Transcriptome profiling of subepithelial PDGFRα cells in colonic mucosa reveals several cell-selective markers.

Subepithelial platelet-derived growth factor receptor alpha (PDGFRα)+ cells found in the colonic mucosal tissue come in close contact with epithelial cells, immune cells, neurons, capillaries, and lymphatic networks. Mucosal subepithelial PDGFRα+ cells (MuPαC) are important regulators in various intestinal diseases including fibrosis and inflammation. However, the transcriptome of MuPαC has not yet been elucidated. Using Pdgfra-eGFP mice and flow cytometry, we isolated colonic MuPαC and obtained their transcriptome data. In analyzing the transcriptome, we identified three novel, and selectively expressed, markers (Adamdec1, Fin1, and Col6a4) found in MuPαC. In addition, we identified a unique set of MuPαC-enriched genetic signatures including groups of growth factors, transcription factors, gap junction proteins, extracellular proteins, receptors, cytokines, protein kinases, phosphatases, and peptidases. These selective groups of genetic signatures are linked to the unique cellular identity and function of MuPαC. Furthermore, we have added this MuPαC transcriptome data to our Smooth Muscle Genome Browser that contains the transcriptome data of jejunal and colonic smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and smooth muscle resident PDGFRα+ cells: (https://med.unr.edu/physio/transcriptome). This online resource provides a comprehensive reference of all currently known genetic transcripts expressed in primary MuPαC in the colon along with smooth muscle resident PDGFRα cells, SMC, and ICC in the murine colon and jejunum.

miR-10b-5p Rescues Diabetes and Gastrointestinal Dysmotility.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICCs) and pancreatic ß cells require receptor tyrosine kinase (KIT) to develop and function properly. Degeneration of ICCs is linked to diabetic gastroparesis. The mechanisms linking diabetes and gastroparesis are unclear, but may involve microRNA (miRNA)-mediated post-transcriptional gene silencing in KIT+ cells. METHODS: We performed miRNA-sequencing analysis from isolated ICCs in diabetic mice and plasma from patients with idiopathic and diabetic gastroparesis. miR-10b-5p target genes were identified and validated in mouse and human cell lines. For loss-of-function studies, we used KIT+ cell-restricted mir-10b knockout mice and KIT+ cell depletion mice. For gain-of-function studies, a synthetic miR-10b-5p mimic was injected in multiple diabetic mouse models. We compared the efficacy of miR-10b-5p mimic treatment vs antidiabetic and prokinetic medicines. RESULTS: miR-10b-5p is highly expressed in ICCs from healthy mice, but drastically depleted in ICCs from diabetic mice. A conditional knockout of mir-10b in KIT+ cells or depletion of KIT+ cells in mice leads to degeneration of ß cells and ICCs, resulting in diabetes and gastroparesis. miR-10b-5p targets the transcription factor Krüppel-like factor 11 (KLF11), which negatively regulates KIT expression. The miR-10b-5p mimic or Klf11 small interfering RNAs injected into mir-10b knockout mice, diet-induced diabetic mice, and TALLYHO polygenic diabetic mice rescue the diabetes and gastroparesis phenotype for an extended period of time. Furthermore, the miR-10b-5p mimic is more effective in improving glucose homoeostasis and gastrointestinal motility compared with common antidiabetic and prokinetic medications. CONCLUSIONS: miR-10b-5p is a key regulator in diabetes and gastrointestinal dysmotility via the KLF11-KIT pathway. Restoration of miR-10b-5p may provide therapeutic benefits for these disorders.

The Piezo2 ion channel is mechanically activated by low-threshold positive pressure.

