Microarray-based approach identifies microRNAs and their target functional patterns in polycystic kidney disease
© Pandey et al; licensee BioMed Central Ltd. 2008
Received: 11 September 2008
Accepted: 23 December 2008
Published: 23 December 2008
MicroRNAs (miRNAs) play key roles in mammalian gene expression and several cellular processes, including differentiation, development, apoptosis and cancer pathomechanisms. Recently the biological importance of primary cilia has been recognized in a number of human genetic diseases. Numerous disorders are related to cilia dysfunction, including polycystic kidney disease (PKD). Although involvement of certain genes and transcriptional networks in PKD development has been shown, not much is known how they are regulated molecularly.
Given the emerging role of miRNAs in gene expression, we explored the possibilities of miRNA-based regulations in PKD. Here, we analyzed the simultaneous expression changes of miRNAs and mRNAs by microarrays. 935 genes, classified into 24 functional categories, were differentially regulated between PKD and control animals. In parallel, 30 miRNAs were differentially regulated in PKD rats: our results suggest that several miRNAs might be involved in regulating genetic switches in PKD. Furthermore, we describe some newly detected miRNAs, miR-31 and miR-217, in the kidney which have not been reported previously. We determine functionally related gene sets, or pathways to reveal the functional correlation between differentially expressed mRNAs and miRNAs.
We find that the functional patterns of predicted miRNA targets and differentially expressed mRNAs are similar. Our results suggest an important role of miRNAs in specific pathways underlying PKD.
MicroRNAs (miRNAs) are known to regulate the expression of key genes relevant to cancer and potentially other diseases [1–3]. MiRNAs are short noncoding RNAs of about 22 nt length that have recently been shown to play important roles in mammalian gene expression [1, 3–5]. They induce posttranscriptional gene repression by blocking protein translation (by binding to the 3' UTR of their target genes) or by inducing mRNA degradation, and have the potential to play central roles in physiological and pathological conditions. Recently, it has been shown that miRNAs can also increase translation [6, 7]. The physiological conditions of a cell seem to affect the recruitment of regulatory proteins, which can alter the effect of a miRNA.
MiRNAs are transcribed as long primary transcripts (pri-miRNAs), some of them being polycistronic, which are processed in the cell nucleus by an enzyme called Drosha, yielding precursor miRNAs (pre-miRNAs) that exhibit a characteristic stem-loop sequence . These are exported into the cytosol where mature miRNAs are generated by the RNAse Dicer, producing the small single-stranded miRNA . Translational inhibition, which seems to be the major mode of action in animals, is performed by a riboprotein complex called RNA-induced silencing complex (RISC) consisting of the miRNA and proteins of the argonaute family [9, 10].
MiRNAs are involved in several cellular processes, including cellular differentiation [11, 12], organism development [13, 14], and apoptosis [15, 16]. While all of these are conserved in metazoans, the number of conserved miRNAs between mammals suggests that there are additional functions only found in vertebrates , e.g. controlling hematopoietic differentiation . Recent studies provide growing evidence for the involvement of miRNAs in cancer pathomechanisms [18–21]. However, to date nothing is known regarding miRNAs in the context of Polycystic Kidney Diseases.
Cilia and flagella are ancient, evolutionary conserved organelles that project from cell surfaces to perform diverse biological roles, including whole-cell locomotion, movement of fluid, chemo-, mechano-, and photosensation, and sexual reproduction. The concept of ciliopathies has helped in advancing a unifying theory of cystic kidney diseases . This theory states that the products of all genes that are mutated in cystic kidney diseases in humans, mice, or zebrafish are expressed in primary cilia or centrosomes of renal epithelial cells .
There are numerous disorders linked to basal body and/or cilia dysfunction, including polycystic kidney disease (PKD), primary ciliary dyskinesia (PCD), nephronophthisis (NPHP1–9) , Senior-Loken syndrome, Joubert syndrome, Meckel syndrome, oral-facial-digital syndrome, Alström syndrome and Bardet-Biedl syndrome . These syndromes are typically associated with one or more of the symptoms like cystic kidneys , retinal degeneration and retinitis pigmentosa , situs inversus [26, 27], anosmia , respiratory problems , infertility , hydrocephalus , other ailments like obesity, diabetes, liver fibrosis, hypertension, heart malformations, skeletal anomalies (e.g. polydactyly), cognitive impairment and developmental defects such as exencephaly [16, 26].
