Alternative splicing in shaping the molecular landscape of the cochlea.

The cochlea is a complex organ comprising diverse cell types with highly specialized morphology and function. Until now, the molecular underpinnings of its specializations have mostly been studied from a transcriptional perspective, but accumulating evidence points to post-transcriptional regulation as a major source of molecular diversity. Alternative splicing is one of the most prevalent and well-characterized post-transcriptional regulatory mechanisms. Many molecules important for hearing, such as cadherin 23 or harmonin, undergo alternative splicing to produce functionally distinct isoforms. Some isoforms are expressed specifically in the cochlea, while some show differential expression across the various cochlear cell types and anatomical regions. Clinical phenotypes that arise from mutations affecting specific splice variants testify to the functional relevance of these isoforms. All these clues point to an essential role for alternative splicing in shaping the unique molecular landscape of the cochlea. Although the regulatory mechanisms controlling alternative splicing in the cochlea are poorly characterized, there are animal models with defective splicing regulators that demonstrate the importance of RNA-binding proteins in maintaining cochlear function and cell survival. Recent technological breakthroughs offer exciting prospects for overcoming some of the long-standing hurdles that have complicated the analysis of alternative splicing in the cochlea. Efforts toward this end will help clarify how the remarkable diversity of the cochlear transcriptome is both established and maintained.

Hedgehog signaling regulates Wolffian duct development through the primary cilium†.

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). Although mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; IFT88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix, mesenchymal-epithelial transition, canonical Hh and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

Follistatin regulates the specification of the apical cochlea responsible for low-frequency hearing in mammals.

The cochlea's ability to discriminate sound frequencies is facilitated by a special topography along its longitudinal axis known as tonotopy. Auditory hair cells located at the base of the cochlea respond to high-frequency sounds, whereas hair cells at the apex respond to lower frequencies. Gradual changes in morphological and physiological features along the length of the cochlea determine each region's frequency selectivity, but it remains unclear how tonotopy is established during cochlear development. Recently, sonic hedgehog (SHH) was proposed to initiate the establishment of tonotopy by conferring regional identity to the primordial cochlea. Here, using mouse genetics, we provide in vivo evidence that regional identity in the embryonic cochlea acts as a framework upon which tonotopy-specific properties essential for frequency selectivity in the mature cochlea develop. We found that follistatin (FST) is required for the maintenance of apical cochlear identity, but dispensable for its initial induction. In a fate-mapping analysis, we found that FST promotes expansion of apical cochlear cells, contributing to the formation of the apical cochlear domain. SHH, in contrast, is required both for the induction and maintenance of apical identity. In the absence of FST or SHH, mice produce a short cochlea lacking its apical domain. This results in the loss of apex-specific anatomical and molecular properties and low-frequency-specific hearing loss.

Tissue-specific requirement of sodium channel and clathrin linker 1 (Sclt1) for ciliogenesis during limb development.

Primary cilia have essential roles as signaling centers during development and adult homeostasis. Disruption of ciliary structure or function causes congenital human disorders called ciliopathies. Centriolar distal appendage (DAP) proteins are important for anchoring cilia to the membrane. However, the exact functions of DAP during in vivo ciliogenesis and animal development remain poorly understood. Here, we showed that the DAP component sodium channel and clathrin linker 1 (Sclt1) mutant mice had abnormal craniofacial and limb development with postnatal lethality. In mutant embryos, most of the affected tissues had defects in DAP recruitment to the basal body and docking to the membrane that resulted in reduced ciliogenesis and disrupted hedgehog (Hh) signaling in limb bud mesenchymal cells. However, limb digit formation and ciliogenesis in Sclt1 mutant mice were differentially affected between the fore- and hindlimb buds. The forelimbs developed normally in Sclt1 mutants, but the hindlimbs had preaxial polydactyly. Heterozygous loss of Cep83, another core DAP component, in Sclt1 mutant mice, caused forelimb and hindlimb polydactyly. These findings revealed the tissue-specific differential requirement of DAPs. Taken together, these results indicated that during limb development the ciliary base components, DAPs, play an essential role in ciliogenesis and Hh signaling in vivo in a position-dependent manner.