Recent parallel studies clearly indicated that Merkel cells and the mechanosensitive piezo2 ion channel play critical roles in the light-touch somatosensation. Moreover, piezo2 was suggested to be a light-touch sensing ion channel without a role in pain sensing in mammals. However, biophysical characteristics of piezo2, such as single channel conductance and sensitivities to various mechanical stimuli, are unclear, hampering a precise understanding of its role in touch sensation. Here, we describe the biophysical properties of piezo2 in human Merkel cell carcinoma (MCC)-13 cells; piezo2 is a low-threshold, positive pressure-specific, curvature-sensitive, mechanically activated cation channel with a single channel conductance of ~28.6 pS. Application of step indentations under the whole-cell mode of the patch-clamp technique, and positive pressures ≥5 mmHg under the cell-attached mode, activated piezo2 currents in MCC-13 and human embryonic kidney 293 T cells where piezo2 was overexpressed. By contrast, application of a negative pressure failed to activate piezo2 in these cells, whereas both positive and negative pressure activated piezo1 in a similar manner. Our results are the first to demonstrate single channel recordings of piezo2. We anticipate that our findings will be a starting point for a more sophisticated understanding of piezo2 roles in light-touch sensation.

Clinical Outcomes and Recurrence Rates After Arthroscopic Stabilization Procedures in Young Patients With a Glenoid Bone Erosion: A Comparative Study Between Glenoid Erosion More and Less Than 20.

PURPOSE: To evaluate the clinical outcomes and recurrence rates of arthroscopic stabilization procedures in young patients who had recurrent anterior shoulder instability with a glenoid bone erosion more than 20%, and to compare with those in patients with a glenoid bone erosion less than 20%. METHODS: A total of 161 patients who underwent an arthroscopic stabilization procedure for recurrent anterior shoulder instability with anterior glenoid bone erosions and at least 2 years of follow-up were included. Patients were divided into 2 groups based on the glenoid defect size (group I [32 patients]: erosion >20%, group II [129 patients]: erosion <20%). The clinical outcomes were compared using the American Shoulder Elbow Surgeons (ASES) score, Rowe score, and sports/recreation activity level between the 2 groups. Postoperative complications including instability recurrence were documented. RESULTS: The mean glenoid defect size was 22.1 ± 2.1% in group I, and 12.2 ± 3.7% in group II. In group I, clinical outcomes were significantly improved after operation (ASES score: 57.9 ± 14.3 at initial, 88.9 ± 6.2 at the last visit, P = .001; Rowe score: 42.1 ± 15.6 at initial, 87.4 ± 7.6 at the last visit, P = .001). These results were inferior to the clinical outcomes of patients in group II (ASES score: 91.5 ± 12.7, P < .001; Rowe score: 89.3 ± 12.4, P = .01). Postoperative recurrences occurred in 5 patients (15.6%) in group I, whereas patients in group II showed 5.4% of recurrence rate (P = .05). Competent recoveries to sports/recreation activity were achieved in 84.4% of patients in group I. CONCLUSIONS: Arthroscopic stabilization procedures for recurrent anterior shoulder instability in young patients with glenoid bone erosions more than 20% showed satisfactory clinical outcomes and recurrence rate, although these results were inferior to those of patients with glenoid erosions less than 20%. Arthroscopic stabilization procedures can be applied as the primary treatment of recurrent anterior shoulder instability with a large glenoid bone erosion for functional restoration and return to previous sports activity level. LEVEL OF EVIDENCE: Level III, retrospective comparative study.

Transcriptome analysis of PDGFRα+ cells identifies T-type Ca2+ channel CACNA1G as a new pathological marker for PDGFRα+ cell hyperplasia.

Platelet-derived growth factor receptor alpha (PDGFRα)+ cells are distributed into distinct morphological groups within the serosal, muscular, and submucosal layers as well as the myenteric and deep muscular plexi. PDGFRα+ cells directly interact with interstitial cells of Cajal (ICC) and smooth muscle cells (SMC) in gastrointestinal smooth muscle tissue. These three cell types, SMC, ICC, and PDGFRα+ cells (SIP cells), form an electrical syncytium, which dynamically regulates gastrointestinal motility. We have previously reported the transcriptomes of SMC and ICC. To complete the SIP cell transcriptome project, we obtained transcriptome data from jejunal and colonic PDGFRα+ cells. The PDGFRα+ cell transcriptome data were added to the Smooth Muscle Genome Browser that we previously built for the genome-scale gene expression data of ICC and SMC. This browser provides a comprehensive reference for all transcripts expressed in SIP cells. By analyzing the transcriptomes, we have identified a unique set of PDGFRα+ cell signature genes, growth factors, transcription factors, epigenetic enzymes/regulators, receptors, protein kinases/phosphatases, and ion channels/transporters. We demonstrated that the low voltage-dependent T-type Ca2+ channel Cacna1g gene was particularly expressed in PDGFRα+ cells in the intestinal serosal layer in mice. Expression of this gene was significantly induced in the hyperplasic PDGFRα+ cells of obstructed small intestine in mice. This gene was also over-expressed in colorectal cancer, Crohn's disease, and diverticulitis in human patients. Taken together, our data suggest that Cacna1g exclusively expressed in serosal PDGFRα+ cells is a new pathological marker for gastrointestinal diseases.

