‐Met, cyclin D1 and cyclin‐dependent kinase 6 (CDK6). The miR‐34a might also inhibit the expression of sirtuin 1 (Sirt1) and its target, B‐cell lymphoma 2. Interestingly, the expression of miR‐34a reverses the suppressive effects of ethanol on cell growth. But, miR‐34a promotes hepatocyte senescence and apoptosis. Although the miR‐34a‐mediated down‐regulation of cell growth‐associated genes may contribute to cell growth retardation, other miR‐34a targets, such as Sirt1, may reverse this phenotype. Future studies will be needed to clarify the role of miR‐34a in ALD progression.