肝脏特异性miR-122的分子生物学特性及功能

miR-122是在肝脏特异高表达的一种microRNA。研究表明：生理状态下,miR-122 在调控肝脏的细胞发育、诱导细胞分化、调节细胞代谢、参与肝细胞应急应答等生命活动过程中发挥重要作用;而在病理状态下,miR-122 与丙型肝炎病毒（HCV）和肝细胞肝癌（HCC）密切相关,可能促进HCV RNA 复制,并在HCC发生、发展过程中发挥抑癌基因样作用,可能对HCC 临床诊断和预后具有重要价值。鉴于miR-122 参与调控肝脏生理及肝脏重大疾病的发生、发展等过程,文章详细阐述并讨论miR-122 在肝脏中的生物学特性和功能,以及可能的作用机制。肝脏特异性miR-122 有可能作为治疗人类肝脏疾病的关键靶点。     

Regulation and biological function of the liver-specific miR-122

miRNAs (microRNAs) are important regulators of gene expression. In higher eukaryotes, the tightly controlled expression of different miRNAs, each of which regulates multiple target mRNAs, is crucial for the maintenance of tissue type and the control of differentiation. miR-122 is a highly liver-specific miRNA that is important in hepatitis C virus infection, cholesterol metabolism and hepatocellular carcinoma. In the present review, we discuss the effects of miR-122 on liver physiology and pathology. Recent evidence of pathways involved in the regulation of miR-122 expression is also considered.     

Liver-specific microRNA-122: Biogenesis and function

microRNA-122 (miR-122) was one of the first examples of a tissue-specific miRNA. It is highly expressed in liver, where it constitutes 70% of the total miRNA pool. miR-122 expression is specific to the vertebrate lineage, where the sequence of the mature miRNA is completely conserved. miR-122 is a target for extensive study due to its association with cholesterol metabolism and hepatocellular carcinoma, and its important role in promoting hepatitis C virus (HCV) replication. This review will discuss the biogenesis and function of miR-122.     

MicroRNA 122对肝癌细胞基因表达谱的影响

为研究microRNA（miR-122）对肝癌细胞Hep3B基因表达谱的影响,并探讨其在肝癌发过程中的可能作用,构建了miR-122稳定高表达的Hep3B细胞,利用基因表达谱芯片技术筛选得到和对照组细胞比较的差异表达基因.研究结果显示,2倍以上变化的差异表达基因有490个,其中上调的有345个,下调的有145个.这些基因中有16个与肿瘤发生相关,其它基因涉及细胞周期、信号转导、细胞凋亡和细胞增殖分化等众多生物学过程.这些结果提示,miR-122可能在肝癌发生的过程中发挥作用,并可能与这些差异表达基因密切相关.另外,还结合生物信息学方法,在下调表达的基因中预测了miR-122可能直接作用的靶基因.本研究初步探讨了miR-122在肝癌细胞中的生物学功能,为进一步研究miR-122在肝癌发生中的作用奠定了基础,同时也为miRNA的生物学功能及其作用机制的研究提供了一些参考.     

Delivery of microRNA-21-sponge and pre-microRNA-122 by MS2 virus-like particles to therapeutically target hepatocellular carcinoma cells

MicroRNAs are related to the development of hepatocellular carcinoma and can serve as potential therapeutic targets. Therapeutic strategies increasing tumor-suppressive microRNAs and reducing oncogenic microRNAs have been developed. Herein, the effects of simultaneously altering two microRNAs using MS2 virus-like particles were studied. The sequences of microRNA-21-sponge and pre-microRNA-122 were connected and cloned into a virus-like particle expression vector. Virus-like particles containing microRNA-21-sponge and pre-microRNA-122 sequences were prepared and crosslinked with a cell-specific peptide targeting hepatocellular carcinoma cells. Delivery effects were studied using RT-qPCR and functional assays to investigate the level of target mRNAs, cell toxicity, and the effects of proliferation, invasion, and migration. Virus-like particles delivered miR-21-sponge into cells, with the Ct value reaching 10 at most. The linked pre-miR-122 was processed into mature miR-122. The mRNA targets of miR-21 were derepressed as predicted and upregulated 1.2–2.8-fold, and the expression of proteins was elevated correspondingly. Proliferation, migration, and invasion of HCC cells were inhibited by miR-21-sponge. Simultaneous delivery of miR-21-sponge and miR-122 further decreased proliferation, migration, and invasion by up to 34%, 63%, and 65%, respectively. And the combination promoted the apoptosis of HCC cells. In conclusion, delivering miR-21-sponge and miR-122 using virus-like particles modified by cell-specific peptides is an effective and convenient strategy to correct microRNA dysregulation in hepatocellular carcinoma cells and is a promising therapeutic strategy for hepatocellular carcinoma.     

