A role for microRNA in cystic liver and kidney diseases

The polycystic liver and kidney diseases are a family of disorders with heterogeneous etiologies. Proposed mechanisms of disease include ciliary dysfunction, excess cell proliferation, and altered cell-cell or cell-matrix interactions. In this issue of the JCI, Lee and colleagues provide data to support a novel mechanism for cystogenesis involving microRNA (miRNA) (see the related article beginning on page 3714). They demonstrate that levels of the miRNA miR15a are decreased in livers of patients with autosomal recessive and autosomal dominant polycystic kidney disease (ARPKD and ADPKD, respectively) and congenital hepatic fibrosis as well as in the PKC rat model of ARPKD. This results in increased expression of the cell-cycle regulator Cdc25A, which is a direct target of miR15a, and increased cellular proliferation and cystogenesis in vitro. These findings suggest that other miRNAs may also participate in the molecular pathogenesis of cystic liver and kidney diseases.     

脑胶质瘤恶性进展关键miRNA表达的研究

目的寻找影响胶质瘤恶性进展的关键微小RNA(miRNA)。方法使用miRNA表达谱芯片检测198例不同级别胶质瘤组织标本miRNA的表达情况,挑选代表性差异表达miRNA,采用荧光定量PCR方法进行验证。在人胶质瘤细胞系,通过MTT试验、平板克隆形成试验、细胞周期试验,探讨关键miRNA对肿瘤细胞增殖能力的影响。结果相对于Ⅱ级胶质瘤组织,Ⅲ级胶质瘤组织中miR-374a、miR-590-3p、miR-374b、miR-29c、miR-153表达上调,而let-7c、miR-544、let-7a、miR-132、miR-7d等表达下调。对miR-374a、miR-544表达水平进行验证,结果与芯片方法一致。人胶质瘤H4细胞转染miR-374amimics后,细胞生长率明显增高,细胞克隆数量明显增加。H4细胞转染miR-544mimics后,细胞生长率明显降低,细胞克隆数量明显减少。与Ⅱ级或Ⅲ级胶质瘤组织相比,miR-196b在Ⅳ级胶质母细胞瘤组织中表达水平明显增高,转染miR-196b可明显增加U251细胞的生长率,S期细胞比例增加。结论在不同级别胶质瘤中差异表达的miR-374a、miR-544、miR-196b,可能是通过影响细胞增殖能力推动胶质瘤恶性进程。     

Systemic Delivery of Synthetic MicroRNA-16 Inhibits the Growth of Metastatic Prostate Tumors via Downregulation of Multiple Cell-cycle Genes

Recent reports have linked the expression of specific microRNAs (miRNAs) with tumorigenesis and metastasis. Here, we show that microRNA (miR)-16, which is expressed at lower levels in prostate cancer cells, affects the proliferation of human prostate cancer cell lines both in vitro and in vivo. Transient transfection with synthetic miR-16 significantly reduced cell proliferation of 22Rv1, Du145, PPC-1, and PC-3M-luc cells. A prostate cancer xenograft model revealed that atelocollagen could efficiently deliver synthetic miR-16 to tumor cells on bone tissues in mice when injected into tail veins. In the therapeutic bone metastasis model, injection of miR-16 with atelocollagen via tail vein significantly inhibited the growth of prostate tumors in bone. Cell model studies indicate that miR-16 likely suppresses prostate tumor growth by regulating the expression of genes such as CDK1 and CDK2 associated with cell-cycle control and cellular proliferation. There is a trend toward lower miR-16 expression in human prostate tumors versus normal prostate tissues. Thus, this study indicates the therapeutic potential of miRNA in an animal model of cancer metastasis with systemic miRNA injection and suggest that systemic delivery of miR-16 could be used to treat patients with advanced prostate cancer.     

miRNA‐1 and miRNA‐133a are involved in early commitment of pluripotent stem cells and demonstrate antagonistic roles in the regulation of cardiac differentiation

