Genome-wide methylation profiling of ADPKD identified epigenetically regulated genes associated with renal cyst development

Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease characterized by the formation of multiple fluid-filled cysts in bilateral kidneys. Although mutations in polycystic kidney disease 1 (PKD1) are predominantly responsible for ADPKD, the focal and sporadic property of individual cystogenesis suggests another molecular mechanism such as epigenetic alterations. To determine the epigenomic alterations in ADPKD and their functional relevance, ADPKD and non-ADPKD individuals were analyzed by unbiased methylation profiling genome-wide and compared with their expression data. Intriguingly, PKD1 and other genes related to ion transport and cell adhesion were hypermethylated in gene-body regions, and their expressions were downregulated in ADPKD, implicating epigenetic silencing as the key mechanism underlying cystogenesis. Especially, in patients with ADPKD, PKD1 was hypermethylated in gene-body region and it was associated with recruitment of methyl-CpG-binding domain 2 proteins. Moreover, treatment with DNA methylation inhibitors retarded cyst formation of Madin-Darby Canine Kidney cells, accompanied with the upregulation of Pkd1 expression. These results are consistent with previous studies that knock-down of PKD1 was sufficient for cystogenesis. Therefore, our results reveal a critical role for hypermethylation of PKD1 and cystogenesis-related regulatory genes in cyst development, suggesting epigenetic therapy as a potential treatment for ADPKD.     

Ginkgolide B inhibits renal cyst development in in vitro and in vivo cyst models

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disease characterized by massive enlargement of fluid-filled cysts in the kidney. However, there is no effective therapy yet for this disease. To examine whether ginkgolide B, a natural compound, inhibits cyst development, a Madin-Darby canine kidney (MDCK) cyst model, an embryonic kidney cyst model, and a PKD mouse model were used. Interestingly, ginkgolide B significantly inhibited MDCK cyst formation dose dependently, with up to 69% reduction by 2 μM ginkgolide B. Ginkgolide B also significantly inhibited cyst enlargement in the MDCK cyst model, embryonic kidney cyst model, and PKD mouse model. To determine the underlying mechanisms, the effect of ginkgolide B on MDCK cell viability, proliferation, apoptosis, chloride transporter CFTR activity, and intracellular signaling pathways were also studied. Ginkgolide B did not affect cell viability, proliferation, and expression and activity of the chloride transporter CFTR that mediates cyst fluid secretion. Ginkgolide B induced cyst cell differentiation and altered the Ras/MAPK signaling pathway. Taken together, our results demonstrate that ginkgolide B inhibits renal cyst formation and enlargement, suggesting that ginkgolide B might be developed into a novel candidate drug for ADPKD.     

Identification of candidate microRNA biomarkers in renal fibrosis: a meta-analysis of profiling studies

Abstract

The prognostic, diagnostic and therapeutic value of microRNA (miRNA) expression aberrations in renal fibrosis has been studied in recent years. However, the miRNA expression profiling efforts have led to inconsistent results between the studies. The aim of this study was to perform a meta-analysis on the renal fibrosis miRNA expression profiling studies to identify candidate diagnostic biomarkers. We performed comprehensive literature searches in several databases to identify miRNA expression studies of renal fibrosis in animal models and humans. The miRNAs expression data were extracted from 20 included studies, and both miRNA vote-counting strategy and Robust Rank Aggregation method were utilized to identify significant miRNA meta-signatures. The predicted and validated targets of miRNA meta-signature were obtained by using MultiMiR package in 11 databases. Then a gene set enrichment analysis (KEGG, PANTHER pathways and GO processes) were carried out with GeneCodis web tool to recognize pathways that are most strongly influenced by modified expressions of these miRNAs. We recognized in both meta-analysis approaches a significant miRNA meta-signature of five up-regulated (miR-142-3p, miR-223-3p, miR-21-5p, miR-142-5p and miR-214-3p) and two down-regulated (miR-29c-3p and miR-200a-3p) miRNAs. Enrichment analysis confirmed that miRNA meta-signature cooperatively target functionally related genes in signalling and developmental pathways in renal fibrosis. This meta-analysis identified seven highly significant and consistently dysregulated miRNAs from 20 datasets, as the focus of future investigations to discover their potential influence to renal fibrosis and their clinical utility as biomarkers and/or as therapeutic mediators against chronic kidney disease..     