High-throughput luciferase reporter assay for small-molecule inhibitors of microRNA function

MicroRNAs (miRNAs) are endogenous, single-stranded, noncoding RNAs of 21 to 23 nucleotides that regulate gene expression, typically by binding the 3' untranslated regions of target messenger RNAs. It is estimated that miRNAs are involved in the regulation of 30% of all genes and almost every genetic pathway. Recently, the misregulation of miRNAs has been linked to various human diseases including cancer and viral infections, identifying miRNAs as potential targets for drug discovery. Thus, small-molecule modifiers of miRNAs could serve as lead structures for the development of new therapeutic agents and be useful tools in the elucidation of detailed mechanisms of miRNA function. As a result, we have developed a high-throughput screen for potential small-molecule regulators of the liver-specific microRNA miR-122, which is involved in hepatocellular carcinoma development and hepatitis C virus infection. Our small-molecule screen employs a Huh7 human hepatoma cell line stably transfected with a Renilla luciferase sensor for endogenous miR-122. The assay was optimized and validated using an miR-122 antisense agent and a previously identified small-molecule miR-122 inhibitor. The described reporter assay will enable the high-throughput screening of small-molecule miR-122 inhibitors and can be readily extended to other miRNAs.     

miR-122 Regulates Tumorigenesis in Hepatocellular Carcinoma by Targeting AKT3

MicroRNAs (miRNAs) have been implicated in the orchestration of diverse cellular processes including differentiation, proliferation, and apoptosis and are believed to play pivotal roles as oncogenes and tumor suppressors. miR-122, a liver specific miRNA, is significantly down-regulated in most hepatocellular carcinomas (HCCs) but its role in tumorigenesis remains poorly understood. Here we identify AKT3 as a novel and direct target of miR-122. Restoration of miR-122 expression in HCC cell lines decreases AKT3 levels, inhibits cell migration and proliferation, and induces apoptosis. These anti-tumor phenotypes can be rescued by reconstitution of AKT3 expression indicating the essential role of AKT3 in miR-122 mediated HCC transformation. In vivo, restoration of miR-122 completely inhibited xenograft growth of HCC tumor in mice. Our data strongly suggest that miR-122 is a tumor suppressor that targets AKT3 to regulate tumorigenesis in HCCs and a potential therapeutic candidate for liver cancer.     

miR-122 Targets Pyruvate Kinase M2 and Affects Metabolism of Hepatocellular Carcinoma

In contrast to normal differentiated cells that depend on mitochondrial oxidative phosphorylation for energy production, cancer cells have evolved to utilize aerobic glycolysis (Warburg’s effect), with benefit of providing intermediates for biomass production. MicroRNA-122 (miR-122) is highly expressed in normal liver tissue regulating a wide variety of biological processes including cellular metabolism, but is reduced in hepatocellular carcinoma (HCC). Overexpression of miR-122 was shown to inhibit cancer cell proliferation, metastasis, and increase chemosensitivity, but its functions in cancer metabolism remains unknown. The present study aims to identify the miR-122 targeted genes and to investigate the associated regulatory mechanisms in HCC metabolism. We found the ectopic overexpression of miR-122 affected metabolic activities of HCC cells, evidenced by the reduced lactate production and increased oxygen consumption. Integrated gene expression analysis in a cohort of 94 HCC tissues revealed miR-122 level tightly associated with a battery of glycolytic genes, in which pyruvate kinase (PK) gene showed the strongest anti-correlation coefficient (Pearson r = −0.6938, p = <0.0001). In addition, reduced PK level was significantly associated with poor clinical outcomes of HCC patients. We found isoform M2 (PKM2) is the dominant form highly expressed in HCC and is a direct target of miR-122, as overexpression of miR-122 reduced both the mRNA and protein levels of PKM2, whereas PKM2 re-expression abrogated the miR-122-mediated glycolytic activities. The present study demonstrated the regulatory role of miR-122 on PKM2 in HCC, having an implication of therapeutic intervention targeting cancer metabolic pathways.     