Although the mechanisms of the cyst formation are not clearly understood, they are postulated to involve improper functioning of several pathways including cell proliferation, apoptosis, cell polarity, and fluid secretion . Woo  has described apoptotic cells in glomeruli, cyst walls, and in both cystic and non-cystic tubules of the polycystic kidneys. Apoptotic loss of renal tissue may be associated with the progressive deterioration of renal function that occurs in patients with autosomal dominant polycystic kidney disease (ADPKD). There is evidence that genes involved in the regulation of cell proliferation, such as p53, c-fos, cyclin D1, and c-myc may be involved in the control of apoptosis . Veis and colleagues have shown overexpression of c-myc in human ADPKD in association with increased levels of apoptosis and cell proliferation . It is clear that pathogenesis of PKD is very complicated and involves multiple molecular pathways with overlapping, complementary, or opposing effects. There are several signalling pathways that have been implicated in ciliary function . The dysregulation of mitogen-activated protein kinases (MAPKs) in the cyst epithelium of pcy (polycystic kidney disease) mice, carrying a missense mutation in NPHP3 , is a downstream consequence of disturbed renal monocilia function . The proteins, implicated in the formation of renal cysts in tuberous sclerosis, have been found at the ciliary base. They form a complex that inhibits mTOR resulting in retarded cyst formation in rats with PKD . Cilia-mediated signalling acts as a switch between canonical and non-canonical Wnt signalling pathways .
Rats or mice have been used as common model systems for the study of PKD. The Hannover rat, Han:Sprague-Dawley (SPRD)-cy rat is an accepted model for human PKD [38, 39] and has been efficiently used since more than a decade. It is an autosomal dominant model for PKD resulting in cyst formation and slowly progressive chronic renal failure . In the current investigation, we explore the transcriptional changes that occur in PKD and investigate if these changes could be related to miRNAs. We use a microarray-based approach to profile the transcriptional (mRNA) changes as well as changes in the miRNA expression patterns in PKD. We use the Han:SPRD cy/+ rat model  for our current investigation. Our results suggest several miRNAs may be involved in regulating the genetic switches in PKD. Furthermore we describe some newly detected miRNAs in the kidney.
Animals and physiological state
Inbred homozygous unaffected (+/+) and heterozygous affected (cy/+) Han:SPRD rats exhibiting PKD were investigated. The Han:SPRD-cy rats used in this study carry a dominant mutation that causes cystic kidneys and is an accepted model for human PKD . After approximately 40 generations of inbreeding this substrain was registered as PKD/Mhm-cy inbred strain of rats: polycystic kidney diseases, Mannheim, Germany hereafter designated as PKD/Mhm. From each of the aforementioned PKD/Mhm (cy/+) and PKD/Mhm (+/+) animals, littermates were investigated. The animals were sacrificed by cervical dislocation at day 36. On the day of sacrifice, body weight was determined. Following dislocation, the left and right kidneys were immediately removed and weights were determined. Kidneys were preserved for histological analyses and the genotypes were confirmed. Histological analysis of pathogenesis was performed using hematoxylin-esosin (HE) staining of the kidney section (3 μm) followed by cyst grading under light microscope [41, 42]; kidneys were graded on 1–5 scale as previously described [41, 42].
We had ethical approval to carry out this work on animals by Regierungspraesidium Nordbaden and Internal Review Board.