Microtubule-associated protein 1 A and tubby act independently in regulating the localization of stereocilin to the tips of inner ear hair cell stereocilia.

Tubby mice exhibit hearing impairment due to the loss of stereocilin from the tip regions that connect the tallest stereocilia of the outer hair cells (OHCs) to the tectorial membrane. Stereocilin is an essential stereociliary protein in the OHCs, the mutation of which in humans causes autosomal recessive non-syndromic deafness. Map1a is a modifier of tubby hearing (moth1), and its wild-type allele, rather than the moth1 allele from the C57BL/6 J strain, restores stereocilin localization to the stereocilia and rescues the hearing impairment of tubby mice. The mechanism by which MAP1A accomplishes this is unclear, partly due to ambiguity regarding whether the tubby mutation is a true null. We therefore generated Tub-null (Tub-/-) mice by deleting exon 3 and found that they exhibit hearing impairment like that of tubby mice, suggesting the tubby mutation is a loss-of-function mutation with regard to hearing. When we crossed Tub-/- mice with AKR mice that have wild-type Map1a alleles, we found that wild-type MAP1A restores stereocilin localization to the tips of stereocilia and rescues hearing impairment. These data suggest MAP1A does not require interaction with tubby protein in maintaining stereocilin at the tips of stereocilia and that OHCs use two independent molecules-MAP1A and tubby-to doubly ensure proper stereocilin localization.

Therapeutic effect of NLRP3 inhibition on hearing loss induced by systemic inflammation in a CAPS-associated mouse model.

BACKGROUND: Cryopyrin-associated periodic syndrome (CAPS) is an inherited autoinflammatory disease caused by a gain-of-function mutation in NLRP3. Although CAPS patients frequently suffer from sensorineural hearing loss, it remains unclear whether CAPS-associated mutation in NLRP3 is associated with the progression of hearing loss. METHODS: We generated a mice with conditional expression of CAPS-associated NLRP3 mutant (D301N) in cochlea-resident CX3CR1 macrophages and examined the susceptibility of CAPS mice to inflammation-mediated hearing loss in a local and systemic inflammation context. FINDINGS: Upon lipopolysaccharide (LPS) injection into middle ear cavity, NLRP3 mutant mice exhibited severe cochlear inflammation, inflammasome activation and hearing loss. However, this middle ear injection model induced a considerable hearing loss in control mice and inevitably caused an inflammation-independent hearing loss possibly due to ear tissue damages by injection procedure. Subsequently, we optimized a systemic LPS injection model, which induced a significant hearing loss in NLRP3 mutant mice but not in control mice. Peripheral inflammation induced by a repetitive low dose of LPS injection caused a blood-labyrinth barrier disruption, macrophage infiltration into cochlea and cochlear inflammasome activation in an NLRP3-dependent manner. Interestingly, both cochlea-infiltrating and -resident macrophages contribute to peripheral inflammation-mediated hearing loss of CAPS mice. Furthermore, NLRP3-specific inhibitor, MCC950, as well as an interleukin-1 receptor antagonist significantly alleviated systemic LPS-induced hearing loss and inflammatory phenotypes in NLRP3 mutant mice. INTERPRETATION: Our findings reveal that CAPS-associated NLRP3 mutation is critical for peripheral inflammation-induced hearing loss in our CAPS mice model, and an NLRP3-specific inhibitor can be used to treat inflammation-mediated sensorineural hearing loss. FUNDING: National Research Foundation of Korea Grant funded by the Korean Government and the Team Science Award of Yonsei University College of Medicine.

Position Specific Alternative Splicing and Gene Expression Profiles Along the Tonotopic Axis of Chick Cochlea.