Transcriptome of interstitial cells of Cajal reveals unique and selective gene signatures.

Transcriptome-scale data can reveal essential clues into understanding the underlying molecular mechanisms behind specific cellular functions and biological processes. Transcriptomics is a continually growing field of research utilized in biomarker discovery. The transcriptomic profile of interstitial cells of Cajal (ICC), which serve as slow-wave electrical pacemakers for gastrointestinal (GI) smooth muscle, has yet to be uncovered. Using copGFP-labeled ICC mice and flow cytometry, we isolated ICC populations from the murine small intestine and colon and obtained their transcriptomes. In analyzing the transcriptome, we identified a unique set of ICC-restricted markers including transcription factors, epigenetic enzymes/regulators, growth factors, receptors, protein kinases/phosphatases, and ion channels/transporters. This analysis provides new and unique insights into the cellular and biological functions of ICC in GI physiology. Additionally, we constructed an interactive ICC genome browser (http://med.unr.edu/physio/transcriptome) based on the UCSC genome database. To our knowledge, this is the first online resource that provides a comprehensive library of all known genetic transcripts expressed in primary ICC. Our genome browser offers a new perspective into the alternative expression of genes in ICC and provides a valuable reference for future functional studies.

Serum response factor regulates smooth muscle contractility via myotonic dystrophy protein kinases and L-type calcium channels.

Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed Dmpk and Cacna1c genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of Srf knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca2+ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract.

Serum Response Factor Is Essential for Prenatal Gastrointestinal Smooth Muscle Development and Maintenance of Differentiated Phenotype.

BACKGROUND/AIMS: Smooth muscle cells (SMCs) characteristically express serum response factor (SRF), which regulates their development. The role of SRF in SMC plasticity in the pathophysiological conditions of gastrointestinal (GI) tract is less characterized. METHODS: We generated SMC-specific Srf knockout mice and characterized the prenatally lethal phenotype using ultrasound biomicroscopy and histological analysis. We used small bowel partial obstruction surgeries and primary cell culture using cell-specific enhanced green fluorescent protein (EGFP) mouse lines to study phenotypic and molecular changes of SMCs by immunofluorescence, Western blotting, and quantitative polymerase chain reaction. Finally we examined SRF change in human rectal prolapse tissue by immunofluorescence. RESULTS: Congenital SMC-specific Srf knockout mice died before birth and displayed severe GI and cardiac defects. Partial obstruction resulted in an overall increase in SRF protein expression. However, individual SMCs appeared to gradually lose SRF in the hypertrophic muscle. Cells expressing low levels of SRF also expressed low levels of platelet-derived growth factor receptor alpha (PDGFRα(low)) and Ki67. SMCs grown in culture recaptured the phenotypic switch from differentiated SMCs to proliferative PDGFRα(low) cells. The immediate and dramatic reduction of Srf and Myh11 mRNA expression confirmed the phenotypic change. Human rectal prolapse tissue also demonstrated significant loss of SRF expression. CONCLUSIONS: SRF expression in SMCs is essential for prenatal development of the GI tract and heart. Following partial obstruction, SMCs down-regulate SRF to transition into proliferative PDGFRα(low) cells that may represent a phenotype responsible for their plasticity. These findings demonstrate that SRF also plays a critical role in the remodeling process following GI injury.

Smooth Muscle Cell Genome Browser: Enabling the Identification of Novel Serum Response Factor Target Genes.