RNA isolation and Affymetrix microarray
Total RNA was extracted using TRIzol method according to manufacturer's protocol (Invitrogen Life Technologies). cDNA synthesis was performed using the SuperScript Choice System (Invitrogen Life Technologies, Invitrogen Corporation) according to manufacturer's protocol. Biotin-labelled cRNA was produced using ENZO BioArray High Yield RNA Transcript Labelling Kit. The standard protocol from Affymetrix (Santa Clara, CA) with 3.3 μL of cDNA was used for the in vitro transcription (IVT). Cleanup of the IVT product was done using CHROMA SPIN-100 columns (Clontech, USA). Spectrophotometric analysis was used for quantification of cRNA with acceptable A260/A280 ratio of 1.9 to 2.1. After that, the cRNA was fragmented using Affymetrix defined protocol. Labelled and fragmented cRNA was hybridized to Affymetrix Rat 230_2 microarrays for 16 hrs at 45°C using Affymetrix defined protocol. cRNA in the range of 15 μg was used for all the 12 microarrays. Microarrays were washed using an Affymetrix fluidics station 450 and stained initially with streptavidin/phycoerytherin. For each sample the signal was further enhanced by incubation with biotinylated goat anti-streptavidin followed by a second incubation with streptavidin/phycoerytherin, and a second round of intensities were measured. Microarrays were scanned with an Affymetrix scanner controlled by the Affymetrix Microarray Suite software. A total of 12 Affymetrix whole genome arrays (from 6 healthy and 6 diseased biological replicates) were performed.
Locked Nucleic Acid (LNA) based miRNA hybridization assays were done as described by Castoldi et al. . Briefly, 5 μg RNA was hybridized to a miRNA microarray (miChip v6.0), containing probes for ~300 miRNAs, based on LNA-modified and Tm-normalized oligonucleotide capture probes (miRCURY Array probes, Exiqon) spotted onto Codelink (GE Healthcare) in multiple replicates, and scanned using an Axon Scanner 4000B. Microarray images were analyzed using the Genepix Pro 4.0 software . As for the Affymetrix whole genome arrays, 6 biologically replicated arrays, each for healthy and diseased animals (total of 12 chips) were performed.
Statistical and bioinformatics analysis of microarray data
Microarray data obtained from Affymetrix chips were analyzed using SAS Micro-Array Solution version 1.3 (SAS, USA). Normalization of raw data was performed by fitting a mixed linear model across all arrays in the experiment. Log2-transformed scores of all spot measures were subjected to normalization. Identification of differentially expressed genes was carried out by Mixed Model Analysis (MMA) using ANOVA approach. A Bonferroni adjustment for multiple testing with α ≤ 0.05 was used to calculate the statistical significance threshold/cut-off [negative log10 (α)]. Custom CDFs version 8 (UniGene CDF files)  were used for annotations. Significantly up-regulated genes in PKD/Mhm (cy/+) animals were defined as those with >0 log-fold-changes in expression and down-regulated as those having <0 log-fold-changes in expression at p-value < 0.05 (adjusted for multiple testing).
The raw data from miRNA chips were also normalized and analyzed using the same modules in SAS Micro-Array Solution version 1.3, as described above. Significantly up-regulated miRNAs in PKD/Mhm (cy/+) animals were defined as those with >0 log-fold-changes in expression and down-regulated as those having <0 log-fold-changes in expression at p-value < 0.05 (adjusted for multiple testing).
Genes previously reported as targets for the significantly regulated miRNAs, and differentially regulated in PKD, were obtained from Argonaute . In addition, targets for the significantly regulated miRNAs, in the genes differentially regulated in PKD, were predicted using two algorithms: TargetScan  and miRanda . To identify the genes commonly predicted by both algorithms, results were intersected using a Perl script; the intersection of TargetScan and miRanda algorithms was used for further analysis.
Identification of pathways possibly affected by miRNAs and their target genes in PKD
The pathways affected in PKD were determined for the significantly regulated genes from KEGG , GeneOntology (GO) , Biocarta http://www.biocarta.com/ and the Molecular Signature database . To check whether significantly regulated genes are overrepresented in a pathway in PKD or not, Fisher's exact test  without correction for multiple testing was used. Pathways showing p-value less than 0.05 were considered as significantly enriched.