Alternative splicing (AS) refers to the production of multiple mRNA isoforms from a single gene due to alternative selection of exons or splice sites during pre-mRNA splicing. It is a primary mechanism of gene regulation in higher eukaryotes and significantly expands the functional complexity of eukaryotic organisms, contributing to animal development and disease. Recent studies have shown that AS also influences functional diversity by affecting the transcriptomic and proteomic profiles in a position-dependent manner in a single organ. The peripheral hearing organ, the cochlea, is organized to detect sounds at different frequencies depending on its location along the longitudinal axis. This unique functional configuration, the tonotopy, is known to be facilitated by differential gene expression along the cochlear duct. We profiled transcriptome-wide gene expression and AS changes that occur within the different positions of chick cochlea. These analyses revealed distinct gene expression profiles and AS, including a splicing program that is unique to tonotopy. Changes in the expression of splicing factors PTBP3, ESRP1, and ESRP2 were demonstrated to contribute to position-specific AS. RNA-binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS at different positions by different RNA-binding proteins. These data, along with gene ontology (GO) analysis, represent a comprehensive analysis of the dynamic regulation of AS at different positions in chick cochlea.

Differential Roles of Tubby Family Proteins in Ciliary Formation and Trafficking.

Cilia are highly specialized organelles that extend from the cell membrane and function as cellular signaling hubs. Thus, cilia formation and the trafficking of signaling molecules into cilia are essential cellular processes. TULP3 and Tubby (TUB) are members of the tubby-like protein (TULP) family that regulate the ciliary trafficking of G-protein coupled receptors, but the functions of the remaining TULPs (i.e., TULP1 and TULP2) remain unclear. Herein, we explore whether these four structurally similar TULPs share a molecular function in ciliary protein trafficking. We found that TULP3 and TUB, but not TULP1 or TULP2, can rescue the defective cilia formation observed in TULP3-knockout (KO) hTERT RPE-1 cells. TULP3 and TUB also fully rescue the defective ciliary localization of ARL13B, INPP5E, and GPR161 in TULP3 KO RPE-1 cells, while TULP1 and TULP2 only mediate partial rescues. Furthermore, loss of TULP3 results in abnormal IFT140 localization, which can be fully rescued by TUB and partially rescued by TULP1 and TULP2. TUB's capacity for binding IFT-A is essential for its role in cilia formation and ciliary protein trafficking in RPE-1 cells, whereas its capacity for PIP2 binding is required for proper cilia length and IFT140 localization. Finally, chimeric TULP1 containing the IFT-A binding domain of TULP3 fully rescues ciliary protein trafficking, but not cilia formation. Together, these two TULP domains play distinct roles in ciliary protein trafficking but are insufficient for cilia formation in RPE-1 cells. In addition, TULP1 and TULP2 play other unknown molecular roles that should be addressed in the future.

Dysregulation of sonic hedgehog signaling causes hearing loss in ciliopathy mouse models.

Defective primary cilia cause a range of diseases known as ciliopathies, including hearing loss. The etiology of hearing loss in ciliopathies, however, remains unclear. We analyzed cochleae from three ciliopathy mouse models exhibiting different ciliogenesis defects: Intraflagellar transport 88 (Ift88), Tbc1d32 (a.k.a. bromi), and Cilk1 (a.k.a. Ick) mutants. These mutants showed multiple developmental defects including shortened cochlear duct and abnormal apical patterning of the organ of Corti. Although ciliogenic defects in cochlear hair cells such as misalignment of the kinocilium are often associated with the planar cell polarity pathway, our results showed that inner ear defects in these mutants are primarily due to loss of sonic hedgehog signaling. Furthermore, an inner ear-specific deletion of Cilk1 elicits low-frequency hearing loss attributable to cellular changes in apical cochlear identity that is dedicated to low-frequency sound detection. This type of hearing loss may account for hearing deficits in some patients with ciliopathies.

Uncovering the secreted signals and transcription factors regulating the development of mammalian middle ear ossicles.

The mammalian middle ear comprises a chain of ossicles, the malleus, incus, and stapes that act as an impedance matching device during the transmission of sound from the tympanic membrane to the inner ear. These ossicles are derived from cranial neural crest cells that undergo endochondral ossification and subsequently differentiate into their final functional forms. Defects that occur during middle ear development can result in conductive hearing loss. In this review, we summarize studies describing the crucial roles played by signaling molecules such as sonic hedgehog, bone morphogenetic proteins, fibroblast growth factors, notch ligands, and chemokines during the differentiation of neural crest into the middle ear ossicles. In addition to these cell-extrinsic signals, we also discuss studies on the function of transcription factor genes such as Foxi3, Tbx1, Bapx1, Pou3f4, and Gsc in regulating the development and morphology of the middle ear ossicles.