MicroRNA-122 Triggers Mesenchymal-Epithelial Transition and Suppresses Hepatocellular Carcinoma Cell Motility and Invasion by Targeting RhoA

The loss of microRNA-122 (miR-122) expression is strongly associated with increased invasion and metastasis, and poor prognosis of hepatocellular carcinoma (HCC), however, the underlying mechanisms remain poorly understood. In the present study, we observed that miR-122 over-expression in HCC cell lines Sk-hep-1 and Bel-7402 triggered the mesenchymal-epithelial transition (MET), as demonstrated by epithelial-like morphological changes, up-regulated epithelial proteins (E-cadherin, ZO-1, α-catenin, occludin, BVES, and MST4), and down-regulated mesenchymal proteins (vimentin and fibronectin). The over-expression of miRNA-122 also caused cytoskeleton disruption, RhoA/Rock pathway inactivation, enhanced cell adhesion, and suppression of migration and invasion of Sk-hep-1 and Bel-7402 cells, whereas, these effects could be reversed through miR-122 inhibition. Additional studies demonstrated that the inhibition of wild-type RhoA function induced MET and inhibited cell migration and invasion, while RhoA over-expression reversed miR-122-induced MET and inhibition of migration and invasion of HCC cells, suggesting that miR-122 induced MET and suppressed the migration and invasion of HCC cells by targeting RhoA. Moreover, our results demonstrated that HNF4α up-regulated its target gene miR-122 that subsequently induced MET and inhibited cell migration and invasion, whereas miR-122 inhibition reversed these HNF4α-induced phenotypes. These results revealed functional and mechanistic links among the tumor suppressors HNF4α, miR-122, and RhoA in EMT and invasive and metastatic phenotypes of HCC. Taken together, our study provides the first evidence that the HNF4α/miR-122/RhoA axis negatively regulates EMT and the migration and invasion of HCC cells.     

Expression of microRNA miR-122 facilitates an efficient replication in nonhepatic cells upon infection with hepatitis C virus

Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly.     

microRNAs在肝病中的研究进展

非编码小分子RNA(miRNA)调节肝脏生物学功能,大量实验表明,小分子RNA(miRNA)对肝脏病理学中有作用。本文概述了miRNA在肝炎、肝硬化肝病领域的进展。microRNA-122是肝细胞中最丰富的微小RNA,在丙型肝炎病毒复制中起着非常明确的作用。实验数据显示,microRNA-122亦可作为一种可行性的靶向治疗。microRNA-122在其他肝脏疾病中亦有作用。大量研究证明,与肝炎有关的其他类型miRNA的重要调节潜力与酒精性肝炎、代谢综合征和自身免疫过程有关。此外,在动物模型和人类研究中,miRNA系列与肝脏纤维化进程有关联。miRNA在肝脏中的功能与细胞分布重要性以及miRNA潜在地作为细胞与器官联系纽带,循环miRNA作为肝损伤、肝癌疾病早期和进展的生物学标志,这些是值得我们讨论的。重要的是:miRNA在肝脏中的调节作用,代表着未来肝病治疗医疗技术中的一种新方法。     

MicroRNA-31 regulated by the extracellular regulated kinase is involved in vascular smooth muscle cell growth via large tumor suppressor homolog 2