miRNA‐1 (miR‐1) and miRNA‐133a (miR‐133a) are muscle‐specific miRNAs that play an important role in heart development and physiopathology. Although both miRNAs have been broadly studied during cardiogenesis, the mechanisms by which miR‐1 and miR‐133a could influence linage commitment in pluripotent stem cells remain poorly characterized. In this study we analysed the regulation of miR‐1 and miR‐133a expression during pluripotent stem cell differentiation [P19.CL6 cells; embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs)] and investigated their role in DMSO and embryoid body (EB)‐mediated mesodermal and cardiac differentiation by gain‐ and loss‐of‐function studies, as well as in vivo, by the induction of teratomas. Gene expression analysis revealed that miR‐1 and miR‐133a are upregulated during cardiac differentiation of P19.CL6 cells, and also during ESC and iPSC EB differentiation. Forced overexpression of both miRNAs promoted mesodermal commitment and a concomitant decrease in the expression of neural differentiation markers. Moreover, overexpression of miR‐1 enhanced the cardiac differentiation of P19.CL6, while miR‐133a reduced it with respect to control cells. Teratoma formation experiments with P19.CL6 cells confirmed the influence of miR‐1 and miR‐133a during in vivo differentiation. Finally, inhibition of both miRNAs during P19.CL6 cardiac differentiation had opposite results to their overexpression. In conclusion, gene regulation involving miR‐1 and miR‐133a controls the mesodermal and cardiac fate of pluripotent stem cells. Copyright © 2014 John Wiley & Sons, Ltd.     

miR-1184 regulates the proliferation and apoptosis of colon cancer cells via targeting CSNK2A1

MicroRNA (miRNA) exerts an important part in colon cancer cell proliferation and apoptosis. Meanwhile, the dysregulation of some miRNAs is detected in colon cancer cells. However, it remains unclear about the underlying mechanism of their effects on tumor pathogenesis. The current work aimed to examine the miR-1184 effect on colon cancer cells. The differentially expressed miRNAs (DEMs), including miR-9-3p, miR-1184, miR-492, miR-92a-1-5p and miR-20a-3p, were obtained from the GSE115108 and GSE132619 data sets using the ‘GEO2R’ online tool. Based on the findings, miR-1184 was significantly down-regulated within colon cancer cells and tissues. Moreover, the experimental results of CCK8, flow cytometry, colony formation and Western blotting assays showed that, miR-1184 over-expression suppressed colon cancer cell proliferation through inhibiting Ki67 expression and promoted their apoptosis through up-regulating cleaved caspase-3 and down-regulating Bcl-2 expression. By contrast, miR-1184 inhibition exerted the opposite effects. A total of 110 target genes of miR-1184 were predicted using the TargetScan and miRTarBase databases, which were then used to construct the protein-protein interaction (PPI) network based on the DAVID and STRING websites and to perform GO and KEGG pathway enrichment analyses. The MCODE plug-in of cytoscape was utilized to verify that CSNK2A1 was the target gene and key gene in significant modules. MiR-1184 directly targets CSNK2A1 via using RNA immunoprecipitation assay and luciferase reporter gene assay. According to the results, CSNK2A1 over-expression reversed the functions of miR‐1184 over-expression in suppressing colon cancer cell proliferation and enhancing their apoptosis. In conclusion, over-expression of miR-1184 inhibits colon cancer cell proliferation but promotes their apoptosis through down-regulating CSNK2A1 expression.     

Therapies to slow polycystic kidney disease

Advances in the understanding of cystogenesis and availability of animal models orthologous to human autosomal dominant polycystic kidney disease (ADPKD) and recessive polycystic kidney disease (ARPKD) will likely facilitate the development of treatments for these diseases. Proteins mutated in ADPKD and ARPKD, as well as in several animal models, are localized to renal primary cilia. These are thought to have a sensory function and contribute to the regulation of the intracellular calcium ([Ca2+]i). It seems likely that the maintenance of a differentiated renal epithelial phenotype, characterized by controlled fluid secretion and cell proliferation, requires precise functional coordination of cAMP and Ras/Raf/MEK/ERK signaling by [Ca2+]i. [Ca2+]i alterations, linked to genetic defects causing polycystic kidney disease, may hinder negative feedback mechanisms that control cAMP and Ras/Raf/MEK/ERK signaling, and result in increased fluid secretion and cell proliferation. cAMP levels, Raf kinase activities and ERK phosphorylation are increased in polycystic kidneys. There is also evidence of abnormal cross-talk between cAMP and MAPK pathways, that can be reproduced in wild-type cells by altering [Ca2+]i. While cAMP inhibits Ras-Raf-1-stimulated phosphorylation of ERK in normal kidney cells, it markedly increases B-Raf kinase activity and ERK phosphorylation in polycystic kidney cells. Treatment strategies should probably be aimed at increasing [Ca2+]i, inhibiting Ras/Raf/MEK/ERK signaling or lowering cAMP in the distal nephron and collecting duct. Vasopressin is the major adenylyl cyclase agonist in the collecting duct principal cells via a V2 receptor. OPC31260, a V2 receptor antagonist, lowers renal cAMP and markedly inhibits cystogenesis in four animal models of polycystic kidney disease, three of which are orthologous to human diseases (PCK rat, ARPKD; pcy mouse, adolescent nephronophthisis; Pkd2WS25/- mouse, ADPKD). The renal selectivity and safety profile of this class of drugs make it an excellent candidate for clinical trials.     