Reverse transcription and quantitative real-time PCR (qPCR) analysis of miRNAs
qPCR assays for miRNAs were performed using "TaqMan MicroRNA Assays" (Applied Biosystems, USA). The reverse transcriptase reactions were performed as per the manufacturer's protocol. Real-time PCR was also performed using a standard TaqMan PCR kit protocol on the Applied Biosystems 7000 Sequence Detection System. 6 biological replicates were used for analysis and all the reactions were run in quadruplicates. The comparative CT method for relative quantitation of gene expression was used to determine miRNA expression levels among the normal animals and PKD/Mhm (cy/+) animals according to manufacturer's protocol. miR-193a was used as an endogenous control to normalize the expression levels of targets. The miR-193a served as a good choice for endogenous control because its expression was almost uniform in all the samples on the miRNA-chips (0.99 ± 0.026) and it was not differentially regulated among the tested samples. miRNAs with ubiquitous and stable expression values are superior normalizers to other RNAs such as 5S rRNA, U6 snRNA or total RNA [52, 53]. Consistency in expression of miR-193a across all the samples was evident in qPCR assays (mean CT-value 29.51 ± 0.19). The significance of differences in relative expression of miRNAs among the two groups was tested by One-way ANOVA method in SAS version 9.1.
Pathological status of PKD/Mhm(cy/+) and healthy PKD/Mhm (+/+) animals
Profiling miRNA expression change during PKD
Significantly regulated miRNAs on Exiqon chips from SAS analysis
Name of miRNAs on chip
miRNA name in rat*
Lsmean animal PKD
Lsmean animal Control
Log Fold Change PKD_Ctrl
-log10(p-value) for log fold change of PKD_Ctrl
Microarray analysis of genes involved in PKD
Top 25 up-regulated and 25 down-regulated genes on Affymetrix chips from SAS analysis
Log_fold_change (>0 = up-regulated; <0 = down-regulated)
-log10(p-value) for Estimate of diseased_healthy
1368821_at, 1368822_at, 1371331_at, 1394119_at
1389189_at, 1396539_at, 1398294_at
1367823_at, 1375144_at, 1386940_at, 1388312_at
1369400_a_at, 1388044_at, 1388063_a_at, 1398320_at
1367571_a_at, 1371206_a_at, 1398322_at
Identifying miRNA-target interaction
miRNAs and their targets
a: miRNAs and their previously reported targets (from Argonaute database). miRNAs and their corresponding targets are both differentially regulated during PKD.
ABCB9, BTG1, DSCR1, RASD1
ABC transporters General
Adherens junction, Maturity onset diabetes of the, Focal adhesion, **Long term depression
ABCB9, CPB2, IRS1, MAPK10
ABC transporters General, Complement and coagulation cas, Adipocytokine signaling pathwa, Insulin signaling pathway, Type II diabetes mellitus, Fc epsilon RI signaling pathwa, Focal adhesion, **GnRH signaling pathway, **MAPK signaling pathway, Toll like receptor signaling p, Wnt signaling pathway
BTG1, SARA1, YWHAB
**Long term depression
Adherens junction, Axon guidance, Focal adhesion, Leukocyte transendothelial mig, Regulation of actin cytoskelet, TGF beta signaling pathway, T cell receptor signaling path, Tight junction, Wnt signaling pathway
ATP2B2, DNM1L, EGR3, PPP1R9B, YWHAB
**Calcium signaling pathway, Cell cycle
HRH3, NCDN, SLC23A2
**Neuroactive ligand receptor in
b: miRNAs and their targets (from TargetScan and miRanda). miRNAs and their corresponding targets are both differentially regulated during PKD.