Distinct roles of stereociliary links in the nonlinear sound processing and noise resistance of cochlear outer hair cells.

Outer hair cells (OHCs) play an essential role in hearing by acting as a nonlinear amplifier which helps the cochlea detect sounds with high sensitivity and accuracy. This nonlinear sound processing generates distortion products, which can be measured as distortion-product otoacoustic emissions (DPOAEs). The OHC stereocilia that respond to sound vibrations are connected by three kinds of extracellular links: tip links that connect the taller stereocilia to shorter ones and convey force to the mechanoelectrical transduction channels, tectorial membrane-attachment crowns (TM-ACs) that connect the tallest stereocilia to one another and to the overlying TM, and horizontal top connectors (HTCs) that link adjacent stereocilia. While the tip links have been extensively studied, the roles that the other two types of links play in hearing are much less clear, largely because of a lack of suitable animal models. Here, while analyzing genetic combinations of tubby mice, we encountered models missing both HTCs and TM-ACs or HTCs alone. We found that the tubby mutation causes loss of both HTCs and TM-ACs due to a mislocalization of stereocilin, which results in OHC dysfunction leading to severe hearing loss. Intriguingly, the addition of the modifier allele modifier of tubby hearing 1 in tubby mice selectively rescues the TM-ACs but not the HTCs. Hearing is significantly rescued in these mice with robust DPOAE production, indicating an essential role of the TM-ACs but not the HTCs in normal OHC function. In contrast, the HTCs are required for the resistance of hearing to damage caused by noise stress.

rMAPS2: an update of the RNA map analysis and plotting server for alternative splicing regulation.

The rMAPS2 (RNA Map Analysis and Plotting Server 2) web server, freely available at http://rmaps.cecsresearch.org/, has provided the high-throughput sequencing data research community with curated tools for the identification of RNA binding protein sites. rMAPS2 analyzes differential alternative splicing or CLIP peak data obtained from high-throughput sequencing data analysis tools like MISO, rMATS, Piranha, PIPE-CLIP and PARalyzer, and then, graphically displays enriched RNA-binding protein target sites. The initial release of rMAPS focused only on the most common alternative splicing event, skipped exon or exon skipping. However, there was a high demand for the analysis of other major types of alternative splicing events, especially for retained intron events since this is the most common type of alternative splicing in plants, such as Arabidopsis thaliana. Here, we expanded the implementation of rMAPS2 to facilitate analyses for all five major types of alternative splicing events: skipped exon, mutually exclusive exons, alternative 5' splice site, alternative 3' splice site and retained intron. In addition, by employing multi-threading, rMAPS2 has vastly improved the user experience with significant reductions in running time, ∼3.5 min for the analysis of all five major alternative splicing types at once.

CXCL12 is required for stirrup-shaped stapes formation during mammalian middle ear development.

BACKGROUND: The mammalian middle ear comprises a chain of three ossicles-the malleus, incus, and stapes-each of which has a unique morphology for efficiently transmitting sound information. In particular, the stapes, which is attached to the inner ear, is stirrup-shaped with a head and base connected by two crural arches, forming the stapedial foramen, through which the stapedial artery passes. However, how the stapes acquires this critical stirrup shape for association with the stapedial artery during development is not clear. RESULTS: C-X-C motif chemokine ligand 12 (CXCL12) is a chemoattractant essential for cellular movement and angiogenesis. In Cxcl12 -/- embryos, migration of neural crest cells into the prospective middle ear regions and their mesenchymal condensation to form the three ossicles proceed normally in correct alignment with each other and the inner ear. However, in the absence of CXCL12, the stapes loses its stirrup shape and instead exhibits a columnar shape lacking the crural arches and central hole. In addition, although the stapedial artery initially forms during early mesenchymal condensation of the stapes, it degenerates without CXCL12 function. CONCLUSION: CXCL12 plays an essential role in establishing the stirrup-shaped architecture of the stapes, possibly by maintaining the stapedial foramen and stapedial artery throughout development.

A pathogen-derived metabolite induces microglial activation via odorant receptors.