Genome-scale expression data on the absolute numbers of gene isoforms offers essential clues in cellular functions and biological processes. Smooth muscle cells (SMCs) perform a unique contractile function through expression of specific genes controlled by serum response factor (SRF), a transcription factor that binds to DNA sites known as the CArG boxes. To identify SRF-regulated genes specifically expressed in SMCs, we isolated SMC populations from mouse small intestine and colon, obtained their transcriptomes, and constructed an interactive SMC genome and CArGome browser. To our knowledge, this is the first online resource that provides a comprehensive library of all genetic transcripts expressed in primary SMCs. The browser also serves as the first genome-wide map of SRF binding sites. The browser analysis revealed novel SMC-specific transcriptional variants and SRF target genes, which provided new and unique insights into the cellular and biological functions of the cells in gastrointestinal (GI) physiology. The SRF target genes in SMCs, which were discovered in silico, were confirmed by proteomic analysis of SMC-specific Srf knockout mice. Our genome browser offers a new perspective into the alternative expression of genes in the context of SRF binding sites in SMCs and provides a valuable reference for future functional studies.

The mitochondrial genome encodes abundant small noncoding RNAs.

Small noncoding RNAs identified thus far are all encoded by the nuclear genome. Here, we report that the murine and human mitochondrial genomes encode thousands of small noncoding RNAs, which are predominantly derived from the sense transcripts of the mitochondrial genes (host genes), and we termed these small RNAs mitochondrial genome-encoded small RNAs (mitosRNAs). DICER inactivation affected, but did not completely abolish mitosRNA production. MitosRNAs appear to be products of currently unidentified mitochondrial ribonucleases. Overexpression of mitosRNAs enhanced expression levels of their host genes in vitro, and dysregulated mitosRNA expression was generally associated with aberrant mitochondrial gene expression in vivo. Our data demonstrate that in addition to 37 known mitochondrial genes, the mammalian mitochondrial genome also encodes abundant mitosRNAs, which may play an important regulatory role in the control of mitochondrial gene expression in the cell.

MicroRNAs dynamically remodel gastrointestinal smooth muscle cells.

Smooth muscle cells (SMCs) express a unique set of microRNAs (miRNAs) which regulate and maintain the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal (GI) SMCs in a transgenic animal model. We generated SMC-specific Dicer null animals that express the reporter, green fluorescence protein, in a SMC-specific manner. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs: the mutant mice developed severe dilation of the intestinal tract associated with the thinning and destruction of the smooth muscle (SM) layers; contractile motility in the mutant intestine was dramatically decreased; and SM contractile genes and transcriptional regulators were extensively down-regulated in the mutant SMCs. Profiling and bioinformatic analyses showed that SMC phenotype is regulated by a complex network of positive and negative feedback by SMC miRNAs, serum response factor (SRF), and other transcriptional factors. Taken together, our data suggest that SMC miRNAs are required for the development and survival of SMCs in the GI tract.

Serum response factor-dependent MicroRNAs regulate gastrointestinal smooth muscle cell phenotypes.

BACKGROUND & AIMS: Smooth muscle cells (SMCs) change phenotypes under various pathophysiological conditions. These changes are largely controlled by the serum response factor (SRF), a transcription factor that binds to CC (A/T)6 GG (CArG) boxes in SM contractile genes. MicroRNAs (miRNA) regulate transitions among SMC phenotypes. The SMC miRNA transcriptome (SMC miRNAome) and its regulation by SRF have not been determined. METHODS: We performed massively parallel sequencing to identify gastrointestinal (GI) SMC miRNA transcriptomes in mice and humans. SMC miRNA transcriptomes were mapped to identify all CArG boxes, which were confirmed by SRF knockdown and microarrays. Quantitative polymerase chain reaction was used to identify SMC-phenotypic miRNAs in differentiated and proliferating SMCs. Bioinformatics and target validation analysis showed regulation of SMC phenotype by SRF-dependent, SMC-phenotype miRNAs. RESULTS: We cloned and identified GI miRNA transcriptomes using genome-wide analyses of mouse and human cells. The SM miRNAome consisted of hundreds of unique miRNAs that were highly conserved among both species. We mapped miRNAs CArG boxes and found that many had an SRF-dependent signature in the SM miRNAome. The SM miRNAs CArG boxes had several distinct features. We also identified approximately 100 SMC-phenotypic miRNAs that were induced in differentiated or proliferative SMC phenotypes. We showed that SRF-dependent, SMC-phenotypic miRNAs bind and regulate Srf and its cofactors, myocadin (Myocd) and member of ETS oncogene family Elk1. CONCLUSIONS: The GI SMC phenotypes are controlled by SRF-dependent, SMC-phenotypic miRNAs that regulate expression of SRF, MYOCD, and ELK1.

