Dysregulated microRNAs in the pathogenesis and progression of cervical neoplasm

MicroRNAs (miRNAs) play an important role in a variety of physiological as well as pathophysiological processes, including carcinogenesis. The aim of this study is to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to unveil individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues and compared to normal cervical epithelium. Unsupervised hierarchical clustering of the miRNA expression pattern displayed a distinct separation between the CIN and normal cervical epithelium samples. Supervised analysis identified 12 highly differentially regulated miRNAs, including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203, which distinguished the high-grade CIN specimens from normal cervical epithelium. This miRNA signature was further validated by an independent set of high-grade CIN cases. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma from normal controls. Target prediction analysis revealed that these dysregulated miRNAs mainly control apoptosis signaling pathways and cell cycle regulation. These findings contribute to understanding the role of microRNAs in the pathogenesis and progression of cervical neoplasm at the molecular level.     

MicroRNA-146a modulates TGF-beta1-induced hepatic stellate cell proliferation by targeting SMAD4

Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. MicroRNAs (miRNAs) have recently been shown to regulate cell proliferation, differentiation, and apoptosis. The involvement of miRNAs and their roles in TGF-β1-induced HSC activation remains largely unknown. Our study found that the expression of miR-146a was downregulated in HSC in response to TGF-β1 stimulation in dose-dependent manner by one-step real-time quantitative PCR. Moreover, we sought to examine whether miR-146a became dysregulated in CCl(4)-induced hepatic fibrosis in rats. Our study revealed that miR-146a was downregulated in liver fibrotic tissues. In addition, The HSC transfected with miR-146a mimics exhibited attendated TGF-β1-induced α-smooth muscle actin (α-SMA) expression compared with the control. Furthermore, overexpression of miR-146a suppressed TGF-β-induced HSC proliferation, and increased HSC apoptosis. Bioinformatics analyses predict that SMAD4 is the potential target of miR-146a. MiR-146a overexpression in TGF-β1-treated HSC did not decrease target mRNA levels, but significantly reduced target protein expression. These results suggested that miR-146a may function as a novel regulator to modulate HSC activation during TGF-β1 induction by targeting SMAD4.     

MicroRNA signatures associated with thioacetamide-induced liver fibrosis in mice

Multiple etiologies of liver injury are associated with fibrosis in which the key event is the activation of hepatic stellate cells (HSCs). Although microRNAs (miRNAs) are reportedly involved in fibrogenesis, the complete array of miRNA signatures associated with the disease has yet to be elucidated. Here, deep sequencing analysis revealed that compared to controls, 80 miRNAs were upregulated and 21 miRNAs were downregulated significantly in the thioacetamide (TAA)-induced mouse fibrotic liver. Interestingly, 58 of the upregulated miRNAs were localized to an oncogenic miRNA megacluster upregulated in liver cancer. Differential expression of some of the TAA-responsive miRNAs was confirmed, and their human orthologs were similarly deregulated in TGF-β1-activated HSCs. Moreover, a functional analysis of the experimentally validated high-confidence miRNA targets revealed significant enrichment for the GO terms and KEGG pathways involved in HSC activation and liver fibrogenesis. This is the first comprehensive report of miRNAs profiles during TAA-induced mouse liver fibrosis.     

Differential expression of microRNAs in mouse liver under aberrant energy metabolic status

Despite years of effort, exact pathogenesis of nonalcoholic fatty liver disease (NAFLD) remains obscure. To gain an insight into the regulatory roles of microRNAs (miRNAs) in aberrant energy metabolic status and pathogenesis of NAFLD, we analyzed the expression of miRNAs in livers of ob/ob mice, streptozotocin (STZ)-induced type 1 diabetic mice, and normal C57BL/6 mice by miRNA microarray. Compared with normal C57BL/6 mice, ob/ob mice showed upregulation of eight miRNAs and downregulation of four miRNAs in fatty livers. Upregulation of miR-34a and downregulation of miR-122 was found in livers of STZ-induced diabetic mice. These results demonstrate that distinct miRNAs are strongly dysregulated in NAFLD and hyperglycemia. Comparison between miRNA expressions in livers of ob/ob mice and STZ-administered mice further revealed upregulation of four miRNAs and downregulation of two miRNAs in livers of ob/ob mice, indicating that these miRNAs may represent a molecular signature of NAFLD. A distinctive miRNA expression pattern was identified in ob/ob mouse liver, and hierarchical clustering of this pattern could clearly discriminate ob/ob mice from either normal C57BL/6 mice or STZ-administered mice. These findings suggest an important role of miRNAs in hepatic energy metabolism and implicate the participation of miRNAs in the pathophysiological processes of NAFLD.     