Microglia (MG), the principal neuroimmune sentinels in the brain, continuously sense changes in their environment and respond to invading pathogens, toxins, and cellular debris, thereby affecting neuroinflammation. Microbial pathogens produce small metabolites that influence neuroinflammation, but the molecular mechanisms that determine whether pathogen-derived small metabolites affect microglial activation of neuroinflammation remain to be elucidated. We hypothesized that odorant receptors (ORs), the largest subfamily of G protein-coupled receptors, are involved in microglial activation by pathogen-derived small metabolites. We found that MG express high levels of two mouse ORs, Olfr110 and Olfr111, which recognize a pathogenic metabolite, 2-pentylfuran, secreted by Streptococcus pneumoniae. These interactions activate MG to engage in chemotaxis, cytokine production, phagocytosis, and reactive oxygen species generation. These effects were mediated through the Gαs -cyclic adenosine monophosphate-protein kinase A-extracellular signal-regulated kinase and Gßγ -phospholipase C-Ca2+ pathways. Taken together, our results reveal a novel interplay between the pathogen-derived metabolite and ORs, which has major implications for our understanding of microglial activation by pathogen recognition. DATABASE: Model data are available in the PMDB database under the accession number PM0082389.

Gene therapy for hereditary hearing loss by SLC26A4 mutations in mice reveals distinct functional roles of pendrin in normal hearing.

Rationale: Mutations of SLC26A4 that abrogate pendrin, expressed in endolymphatic sac, cochlea and vestibule, are known to cause autosomal recessive sensorineural hearing loss with enlargement of the membranous labyrinth. This is the first study to demonstrate the feasibility of gene therapy for pendrin-related hearing loss. Methods: We used a recombinant viral vector to transfect Slc26a4 cDNA into embryonic day 12.5 otocysts of pendrin-deficient knock-out (Slc26a4∆/∆ ) and pendrin-deficient knock-in (Slc26a4tm1Dontuh/tm1Dontuh ) mice. Results: Local gene-delivery resulted in spatially and temporally limited pendrin expression, prevented enlargement, failed to restore vestibular function, but succeeded in the restoration of hearing. Restored hearing phenotypes included normal hearing as well as sudden, fluctuating, and progressive hearing loss. Conclusion: Our study illustrates the feasibility of gene therapy for pendrin-related hearing loss, suggests differences in the requirement of pendrin between the cochlea and the vestibular labyrinth, and documents that insufficient pendrin expression during late embryonal and early postnatal development of the inner ear can cause sudden, fluctuating and progressive hearing loss without obligatory enlargement of the membranous labyrinth.

Activation of sonic hedgehog signaling by a Smoothened agonist restores congenital defects in mouse models of endocrine-cerebro-osteodysplasia syndrome.

BACKGROUND: Endocrine-cerebro-osteodysplasia (ECO) syndrome is a genetic disorder associated with congenital defects of the endocrine, cerebral, and skeletal systems in humans. ECO syndrome is caused by mutations of the intestinal cell kinase (ICK) gene, which encodes a mitogen-activated protein (MAP) kinase-related kinase that plays a critical role in controlling the length of primary cilia. Lack of ICK function disrupts transduction of sonic hedgehog (SHH) signaling, which is important for development and homeostasis in humans and mice. Craniofacial structure abnormalities, such as cleft palate, are one of the most common defects observed in ECO syndrome patients, but the role of ICK in palatal development has not been studied. METHODS: Using Ick-mutant mice, we investigated the mechanisms by which ICK function loss causes cleft palate and examined pharmacological rescue of the congenital defects. FINDINGS: SHH signaling was compromised with abnormally elongated primary cilia in the developing palate of Ick-mutant mice. Cell proliferation was significantly decreased, resulting in failure of palatal outgrowth, although palatal adhesion and fusion occurred normally. We thus attempted to rescue the congenital palatal defects of Ick mutants by pharmacological activation of SHH signaling. Treatment of Ick-mutant mice with an agonist for Smoothened (SAG) rescued several congenital defects, including cleft palate. INTERPRETATIONS: The recovery of congenital defects by pharmacological intervention in the mouse models for ECO syndrome highlights prenatal SHH signaling modulation as a potential therapeutic measure to overcome congenital defects of ciliopathies.