ABCB9, BTG2, CACNB2, CNR1, COL3A1, GLRA2, IRS1, NEK6, PDE7B, PTPN5, SV2A, SYT4, YWHAB, NR5A2, NTRK3
ABC transporters General, **MAPK signaling pathway, **Neuroactive ligand receptor in, **Cell Communication, **ECM receptor interaction, Focal adhesion, Adipocytokine signaling pathwa, Insulin signaling pathway, Type II diabetes mellitus, Purine metabolism, Cell cycle, Maturity onset diabetes of the
ADAMTS1, ATP2B2, CACNB2, CDH13, CNR1, DUSP6, EGR3, EPHA7, GRIK2, GRM5, GRM7, HOXA1, MMP14
**Calcium signaling pathway, **MAPK signaling pathway, **Neuroactive ligand receptor in, Axon guidance, Gap junction, **Long term depression, Long term potentiation, **GnRH signaling pathway
ABCB9, COL3A1, EPHA7, GAS7, OTX1
ABC transporters General, **Cell Communication, **ECM receptor interaction, Focal adhesion, Axon guidance
Cell cycle, **MAPK signaling pathway, TGF beta signaling pathway
CLU, FN1, GRIK2, KCNH5, NR4A2, RAP1B
**Cell Communication, **ECM receptor interaction, Focal adhesion, Regulation of actin cytoskelet, **Neuroactive ligand receptor in, Leukocyte transendothelial mig, Long term potentiation, **MAPK signaling pathway
**Calcium signaling pathway
ATP2B2, CNR1, SNF1LK
**Calcium signaling pathway, **Neuroactive ligand receptor in
**Calcium signaling pathway
CCKBR, COLEC12, EPHA7, GRM7, JUN, KCNAB1, LIN7A, MAMDC1, PRRX1
**Calcium signaling pathway, **Neuroactive ligand receptor in, Axon guidance, B cell receptor signaling path, Focal adhesion, **GnRH signaling pathway, **MAPK signaling pathway, T cell receptor signaling path, Toll like receptor signaling p, Wnt signaling pathway
**Neuroactive ligand receptor in
ATP2B2, NR4A2, SNF1LK, RET, SLC11A2
**Calcium signaling pathway
COLEC12, DNM3, GRIK2, IGF1R, IL12B, KCNH7, KCNIP4, SCN3A, SCN5A
**Neuroactive ligand receptor in, Adherens junction, Focal adhesion, **Long term depression, **Cytokine cytokine receptor int, **Jak STAT signaling pathway, Toll like receptor signaling p, **Type I diabetes mellitus
BTG2, CALCR, DLL1, MYRIP
**Neuroactive ligand receptor in, Notch signaling pathway
Amyotrophic lateral sclerosis, Apoptosis, **Jak STAT signaling pathway, Neurodegenerative Disorders
Secondly, we mapped the differentially regulated genes for miRNA targets for the differentially regulated miRNAs using two tools, TargetScan  and miRanda . Only commonly predicted targets for different miRNAs were taken for further analysis. A number of 65 genes were identified as miRNA targets, common from TargetScan as well as miRanda (Table 3b), out of which 31 genes participate in regulating 33 pathways, which could further be grouped into 13 functional categories (Figure 6b). The intersection of TargetScan and miRanda results gave target genes for 20 miRNAs out of 30 differentially regulated miRNAs. This shows that available prediction tools are not yet fully optimal. Altogether, we obtained 89 target genes differentially expressed on Affymetrix chips (Figure 2). These were associated with 36 pathways, out of which 10 were significantly enriched by ORA (Table 3a and 3b; Figure 2).