Hepato-specific microRNA-122 facilitates accumulation of newly synthesized miRNA through regulating PRKRA

microRNAs (miRNAs) are a versatile class of non-coding RNAs involved in regulation of various biological processes. miRNA-122 (miR-122) is specifically and abundantly expressed in human liver. In this study, we employed 3'-end biotinylated synthetic miR-122 to identify its targets based on affinity purification. Quantitative RT-PCR analysis of the affinity purified RNAs demonstrated a specific enrichment of several known miR-122 targets such as CAT-1 (also called SLC7A1), ADAM17 and BCL-w. Using microarray analysis of affinity purified RNAs, we also discovered many candidate target genes of miR-122. Among these candidates, we confirmed that protein kinase, interferon-inducible double-stranded RNA-dependent activator (PRKRA), a Dicer-interacting protein, is a direct target gene of miR-122. miRNA quantitative-RT-PCR results indicated that miR-122 and small interfering RNA against PRKRA may facilitate the accumulation of newly synthesized miRNAs but did not detectably affect endogenous miRNAs levels. Our findings will lead to further understanding of multiple functions of this hepato-specific miRNA. We conclude that miR-122 could repress PRKRA expression and facilitate accumulation of newly synthesized miRNAs.     

Hypoxia-induced down-regulation of microRNA-34a promotes EMT by targeting the Notch signaling pathway in tubular epithelial cells

Background

Hypoxia-induced renal tubular cell epithelial–mesenchymal transition (EMT) is an important event leading to renal fibrosis. MicroRNAs (miRNAs) are small non-coding RNA molecules that bind to their mRNA targets, thereby leading to translational repression. The role of miRNA in hypoxia-induced EMT is largely unknown.

Methodology/Principal Findings

miRNA profiling was performed for the identification of differentially expressed miRNAs in HK-2 cells under normal and low oxygen, and the results were then verified by quantitative real time RT-PCR (qRT-PCR). The function of miRNAs in hypoxia-induced renal tubular cell EMT was assessed by the transfection of specific miRNA inhibitors and mimics. Luciferase reporter gene assays and western blot analysis were performed to validate the target genes of miR-34a. siRNA against Jagged1 was designed to investigate the role of the miR-34a-Notch pathway in hypoxia induced renal tubular cell EMT. miRNA-34a was identified as being downregulated in hypoxic renal tubular epithelial cells. Inhibition of miR-34a expression in HK-2 cells, which highly express endogenous miR-34a, promoted a mesenchymal phenotype accompanied by reduced expression of the epithelial marker Z0-1, E-cadherin and increased expression of the mesenchymal markers α-SMA and vimentin. Conversely, miR-34a mimics effectively prevented hypoxia-induced EMT. Transfection of miRNA-34a in HK-2 cells under hypoxia abolished hypoxia-induced expression of Notch1 and Jagged1 as well as Notch downstream signals, such as snail. Western blot analysis and luciferase reporter gene assays showed direct evidence for miR-34a targeting Notch1 and Jagged1. siRNAs against Jagged1 or Notch1 effectively prevented miR-34a inhibitor-induced tubular epithelial cell EMT.

Conclusions/Significance

Our study provides evidence that the hypoxia-induced decrease of miR-34a expression could promote EMT in renal tubular epithelial cells by directly targeting Notch1 and Jagged1, and subsequently, Notch downstream signaling.     

miR-15b and miR-16 regulate TNF mediated hepatocyte apoptosis via BCL2 in acute liver failure

Acute liver failure (ALF) still has an unacceptable high mortality rate, despite substantial improvements with multidisciplinary care. The precise underlying mechanism of ALF remains to be explored. It has been reported that microRNAs (miRNAs) are novel regulators in a number of liver diseases, but the role of miRNAs in the development of ALF is not fully understood. An ALF murine model was generated by ip injection of D: -GalN/LPS, which was confirmed with histopathology and biochemistry. The hepatic miRNA expression profile in ALF was determined by microarray and verified by qRT-PCR. The functions and signal pathways of the targeted genes of these deregulated miRNAs were predicted, using bioinformatics analysis. The possible underlying mechanism was investigated by exploring the relationship between miRNA modification and hepatocyte apoptosis. There were a total of 95 significantly changed miRNAs in ALF compared to mock-treated (P < 0.01). Among these 95 miRNAs, 20 were up-regulated and 26 were down-regulated at both 5 and 7 h time points. Bioinformatics analysis predicted that some of these 46 miRNAs were involved in apoptosis. Among the up-regulated miRNAs involved in apoptosis, miR-15b and miR-16 showed the highest enrichment and targeted the common anti-apoptotic gene, BCL2. Our in vitro data demonstrated that miR-15b and/or miR-16 regulated BCL2 at the protein level. Inhibition of miR-15b and/or miR-16 reduced hepatic apoptosis and TNF production. These data suggest that miR-15b and miR-16 regulate TNF mediated hepatic apoptosis via BCL2 during ALF, and may shed light on the development of a therapeutic strategy for treatment of ALF.     