Polycystic kidney disease: novel insights into polycystin function

Autosomal dominant polycystic kidney disease (ADPKD) is a life-threatening monogenic disease caused by mutations in PKD1 and PKD2 that encode polycystin 1 (PC1) and polycystin 2 (PC2). PC1/2 localize to cilia of renal epithelial cells, and their function is believed to embody an inhibitory activity that suppresses the cilia-dependent cyst activation (CDCA) signal. Consequently, PC deficiency results in activation of CDCA and stimulates cyst growth. Recently, re-expression of PCs in established cysts has been shown to reverse PKD. Thus, the mode of action of PCs resembles a ‘counterbalance in cruise control’ to maintain lumen diameter within a designated range. Herein we review recent studies that point to novel arenas for future PC research with therapeutic potential for ADPKD.     

miR-27b synergizes with anticancer drugs via p53 activation and CYP1B1 suppression

Liver and kidney cancers are notorious for drug resistance. Due to the complexity, redundancy and interpatient heterogeneity of resistance mechanisms, most efforts targeting a single pathway were unsuccessful. Novel personalized therapies targeting multiple essential drug resistance pathways in parallel hold a promise for future cancer treatment. Exploiting the multitarget characteristic of microRNAs (miRNAs), we developed a new therapeutic strategy by the combinational use of miRNA and anticancer drugs to increase drug response. By a systems approach, we identified that miR-27b, a miRNA deleted in liver and kidney cancers, sensitizes cancer cells to a broad spectrum of anticancer drugs in vitro and in vivo. Functionally, miR-27b enhances drug response by activating p53-dependent apoptosis and reducing CYP1B1-mediated drug detoxification. Notably, miR-27b promotes drug response specifically in patients carrying p53-wild-type or CYP1B1-high signature. Together, we propose that miR-27b synergizes with anticancer drugs in a defined subgroup of liver and kidney cancer patients.     

Identification of endogenous microRNA references in porcine serum for quantitative real-time PCR normalization

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate the expression of genes, and they affect important biological and physiological states. Circulating miRNAs in blood are useful markers of metabolism and economic traits. Expression levels of circulating miRNAs have been estimated using quantitative real-time PCR (qPCR). Proper normalization is critical for accurate miRNA expression analysis. However, there is no study which systematically presented endogenous reference genes for evaluating circulating miRNA expression in pigs. In this study, ten porcine miRNAs (let-7a, miR-16, miR-17, miR-23a, miR-26a, miR-93, miR-103, miR-107, miR-127 and miR-191), based on the literature, were chosen as candidate reference miRNAs in serum. We evaluated the expression stability value of these miRNAs in Berkshire, Duroc, Landrace and Yorkshire pigs using geNorm and NormFinder. We determined the optimal combination of reference miRNAs for qPCR experiments: miR-127 and miR-17 in Berkshire pigs; miR-127 and miR-93 in Duroc and Landrace pigs; miR-127 and miR-16 in Yorkshire pigs. miR-127 was the best reference gene in pigs, regardless of the breed. Our study is crucial for the discovery of novel biomarkers in pigs. The reference miRNAs presented in this study could be used as appropriate reference genes for the measurement of circulating miRNA levels in studies of physiological blood metabolites.

     

Comprehensive miRNA sequence analysis reveals survival differences in diffuse large B-cell lymphoma patients

BackgroundDiffuse large B-cell lymphoma (DLBCL) is an aggressive disease, with 30% to 40% of patients failing to be cured with available primary therapy. microRNAs (miRNAs) are RNA molecules that attenuate expression of their mRNA targets. To characterize the DLBCL miRNome, we sequenced miRNAs from 92 DLBCL and 15 benign centroblast fresh frozen samples and from 140 DLBCL formalin-fixed, paraffin-embedded tissue samples for validation.ResultsWe identify known and candidate novel miRNAs, 25 of which are associated with survival independently of cell-of-origin and International Prognostic Index scores, which are established indicators of outcome. Of these 25 miRNAs, six miRNAs are significantly associated with survival in our validation cohort. Abundant expression of miR-28-5p, miR-214-5p, miR-339-3p, and miR-5586-5p is associated with superior outcome, while abundant expression of miR-324-5p and NOVELM00203M is associated with inferior outcome. Comparison of DLBCL miRNA-seq expression profiles with those from other cancer types identifies miRNAs that were more abundant in B-cell contexts. Unsupervised clustering of miRNAs identifies two clusters of patients that have distinct differences in their outcomes. Our integrative miRNA and mRNA expression analyses reveal that miRNAs increased in abundance in DLBCL appear to regulate the expression of genes involved in metabolism, cell cycle, and protein modification. Additionally, these miRNAs, including one candidate novel miRNA, miR-10393-3p, appear to target chromatin modification genes that are frequent targets of somatic mutation in non-Hodgkin lymphomas.ConclusionsOur comprehensive sequence analysis of the DLBCL miRNome identifies candidate novel miRNAs and miRNAs associated with survival, reinforces results from previous mutational analyses, and reveals regulatory networks of significance for lymphomagenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0568-y) contains supplementary material, which is available to authorized users.     