Selected inverse miRNA-target relation identified
Log fold change miRNA
Log fold change gene
**Cell Communication, **ECM receptor interaction, Focal adhesion
**Cell Communication, **ECM receptor interaction, Focal adhesion
Adherens junction, Focal adhesion, **Long term depression
**GnRH signaling pathway
**MAPK signaling pathway
**Neuroactive ligand receptor in
**Cell Communication, **ECM receptor interaction, Focal adhesion
Cell cycle, **MAPK signaling pathway, TGF beta signaling pathway
Adherens junction, Axon guidance, Focal adhesion, Leukocyte transendothelial mig, Regulation of actin cytoskelet, T cell receptor signaling path, TGF beta signaling pathway, Tight junction, Wnt signaling pathway
**Cell Communication, **ECM receptor interaction, Focal adhesion, Regulation of actin cytoskelet
Focal adhesion, Leukocyte transendothelial mig, Long term potentiation, **MAPK signaling pathway
**Neuroactive ligand receptor in
B cell receptor signaling path, Focal adhesion, **GnRH signaling pathway, **MAPK signaling pathway, T cell receptor signaling path, Toll like receptor signaling p, Wnt signaling pathway
Adherens junction, Focal adhesion, **Long term depression
**Neuroactive ligand receptor in
In the current investigation, we explore the possible involvement of miRNAs in PKD. In a well characterized rat model system (Han:SPRD cy/+ rat model; ), we used a combinatorial approach involving data mining and microarray analysis, to profile the miRNAs involved in PKD. In parallel to the miRNA expression profiling, we also describe the changes in mRNA transcript patterns in PKD using genome-wide Affymetrix arrays. Whereas, the gene expression arrays e.g. Affymetrix arrays have already established themselves for measuring large-scale differential regulation of mRNAs, microarrays have recently been developed to measure miRNA expression changes accurately [43, 62]. We used arrays based on LNA technology . The cluster analysis of the control and PKD miRNA-microarrays showed tight clustering PKD samples (Figure 3). Few studies have appeared showing the use of proteomics based approaches for identifying specific miRNA targets [63–66]. Several of these studies also applied mRNA-expression arrays to derive correlations between the protein- and messenger- turndown. Over-expressing miRNAs resulted in repressed targets, both at protein and messenger levels: how much both processes contribute to down-regulation of targets depends on individual miRNA-mRNA pairs . These studies show that mRNA profiling are indeed valuable tools for studying mRNA-miRNA interaction, although additional information on fine-tuning of proteome-expression may be obtained by including proteomics approaches.
Our study, profiling the changes in the mRNA transcripts in PKD, revealed large-scale changes. Although some transcription factors, such as SP1 , JUN and c-myc  have been implicated to play a role in PKD, the magnitude of transcriptional changes (>900 genes) suggest involvement of other regulators. Moreover, the regulatory basis of changes in the expression of transcription factors involved in PKD remains poorly understood. On the other hand, miRNAs have emerged recently as key regulators of gene expression in many developmental [69, 70] and disease events [71, 72]. Our miRNA study shows that changes in gene expression during PKD also involve miRNAs.
When we compared the mRNA and miRNA profiles, differentially regulated in PKD, with Argonaute (a comprehensive database on miRNAs; [45, 71]), there were few genes reported as miRNA target like tropomyosin 1, alpha (TPM1) as a target of miR-21, the beta polypeptide of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAB), regulatory subunit 9B of protein phosphatase 1 (PPP1R9B), early growth response 3 (EGR3) and dynamin 1-like (DNM1L) as targets of miR-31, plysia ras-related homolog A2 (RHOA) as targets of miR-217, etc. (Table 3a). But none of these genes had previously been associated with PKD. Over-representation analysis showed the enrichment of five pathways namely, GnRH signaling pathway, MAPK signaling pathway, Long term depression, Calcium signaling pathway and Neuroactive ligand receptor interaction. The comparison of differentially regulated genes/miRNAs to Argonaute database indicates that previously known miRNA-target interactions (Table 3a) could play important roles in PKD.
Parallel profiling of the transcripts as well as miRNAs on the same set of samples gives us insight into potential interactions between miRNAs and differentially expressed targets in PKD. This parallel profiling allowed us to scrutinize for the negative expression patterns for the miRNA-target relation at a given time point (Table 4). A negative relationship of the expression patterns between miRNA and its target mRNA is an important parameter for determining their interactions because miRNA, in general, are regarded as negative regulators of their targets.
Microarray analysis of miRNAs revealed down regulation of 29 out of 30 differentially regulated miRNAs, which corresponded to increased expression of several genes related to pathways upregulated during PKD. To further uncover the functional correlation between differentially expressed mRNAs and miRNAs, we determined functionally related gene sets, or pathways. The differentially regulated mRNAs were associated with 24 functional categories, which included several enriched pathways important to renal diseases. Previous studies of cystic kidneys implicated several pathways thought to contribute to the pathogenesis of renal cysts' formation like mTOR signalling, MAPK (Mitogen-Activated Protein Kinase) signalling, Wnt signalling, and the TGF-β (Transforming Growth Factor-β) pathway.