MicroRNA-199a-3p is downregulated in human osteosarcoma and regulates cell proliferation and migration

microRNAs (miRNA, miR) play an important role in cancer cell growth and migration; however, the potential roles of miRNAs in osteosarcoma remain largely uncharacterized. By applying a miRNA microarray platform and unsupervised hierarchical clustering analysis, we found that several miRNAs have altered expression levels in osteosarcoma cell lines and tumor tissues when compared with normal human osteoblasts. Three miRNAs, miR-199a-3p, miR-127-3p, and miR-376c, were significantly decreased in osteosarcoma cell lines, whereas miR-151-3p and miR-191 were increased in osteosarcoma cell lines in comparison with osteoblasts. Transfection of precursor miR-199a-3p into osteosarcoma cell lines significantly decreased cell growth and migration, thus indicating that the inhibition effect is associated with an increase in the G(1)-phase and a decrease of the S-phase cell population. In addition, we observed decreased mTOR and Stat3 expression in miR-199a-3p transfected cells. This study provides new insights for miRNAs in osteosarcoma and suggests that miR-199a-3p may play a functional role in osteosarcoma cell growth and proliferation. Restoring miR-199a-3p's function may provide therapeutic benefits in osteosarcoma.     

Pkd1 regulates immortalized proliferation of renal tubular epithelial cells through p53 induction and JNK activation

Autosomal dominant polycystic kidney disease (ADPKD) is the most common human monogenic genetic disorder and is characterized by progressive bilateral renal cysts and the development of renal insufficiency. The cystogenesis of ADPKD is believed to be a monoclonal proliferation of PKD-deficient (PKD(-/-)) renal tubular epithelial cells. To define the function of Pkd1, we generated chimeric mice by aggregation of Pkd1(-/-) ES cells and Pkd1(+/+) morulae from ROSA26 mice. As occurs in humans with ADPKD, these mice developed cysts in the kidney, liver, and pancreas. Surprisingly, the cyst epithelia of the kidney were composed of both Pkd1(-/-) and Pkd1(+/+) renal tubular epithelial cells in the early stages of cystogenesis. Pkd1(-/-) cyst epithelial cells changed in shape from cuboidal to flat and replaced Pkd1(+/+) cyst epithelial cells lost by JNK-mediated apoptosis in intermediate stages. In late-stage cysts, Pkd1(-/-) cells continued immortalized proliferation with downregulation of p53. These results provide a novel understanding of the cystogenesis of ADPKD patients. Furthermore, immortalized proliferation without induction of p53 was frequently observed in 3T3-type culture of mouse embryonic fibroblasts from Pkd1(-/-) mice. Thus, Pkd1 plays a role in preventing immortalized proliferation of renal tubular epithelial cells through the induction of p53 and activation of JNK.     

The effects of GM-CSF, steel factor and MIP-1alpha on the expression and activation of Cdc25A phosphatase in Mo7e cells

Active cyclin-dependent kinases (CDKs) are required for progression through the G1 phase of the cell cycle and entry into S phase. Activity of G1 CDKs is controlled by mechanisms including phosphorylation of Thr14 and Tyr15 residues. Removal of inhibitory phosphates on these amino acid residues is required for G1 CDK activation, and is mediated by the Cdc25A phosphatase. Regulation of active Cdc25A phosphatase levels may be important for the proliferation of hematopoietic progenitor cells, effects assessed in the human growth-factor-dependent cell line Mo7e. Constitutive Cdc25A protein levels were enhanced with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus steel factor (SF). Cdc25A is thought to exert its activity in the nucleus, and nuclear protein levels of Cdc25A were also enhanced with GM-CSF and SF. GM-CSF plus SF promote synergistic growth of Mo7e cells. Pretreatment with macrophage inflammatory protein (MIP-1alpha) inhibited GM-CSF- plus SF-induced growth and upregulation of Cdc25A protein levels. Stimulation with GM-CSF and SF also rapidly increased Cdc25A phosphatase activity, an effect suppressed by MIP-1alpha. A concomitant inhibition of increased CDK4 kinase activity correlated with increased phosphotyrosine levels on CDK4 when cells were pretreated with MIP-1alpha prior to GM-CSF and SF. These data suggest that Cdc25A expression and activity are regulated during proliferation of Mo7e cells.     