Targeted Gene Delivery into the Mammalian Inner Ear Using Synthetic Serotypes of Adeno-Associated Virus Vectors.

Targeting specific cell types in the mammalian inner ear is important for treating genetic hearing loss due to the different cell type-specific functions. Adeno-associated virus (AAV) is an efficient in vivo gene transfer vector, and it has demonstrated promise for treating genetic hearing loss. Although more than 100 AAV serotypes have been identified, few studies have investigated whether AAV can be distributed to specific inner ear cell types. Here we screened three EGFP-AAV reporter constructs (serotypes DJ, DJ8, and PHP.B) in the neonatal mammalian inner ear by injection via the round window membrane to determine the cellular specificity of the AAV vectors. Sensory hair cells, supporting cells, cells in Reissner's membrane, interdental cells, and root cells were successfully transduced. Hair cells in the cochlear sensory epithelial region were the most frequently transduced cell type by all tested AAV serotypes. The recombinant DJ serotype most effectively transduced a range of cell types at a high rate. Our findings provide a basis for improving treatment of hereditary hearing loss using targeted AAV-mediated gene therapy.

Correction to: Exocyst Complex Member EXOC5 Is Required for Survival of Hair Cells and Spiral Ganglion Neurons and Maintenance of Hearing.

The original article contains an error for a grant number in the Acknowledgements section.

Region-specific endodermal signals direct neural crest cells to form the three middle ear ossicles.

Defects in the middle ear ossicles - malleus, incus and stapes - can lead to conductive hearing loss. During development, neural crest cells (NCCs) migrate from the dorsal hindbrain to specific locations in pharyngeal arch (PA) 1 and 2, to form the malleus-incus and stapes, respectively. It is unclear how migratory NCCs reach their proper destination in the PA and initiate mesenchymal condensation to form specific ossicles. We show that secreted molecules sonic hedgehog (SHH) and bone morphogenetic protein 4 (BMP4) emanating from the pharyngeal endoderm are important in instructing region-specific NCC condensation to form malleus-incus and stapes, respectively, in mouse. Tissue-specific knockout of Shh in the pharyngeal endoderm or Smo (a transducer of SHH signaling) in NCCs causes the loss of malleus-incus condensation in PA1 but only affects the maintenance of stapes condensation in PA2. By contrast, knockout of Bmp4 in the pharyngeal endoderm or Smad4 (a transducer of TGFß/BMP signaling) in the NCCs disrupts NCC migration into the stapes region in PA2, affecting stapes formation. These results indicate that region-specific endodermal signals direct formation of specific middle ear ossicles.

Impact of miR-192 and miR-194 on cyst enlargement through EMT in autosomal dominant polycystic kidney disease.

Altered miRNA (miR) expression occurs in various diseases. However, the therapeutic effect of miRNAs in autosomal dominant polycystic kidney disease (ADPKD) is unclear. Genome-wide analyses of miRNA expression and DNA methylation status were conducted to identify crucial miRNAs in end-stage ADPKD. miR-192 and -194 levels were down-regulated with hypermethylation at these loci, mainly in the intermediate and late stages, not in the early stage, of cystogenesis, suggesting their potential impact on cyst expansion. Cyst expansion has been strongly associated with endothelial-mesenchymal transition (EMT). Zinc finger E-box-binding homeobox-2 and cadherin-2, which are involved in EMT, were directly regulated by miR-192 and -194. The therapeutic effect of miR-192 and -194 in vivo and in vitro were assessed. Restoring these miRs by injection of precursors influenced the reduced size of cysts in Pkd1 conditional knockout mice. miR-192 and -194 may act as potential therapeutic targets to control the expansion and progression of cysts in patients with ADPKD.-Kim, D. Y., Woo, Y. M., Lee, S., Oh, S., Shin, Y., Shin, J.-O., Park, E. Y., Ko, J. Y., Lee, E. J., Bok, J., Yoo, K. H., Park, J. H. Impact of miR-192 and miR-194 on cyst enlargement through EMT in autosomal dominant polycystic kidney disease.