MAPKs play important roles in the cell by transmitting extracellular signals from the cell membrane to the nucleus . MAPKs are activated by various stimuli, influencing cell proliferation, differentiation, and apoptosis. Aberrant regulation of MAPKs and other signalling pathways has been reported to be consistent with altered regulation of cell proliferation and differentiation observed in renal cystic disease . Sustained activation of MAPKs in kidney epithelial cells inhibits normal epithelial phenotype and formation of adherens junctions . The expression of genes involved in MAPK signalling pathway like MAP3K1, JUN, and MYC was significantly higher in the PKD animals, indicating the activation of MAPK signalling pathway.
Wnt signalling is essential for renal development. Recent research has revealed an unexpected intersection between Wnt signalling and PKD . It has been reported that canonical Wnt signalling seemed mandatory for early renal development, but persistent β-catenin signalling seemed to trigger cyst formation at later developmental stages . Components of the Wnt signalling pathway like FZD2, JUN, MYC, and RHOA were significantly upregulated in our PKD animals.
TGF-β promotes renal cell hypertrophy and stimulates extracellular matrix accumulation in several renal diseases, including diabetic nephropathy  and PKD . It activates the inhibitors of the proteases e.g. tissue inhibitors of metalloproteinases and plasminogen activator inhibitor 1 . TGF-β 1, TGF-β 2 and TGF-β 3 were consistently upregulated in PKD-affected animals. The expression of extracellular matrix components, such as collagen triple helix repeat containing 1 (CTHRC1), and fibronectin 1 (FN1) was significantly higher in animals with PKD. One of the integrins, integrin beta 1 (ITGB1) was upregulated. Therefore, there was a clear activation of the TGF-β signaling pathway in the PKD animals with a significant increase of the synthesis of the extracellular matrix components and inhibition of the proteases that digest this matrix. Recently Kato et al. showed that miRNA miR-192 has been associated with TGF-β pathway in diabetic kidney glomeruli . Its implication in PKD has not been shown, though.
We could not find negative co-relations between the predicted targets of the only up-regulated miRNA, miR-21, whose expression was verified by qPCR analysis too. All the predicted targets were up-regulated. It should be noted that alternate action mechanisms for miRNAs also exist. The action of miRNAs may not to be reflected on the level of their target mRNAs as they are believed to block or attenuate further translation of mRNAs to protein. In such conditions, miRNAs will exert their regulatory role on the level of translation. Moreover, a new mode of action where the miRNAs act as positive regulators has also been defined recently . Depending upon the state of a cell, a miRNA can act as positive or negative regulator .
The specific cellular pathways that were found to be associated with dysregulated miRNAs or differentially expressed transcripts in PKD can be used to shape some initial hypotheses on how alteration of miRNA expression may be directly involved in the disease. Furthermore, the functional correlation between the differentially expressed mRNAs and miRNAs in PKD revealed a tight posttranscriptional regulation network at both mRNA and protein level.
While this work establishes a multi-tiered approach for investigation of miRNA mode of genetic regulations of PKD, the results await further investigation. PKD is an important disease as 8–10% of all patients on renal replacement therapy including haemodialysis or transplantation, suffer from PKD . The molecular components have just started to become apparent, and we add another layer of regulators, namely miRNAs. We predict that several of the differentially regulated genes are miRNA targets and miR-21, miR-31, miR-128, miR-147 and miR-217 may be the important players in such interaction. It is interesting to note that miR-31 and miR-217 have not been previously reported in kidney. miR-21 has been reported in kidney  but its function has yet not been established. It has been speculated in previous studies that a single miRNA could target more than hundred genes and one gene could be the target of several miRNAs [61, 80]. Knockout and over-expression studies will provide further insight into the regulatory interaction between these miRNAs and their targets in order to properly understand PKD development and design new therapeutic measures.
We thank Dr. Xiaolei Yu for curated pathway lists, Dr. Mirco Castoldi for performing Exiqon miRNA arrays at EMBL, Heidelberg, Germany and Maria Saile for the technical support. This work was supported by the DFG research Training Group 886 'Molecular Imaging Methods for the analysis of Gene and Protein Expression'. There is no kind of conflict of interest for any of the authors of this paper.
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