Aberrant growth of vascular smooth muscle cells (VSMCs) is a major cellular event in the pathogenesis of many proliferative vascular diseases. Recently, microRNA-31 (miR-31) has been found to play a critical role in cancer cell proliferation. However, the biological role of miR-31 in VSMC growth and the mechanisms involved are currently unknown. In the present study, the expression of rat mature miR-31 (rno-miR-31) was determined in cultured VSMCs and in rat carotid arteries. We identified that rno-miR-31 is an abundant miRNA in VSMCs, and its expression was significantly increased in proliferative VSMCs and in vascular walls with neointimal growth. The up-regulation of rno-miR-31 in proliferative VSMCs was inhibited by the inhibitor of mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK). By both gain-of-function and loss-of-function approaches, we demonstrated that rno-miR-31 had a proproliferative effect on VSMCs. We further identified that LATS2 (large tumor suppressor homolog 2) is a downstream target gene product of rno-miR-31 that is involved in rno-miR-31-mediated effect on VSMC proliferation. The LATS2 as a target gene protein of rno-miR-31 is verified in vivo in balloon-injured rat carotid arteries. The results suggest that MAPK/ERK/miR-31/LATS2 may represent a novel signaling pathway in VSMC growth. miR-31 is able to enhance VSMC proliferation via its downstream target gene product, LATS2.     

A two-dimensional electrophoresis preliminary approach to human hepatocarcinoma differentiation induced by PPAR-agonists

Adopting biochemical and proteomic approaches, we investigated the effect of some PPAR-agonists, a new class of differentiating agents, on human hepatocellular carcinoma Hep-G2 cell line. Cancer differentiation was assayed by checking albumin, transferrin and alpha-fetoprotein synthesis. Cell metabolism was studied by NMR spectroscopy of cell culture supernatants and by evaluation of mitochondrial respiratory chain enzyme activities. The two dimensional electrophoresis approach was employed to analyze modifications in the expression of cellular proteins linked to cell phenotype differentiation in the attempt to identify potential diagnostic and prognostic biomarkers of hepatocellular carcinoma. Results indicate that PPAR-agonists are able to act as differentiating inducers in human hepatocellular carcinoma Hep-G2 cell line as well as to inhibit mitochondrial respiratory chain Complex I, provoking a selective derangement of cellular oxidative metabolism. Lastly, two dimensional electrophoresis showed interesting modifications in the pattern of expression of cellular proteins that confirm biochemical data (increase in albumin and transferrin, decrease of alpha-fetoprotein synthesis) and, moreover, emphasize the meaning of these data by the increase of spots indicatively ascribed to HSP70 and catalase.     

miR-224 在原发性肝癌患者血清中的表达及意义

目的：原发性肝癌(primary hepatocellular carcinoma, PHC) 作为常见的恶性程度极高的肿瘤,严重威胁着人类的生命。 miR-224 是近年来发现的一个肿瘤相关miRNA 分子,在肿瘤的发生及发展过程中发挥着重要的作用。本研究通过测定原发性肝 癌患者血清中miR-224 的表达水平,探讨血清miR-224 与原发性肝癌预后的关系。方法：采用实时荧光定量RT-PCR (Real-time RT-PCR)方法,分别检测40 例原发性肝癌患者,20例慢性肝炎患者,20 例慢性肝硬化患者及20 例正常人的血清标本中miR-224 的表达水平。分析血清miR-224 的表达水平与AFP和MMP-9的相关性。结果：原发性肝癌患者血清miR-224 的表达水平明显高 于正常人、慢性肝炎和慢性肝硬化患者(P<0.05)。皮尔森相关分析结果显示原发性肝癌患者血清miR-224 的表达与AFP 和 MMP-9 呈正相关。血清miR-224 低表达组术后复发/转移率显著低于高表达组,术后生存率则高于高表达组(P<0.01)。结论： miR-224 在原发性肝癌患者的血清中呈高表达,其血清表达水平与原发性肝癌的临床预后密切相关。这提示我们,miR-224 可能 成为新的原发性肝癌检测标记物和潜在的原发性肝癌预后分子标志物。     

Disease-linked microRNA-21 exhibits drastically reduced mRNA binding and silencing activity in healthy mouse liver