miR-93通过靶定PTEN基因增加肝癌细胞对奥沙利铂的耐药性

微小RNA（miRNA）参与了肿瘤的耐药过程.本研究通过建立对奥沙利铂 (oxaliplatin,Oxa)耐药的肝癌细胞系BEL-7402/Oxa和Hep-3B/Oxa,利用miRNA芯片结合实时荧光定量PCR的方法,筛选得到数个参与肝癌细胞对奥沙利铂耐药的miRNA 分子,其中miR-93表达增加最为明显.MTT实验发现,增加肝癌细胞株中miR-93的表达可以增强其对奥沙利铂的耐药性.进一步结合生物信息学、荧光报告载体及免疫印迹实验,证实miR-93通过靶定抑癌基因PTEN增加肝癌细胞对奥沙利铂的耐药性.总之,肝癌耐药细胞系的建立及其miRNA差异表达谱的分析,以及miRNA分子对肝癌细胞发生奥沙利铂耐药的具体作用及其分子机制的研究,不仅有助于理解肝癌细胞发生耐药的分子机制,而且为探索克服肝癌对奥沙利铂耐药性的有效途径提供可靠依据.     

Identification of a common lupus disease-associated microRNA expression pattern in three different murine models of lupus

Background

Recent reports have shown that microRNAs (miRNAs) regulate vital immunological processes and have emerged as key regulators of immune system development and function. Therefore, it is important to determine miRNA dysregulation and its pathogenic contribution in autoimmune diseases, an aspect not adequately addressed thus far.

Methodology/Principal Findings

In this study, we profiled miRNA expressions in splenic lymphocytes from three murine lupus models (MRL-lpr, B6-lpr and NZB/W_F1) with different genetic background by miRNA microarray assays and Real-time RT-PCR. Despite the genetic differences among these three lupus stains, a common set of dysregulated miRNAs (miR-182-96-183 cluster, miR-31, and miR-155) was identified in splenocytes when compared with age-matched control mice. The association of these miRNAs with the disease was highlighted by our observation that this miRNA expression pattern was evident in NZB/W mice only at an age when lupus disease is manifested. Further, we have shown that the miRNA dysregulation in MRL-lpr mice was not simply due to the activation of splenocytes. By Real-time RT-PCR, we confirmed that these miRNAs were upregulated in both purified splenic B and T cells from MRL-lpr mice. miR-127 and miR-379, which were greatly upregulated in splenocytes from lpr mice, were moderately increased in diseased NZB/W mice. In addition, Real-time RT-PCR revealed that miR-146a, miR-101a, and miR-17-92 were also markedly upregulated in splenic T, but not B cells from MRL-lpr mice.

Conclusions/Significance

The identification of common lupus disease-associated miRNAs now forms the basis for the further investigation of the pathogenic contribution of these miRNAs in autoimmune lupus, which will advance our knowledge of the role of miRNAs in autoimmunity. Given that miRNAs are conserved, with regard to both evolution and function, our observation of a common lupus disease-associated miRNA expression pattern in murine lupus models is likely to have significant pathogenic, diagnostic, and/or therapeutic implications in human lupus.     

Grouping Pentylenetetrazol-Induced Epileptic Rats According to Memory Impairment and MicroRNA Expression Profiles in the Hippocampus

Previous studies have demonstrated a close relationship between abnormal regulation of microRNA (miRNA) and various types of diseases, including epilepsy and other neurological disorders of memory. However, the role of miRNA in the memory impairment observed in epilepsy remains unknown. In this study, a model of temporal lobe epilepsy (TLE) was induced via pentylenetetrazol (PTZ) kindling in Sprague-Dawley rats. First, the TLE rats were subjected to Morris water maze to identify those with memory impairment (TLE-MI) compared with TLE control rats (TLE-C), which presented normal memory. Both groups were analyzed to detect dysregulated miRNAs in the hippocampus; four up-regulated miRNAs (miR-34c, miR-374, miR-181a, and miR-let-7c-1) and seven down-regulated miRNAs (miR-1188, miR-770-5p, miR-127-5p, miR-375, miR-331, miR-873-5p, and miR-328a) were found. Some of the dysregulated miRNAs (miR-34c, miR-1188a, miR-328a, and miR-331) were confirmed using qRT-PCR, and their blood expression patterns were identical to those of their counterparts in the rat hippocampus. The targets of these dysregulated miRNAs and other potentially enriched biological signaling pathways were analyzed using bioinformatics. Following these results, the MAPK, apoptosis and hippocampal signaling pathways might be involved in the molecular mechanisms underlying the memory disorders of TLE.     