Silencing of miR20a is crucial for Ngn1-mediated neuroprotection in injured spinal cord

MicroRNAs (miRNAs) compose a relatively new discipline in biomedical research, and many physiological processes in disease have been associated with changes in miRNA expression. Several studies report that miRNAs participate in biological processes such as the control of secondary injury in several disease models. Recently, we identified novel miRNAs that were abnormally up-regulated in a traumatic spinal cord injury (SCI). In the current study, we focused on miR20a, which causes continuing motor neuron degeneration when overexpressed in SCI lesions. Blocking miR20a in SCI animals led to neural cell survival and eventual neurogenesis with rescued expression of the key target gene, neurogenin 1 (Ngn1). Infusion of siNgn1 resulted in functional deficit in the hindlimbs caused by aggressive secondary injury and actively enhanced the inflammation involved in secondary injury progression. The events involving miR20a underlie motor neuron and myelin destruction and pathophysiology and ultimately block regeneration in injured spinal cords. Inhibition of miR20a expression effectively induced definitive motor neuron survival and neurogenesis, and SCI animals showed improved functional deficit. In this study, we showed that abnormal expression of miR20a induces secondary injury, which suggests that miR20a could be a potential target for therapeutic intervention following SCI.     

Identification of miRNAs as potential modulators of tissue factor expression in patients with systemic lupus erythematosus and antiphospholipid syndrome

Summary. Background: Tissue factor (TF) is the main initiator of the coagulation cascade and elements that may upregulate its expression might provoke thrombotic events. Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are autoimmune diseases characterized by a high TF expression in monocytes. Objectives: To examine the role of microRNAs (miRNAs) in TF expression and to evaluate their levels in SLE and APS patients. Methods: An in silico search was performed to find potential putative binding sites of miRNAs in TF mRNA. In vitro validation was performed transfecting cells expressing TF (THP‐1 and MDA‐MB‐231) with oligonucleotide miRNA precursors and inhibitors. Additionally, reporter assays were performed to test for the binding of miR‐20a to TF mRNA. Levels of miRNAs and TF were measured by quantitative (qRT‐PCR) in patients with APS and SLE. Results: Overexpression of miRNA precursors, but not inhibitors, of two of the members of cluster miR‐17～92, for example miR‐19b and miR‐20a, in cells expressing TF decreased TF mRNA, protein levels, and procoagulant activity between 30% and 60%. Reporter assays showed that miR‐20a binds to TF mRNA. Finally, we measured levels of miR‐19b and miR‐20a in monocytes from patients with APS and SLE and observed significantly lower miRNAs levels in comparison with healthy subjects inversely correlated with the levels of TF. Conclusions: Down‐regulation of miR‐19b and miR‐20a observed in patients with SLE and APS could contribute to increased TF expression and thus provoke the hypercoagulable state characteristic of these patients.     

microRNA在淋巴细胞发育分化及淋巴系统恶性肿瘤发生中的作用

microRNA(miRNA)是一类普遍存在的长度为21-25 nt的小分子非编码RNA,通过与靶基因mRNA 3’非编码区不完全互补结合,抑制翻译,在转录后水平调控靶基因的表达。miRNA参与了多种正常细胞发育及肿瘤发生的调控,在淋巴细胞的发育分化以及淋巴系统恶性肿瘤的发生中发挥着重要作用。研究表明,miRNA可作为有效的肿瘤生物标记应用于淋巴系统恶性肿瘤的诊断、预后判断以及对治疗反应的预测。本文就miRNA在淋巴细胞发育分化中的作用及miRNA与淋巴系统恶性肿瘤发生的关系进行了综述。     

miR‐216a promotes breast cancer cell apoptosis by targeting PKCα

Breast cancer (BC) is the most common cause of death in women throughout the world. MicroRNAs (miRNAs, miR) have been identified as key regulators in carcinogenesis of several cancers, including BC. MicroRNA‐216a (miR‐216a) is downregulated in several cancers. Here, we evaluated the effects of miRNA‐216a on breast cancer cells and the underlying mechanisms. miR‐216a level was quantified by real‐time RT‐PCR. Cell viability was analyzed by MTT assay. Wound‐healing assay was performed for detection of cell migration. Apoptosis was detected by TUNEL and caspase‐3 activity assay. Moreover, the level of protein expression was determined by Western blot. We found that miR‐216a expression was remarkably decreased in both human BC tissues and MCF‐7 cells. miR‐216a overexpression dramatically suppressed the migration and promoted the apoptosis in cultured MCF‐7 cells. We validated PKCα (protein kinase C alpha, PRKCA) as a direct target of miR‐216a. Knockdown of PKCα induced apoptosis and inhibited migration in cultured MCF‐7 cells which were reversed by miR‐216a inhibitor. Moreover, the level of miR‐216a is negatively correlated with PKCα in cell lines. Our results collectively suggest that miR‐216a suppressed migration and promoted apoptosis in breast cancer cells by targeting PKCα. These findings indicate that manipulation of miR‐216a expression may represent a novel therapeutic strategy in the treatment of breast cancer.     