Sin3B: An essential regulator of chromatin modifications at E2F target promoters during cell cycle withdrawal

Tubular epithelial cell apoptosis occurs in most animal models of polycystic kidney disease (PKD) and in kidneys from humans with autosomal dominant polycystic kidney disease (ADPKD). Induction of apoptosis in cultured tubular epithelial cells results in cyst formation. Induction of apoptosis in the kidney in Bcl-2 deficient mice results in increased proliferation of tubular epithelium and cyst formation. Caspase inhibition reduces tubular apoptosis and proliferation and slows disease progression in the Han:SPRD rat model of PKD. Thus, there is evidence that both epithelial cell apoptosis and proliferation are dysregulated in ADPKD and may represent a general mechanism for cyst growth.     

Age and sex differences in kidney microRNA expression during the life span of F344 rats

Background

Growing evidence suggests that epigenetic mechanisms of gene regulation may play a role in susceptibilities to specific toxicities and adverse drug reactions. MiRNAs in particular have been shown to be important regulators in cancer and other diseases and show promise as predictive biomarkers for diagnosis and prognosis. In this study, we characterized the global kidney miRNA expression profile in untreated male and female F344 rats throughout the life span. These findings were correlated with sex-specific susceptibilities to adverse renal events, such as male-biased renal fibrosis and inflammation in old age.

Methods

Kidney miRNA expression was examined in F344 rats at 2, 5, 6, 8, 15, 21, 78, and 104 weeks of age in both sexes using Agilent miRNA microarrays. Differential expression was determined using filtering criteria of ≥1.5 fold change and ANOVA or pairwise t-test (FDR <5%) to determine significant age and sex effects, respectively. Pathway analysis software was used to investigate the possible roles of these target genes in age- and sex-specific differences.

Results

Three hundred eleven miRNAs were found to be expressed in at least one age and sex. Filtering criteria revealed 174 differentially expressed miRNAs in the kidney; 173 and 34 miRNAs exhibiting age and sex effects, respectively. Principal component analysis revealed age effects predominated over sex effects, with 2-week miRNA expression being much different from other ages. No significant sexually dimorphic miRNA expression was observed from 5 to 8 weeks, while the most differential expression (13 miRNAs) was observed at 21 weeks. Potential target genes of these differentially expressed miRNAs were identified.

Conclusions

The expression of 56% of detected renal miRNAs was found to vary significantly with age and/or sex during the life span of F344 rats. Pathway analysis suggested that 2-week-expressed miRNAs may be related to organ and cellular development and proliferation pathways. Male-biased miRNA expression at older ages correlated with male-biased renal fibrosis and mononuclear cell infiltration. These miRNAs showed high representation in renal inflammation and nephritis pathways, and included miR-214, miR-130b, miR-150, miR-223, miR-142-5p, miR-185, and miR-296*. Analysis of kidney miRNA expression throughout the rat life span will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13293-014-0019-1) contains supplementary material, which is available to authorized users.     

miR-17 inhibitor suppressed osteosarcoma tumor growth and metastasis via increasing PTEN expression

MicroRNAs (miRNAs) play essential roles in cancer development and progression. Here, we investigated the role of miR-17 in the progression and metastasis of osteosarcoma (OS). miR-17 was frequently increased in OS tissues and cell lines. Inhibition of miR-17 in OS cell lines substantially suppressed cell proliferation, migration, and invasion. Phosphatase and tensin homolog (PTEN) was identified as a target of miR-17, and ectopic expression of miR-17 inhibited PTEN by direct binding to its 3′-untranslated region (3′-UTR). Expression of miR-17 was negatively correlated with PTEN in OS tissues. Together, these findings indicate that miR-17 acts as an oncogenic miRNA and may contribute to the progression and metastasis of OS, suggesting miR-17 as a potential novel diagnostic and therapeutic target of OS.     

miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus

miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes – endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease.     

MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation

In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2.