Deep Sequencing of Small RNA Repertoires in Mice Reveals Metabolic Disorders-Associated Hepatic miRNAs

Obesity and associated metabolic disorders contribute importantly to the metabolic syndrome. On the other hand, microRNAs (miRNAs) are a class of small non-coding RNAs that repress target gene expression by inducing mRNA degradation and/or translation repression. Dysregulation of specific miRNAs in obesity may influence energy metabolism and cause insulin resistance, which leads to dyslipidemia, steatosis hepatis and type 2 diabetes. In the present study, we comprehensively analyzed and validated dysregulated miRNAs in ob/ob mouse liver, as well as miRNA groups based on miRNA gene cluster and gene family by using deep sequencing miRNA datasets. We found that over 13.8% of the total analyzed miRNAs were dysregulated, of which 37 miRNA species showed significantly differential expression. Further RT-qPCR analysis in some selected miRNAs validated the similar expression patterns observed in deep sequencing. Interestingly, we found that miRNA gene cluster and family always showed consistent dysregulation patterns in ob/ob mouse liver, although they had various enrichment levels. Functional enrichment analysis revealed the versatile physiological roles (over six signal pathways and five human diseases) of these miRNAs. Biological studies indicated that overexpression of miR-126 or inhibition of miR-24 in AML-12 cells attenuated free fatty acids-induced fat accumulation. Taken together, our data strongly suggest that obesity and metabolic disturbance are tightly associated with functional miRNAs. We also identified hepatic miRNA candidates serving as potential biomarkers for the diagnose of the metabolic syndrome.     

Participation of miR-200a in TGF-β1-mediated hepatic stellate cell activation

Hepatic stellate cell (HSC) activation is a pivotal event in the initiation and progression of hepatic fibrosis since it mediates transforming growth factor beta 1 (TGF-β1)-driven extracellular matrix (ECM) deposition. MicroRNAs (miRNAs), small non-coding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key factors to regulate cell proliferation, differentiation, and apoptosis. Although the function of miR-200a has been discussed in many cancers and fibrotic diseases, its role in hepatic fibrosis is still poorly understood. The aim of this study is to investigate whether miR-200a could attenuate hepatic fibrosis partly through Wnt/β-catenin and TGF-β-dependant mechanisms. Our study found that the expression of endogenous miR-200a was decreased in vitro in TGF-β1-induced HSC activation as well as in vivo in CCl₄-induced rat liver fibrosis. Overexpression of miR-200a significantly inhibited α-SMA activity and further affected the proliferation of TGF-β1-dependent activation of HSC. In addition, we identified β-catenin and TGF-β2 as two functional downstream targets for miR-200a. Interestingly, miR-200a specifically suppressed β-catenin in the protein level, whereas miR-200a-mediated suppression of TGF-β2 was shown on both mRNA and protein levels. Our results revealed the critical regulatory role of miR-200a in HSC activation and implied miR-200a as a potential candidate for therapy by deregulation of Wnt/β-catenin and TGFβ signaling pathways, at least in part, via decreasing the expression of β-catenin and TGF-β2.     

Profibrotic effect of miR-33a with Akt activation in hepatic stellate cells

MicroRNAs (miRNAs) attract more attention in the pathophysiology of liver fibrosis and miR-33a has been previously demonstrated as involved in the regulation of cholesterol and lipid metabolism. Transforming growth factor-beta1 (TGF-β1) is generally accepted to be the main stimulating factor in the hepatic stellate cells (HSCs) activation, which plays an important role in hepatic fibrosis. However, the involvement and underlying mechanism of miR-33a and its role in TGF-β1-induced hepatic fibrogenesis remains unknown. Here, we investigate the role of miR-33a in the activation of immortalized human HSCs, Lx-2 cells. Our findings have shown that the expression of miR-33a with its host gene sterol regulatory element-binding protein 2 (SREBP2) was more highly expressed in activation of Lx-2 cells than in quiescent cells. The expression of miR-33a on TGF-β1-induced HSCs activation may be modulated via the activation of PI3K/Akt pathway. In addition, miR-33a significantly correlated with TGF-β1-induced expression of α1 (I) collagen (Col1A1) and α-SMA in HSCs. Bioinformatics analyses predict that peroxisome proliferator activated receptor-alpha (PPAR-α) is the potential target of miR-33a. We further found that anti-miR-33a significantly increases target gene PPAR-α mRNA and protein level, suggesting that miR-33a involved in HSCs function might be modulated by targeting PPAR-α. Finally, our results indicate that the expression of miR-33a increased with the progression of liver fibrosis. These results suggested that anti-miR-33a inhibit activation and extracellular matrix production, at least in part, via the activation of PI3K/Akt pathway and PPAR-α and anti sense of miR-33a may be a novel potential therapeutic approach for treating hepatic fibrosis in the future.