Therapeutic Effects of MicroRNA-582-5p and -3p on the Inhibition of Bladder Cancer Progression

Many reports have indicated that the abnormal expression of microRNAs (miRNAs) is associated with the progression of disease and have identified miRNAs as attractive targets for therapeutic intervention. However, the bifunctional mechanisms of miRNA guide and passenger strands in RNA interference (RNAi) therapy have not yet been clarified. Here, we show that miRNA (miR)-582-5p and -3p, which are strongly decreased in high-grade bladder cancer clinical samples, regulate tumor progression in vitro and in vivo. Significantly, the overexpression of miR-582-5p or -3p reduced the proliferation and invasion of UM-UC-3 human bladder cancer cells. Furthermore, transurethral injections of synthetic miR-582 molecule suppressed tumor growth and metastasis in an animal model of bladder cancer. Most interestingly, our study revealed that both strands of miR-582-5p and -3p suppressed the expression of the same set of target genes such as protein geranylgeranyltransferase type I beta subunit (PGGT1B), leucine-rich repeat kinase 2 (LRRK2) and DIX domain containing 1 (DIXDC1). Knockdown of these genes using small interfering RNA (siRNA) resulted in the inhibition of cell growth and invasiveness of UM-UC-3. These findings uncover the unique regulatory pathway involving tumor suppression by both strands of a single miRNA that is a potential therapeutic target in the treatment of invasive bladder cancer.     

miRNA control of apoptotic programs: focus on ovarian cancer

miRNAs are a class of small non-coding RNAs that regulate the stability or translational efficiency of targeted mRNAs. miRNAs are involved in many cellular processes, such as differentiation, proliferation and apoptosis, which are altered in cancer through miRNA expression dysregulation. In this article we will discuss recent findings implicating miRNAs in apoptotic program regulation using ovarian carcinoma as an example. Ovarian cancer is the most lethal gynecological malignancy. Most patients are diagnosed with advanced disease that is conventionally managed with surgical resection followed by platinum-based chemotherapy. Killing of cancer cells by chemotherapeutic agents or by triggering cell-surface death receptors relies on activation of apoptotic programs executed through receptor-mediated extrinsic pathways and mitochondrial-dependent intrinsic pathways. Despite an initial good response to chemotherapy, ovarian cancer patients typically experience disease relapse within 2 years of the initial treatment developing resistance even to structurally different drugs. Thus, also in this pathology, tumor cells are able to evade apoptosis using multiple mechanisms, several of which are dependent on miRNA gene regulation.     

Differential profiling of human red blood cells during storage for 52 selected microRNAs

BACKGROUND: MicroRNAs (miRNAs), the negative regulators of cellular mRNAs, are present in mature red blood cells (RBCs) in abundance relative to other blood cells. So far, there are no studies aimed at identifying large‐scale miRNA profiles during storage of RBCs. STUDY DESIGN AND METHODS: RNA samples from each RBC bag stored at 4°C were collected on Days 0, 20, and 40 and subjected to miRNA profiling by using a membrane‐based array. Fifty‐two selected miRNAs of cellular apoptotic pathway represent the array. Through bioinformatics analyses, we identified potential target genes for selected miRNAs. RESULTS: Differential profiling of RBCs for 52 miRNAs revealed two distinguishable patterns during storage: Forty‐eight miRNAs demonstrated no trend at all, while four miRNAs, miR‐96, miR‐150, miR‐196a, and miR‐197, demonstrated an increase up to Day 20 and subsequently decreased during storage. We selected miR‐96 and subjected it to standard bioinformatics analyses for target gene predictions, which identified several mRNAs including the RBC proapoptotic calpain small subunit‐1 (CAPNS1) as potential targets of miR‐96. To validate these predictions, we selected CAPNS1 mRNA as an example and confirmed its presence in the RBCs. Future experimental verification would help define miR‐96–CAPNS1 interaction, if any, in the stored RBCs. CONCLUSIONS: This study for the first time provided a differential profile of stored RBCs for selected miRNAs related to cellular apoptotic pathway and opened new avenues toward identification of novel in vitro RBC biomarkers of storage lesions. Future studies focusing on target gene‐miRNA interactions in stored RBCs would also unravel underlying mechanisms of storage lesions.