MicroRNAs (miRNAs) bind to mRNAs and fine-tune protein output by affecting mRNA stability and/or translation. miR-21 is a ubiquitous, highly abundant, and stress-responsive miRNA linked to several diseases, including cancer, fibrosis, and inflammation. Although the RNA silencing activity of miR-21 in diseased cells has been well documented, the roles of miR-21 under healthy cellular conditions are not well understood. Here, we show that pharmacological inhibition or genetic deletion of miR-21 in healthy mouse liver has little impact on regulation of canonical seed-matched mRNAs and only a limited number of genes enriched in stress response pathways. These surprisingly weak and selective regulatory effects on known and predicted target mRNAs contrast with those of other abundant liver miRNAs such as miR-122 and let-7. Moreover, miR-21 shows greatly reduced binding to polysome-associated target mRNAs compared to miR-122 and let-7. Bioinformatic analysis suggests that reduced thermodynamic stability of seed pairing and target binding may contribute to this deficiency of miR-21. Significantly, these trends are reversed in human cervical carcinoma (HeLa) cells, where miRNAs including miR-21 show enhanced target binding within polysomes and where miR-21 triggers strong degradative activity toward target mRNAs. Taken together, our results suggest that, under normal cellular conditions in liver, miR-21 activity is maintained below a threshold required for binding and silencing most of its targets. Consequently, enhanced association with polysome-associated mRNA is likely to explain in part the gain of miR-21 function often found in diseased or stressed cells.     

MiR-122 Inhibits Cell Proliferation and Tumorigenesis of Breast Cancer by Targeting IGF1R

miRNAs are emerging as critical regulators in carcinogenesis and tumor progression. Recently, microRNA-122 (miR-122) has been proved to play an important role in hepatocellular carcinoma, but its functions in the context of breast cancer (BC) remain unknown. In this study, we report that miR-122 is commonly downregulated in BC specimens and BC cell lines with important functional consequences. Overexpression of miR-122 not only dramatically suppressed cell proliferation, colony formation by inducing G1-phase cell-cycle arrest in vitro, but also reduced tumorigenicity in vivo. We then screened and identified a novel miR-122 target, insulin-like growth factor 1 receptor (IGF1R), and it was further confirmed by luciferase assay. Overexpression of miR-122 would specifically and markedly reduce its expression. Similar to the restoring miR-122 expression, IGF1R downregulation suppressed cell growth and cell-cycle progression, whereas IGF1R overexpression rescued the suppressive effect of miR-122. To identify the mechanisms, we investigated the Akt/mTOR/p70S6K pathway and found that the expression of Akt, mTOR and p70S6K were suppressed, whereas re-expression of IGF1R which did not contain the 3′UTR totally reversed the inhibition of Akt/mTOR/p70S6K signal pathway profile. We also identified a novel, putative miR-122 target gene, PI3CG, a member of PI3K family, which further suggests miR-122 may be a key regulator of the PI3K/Akt pathway. In clinical specimens, IGF1R was widely overexpressed and its mRNA levels were inversely correlated with miR-122 expression. Taken together, our results demonstrate that miR-122 functions as a tumor suppressor and plays an important role in inhibiting the tumorigenesis through targeting IGF1R and regulating PI3K/Akt/mTOR/p70S6K pathway. Given these, miR-122 may serve as a novel therapeutic or diagnostic/prognostic-target for treating BC.     

MicroRNAs Involved in Skeletal Muscle Differentiation

MicroRNAs (miRNAs) negatively regulate gene expression by promoting degradation of target mRNAs or inhibiting their translation. Previous studies have expanded our understanding that miRNAs play an important role in myogenesis and have a big impact on muscle mass, muscle fiber type and muscle-related diseases. The muscle-specific miRNAs, miR-206, miR-1 and miR-133, are among the most studied and best characterized miRNAs in skeletal muscle differentiation. They have a profound influence on multiple muscle differ-entiation processes, such as alternative splicing, DNA synthesis, and cell apoptosis. Many non-muscle-specific miRNAs are also required for the differentiation of muscle through interaction with myogenic factors. Studying the regulatory mechanisms of these miRNAs in muscle differentiation will extend our knowledge of miRNAs in muscle biology and will improve our understanding of the myogenesis regulation.