A model to study the phenotypic changes of interstitial cells of Cajal in gastrointestinal diseases.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) express the receptor tyrosine kinase, KIT, the receptor for stem cell factor. In the gastrointestinal (GI) tract, ICC are pacemaker cells that generate spontaneous electrical slow waves, and mediate inputs from motor neurons. Absence or loss of ICC are associated with GI motility disorders, including those consequent of diabetes. Studies of ICC have been hampered by the low density of these cells and difficulties in recognizing these cells in cell dispersions. METHODS: Kit(+/copGFP) mice harboring a copepod super green fluorescent protein (copGFP) complementary DNA, inserted at the Kit locus, were generated. copGFP(+) ICC from GI muscles were analyzed using confocal microscopy and flow cytometry. copGFP(+) ICC from the jejunum were purified by a fluorescence-activated cell sorter and validated by cell-specific markers. Kit(+/copGFP) mice were crossbred with diabetic Lep(+/ob) mice to generate compound Kit(+/copGFP);Lep(ob/ob) mutant mice. copGFP(+) ICC from compound transgenic mice were analyzed by confocal microscopy. RESULTS: copGFP in Kit(+/copGFP) mice colocalized with KIT immunofluorescence and thus was predominantly found in ICC. In other smooth muscles, mast cells were also labeled, but these cells were relatively rare in the murine GI tract. copGFP(+) cells from jejunal muscles were Kit(+) and free of contaminating cell-specific markers. Kit(+/copGFP);Lep(ob/ob) mice displayed ICC networks that were dramatically disrupted during the development of diabetes. CONCLUSIONS: Kit(+/copGFP) mice offer a powerful new model to study the function and genetic regulation of ICC phenotypes. Isolation of ICC from animal models will help determine the causes and responses of ICC to therapeutic agents.

Many X-linked microRNAs escape meiotic sex chromosome inactivation.

Meiotic sex chromosome inactivation (MSCI) during spermatogenesis is characterized by transcriptional silencing of genes on both the X and Y chromosomes in mid-to-late pachytene spermatocytes. MSCI is believed to result from meiotic silencing of unpaired DNA because the X and Y chromosomes remain largely unpaired throughout first meiotic prophase. However, unlike X-chromosome inactivation in female embryonic cells, where 25-30% of X-linked structural genes have been reported to escape inactivation, previous microarray- and RT-PCR-based studies of expression of >364 X-linked mRNA-encoding genes during spermatogenesis have failed to reveal any X-linked gene that escapes the silencing effects of MSCI in primary spermatocytes. Here we show that many X-linked miRNAs are transcribed and processed in pachytene spermatocytes. This unprecedented escape from MSCI by these X-linked miRNAs suggests that they may participate in a critical function at this stage of spermatogenesis, including the possibility that they contribute to the process of MSCI itself, or that they may be essential for post-transcriptional regulation of autosomal mRNAs during the late meiotic and early postmeiotic stages of spermatogenesis.

Sertoli cell Dicer is essential for spermatogenesis in mice.

Spermatogenesis requires intact, fully competent Sertoli cells. Here, we investigate the functions of Dicer, an RNaseIII endonuclease required for microRNA and small interfering RNA biogenesis, in mouse Sertoli cell function. We show that selective ablation of Dicer in Sertoli cells leads to infertility due to complete absence of spermatozoa and progressive testicular degeneration. The first morphological alterations appear already at postnatal day 5 and correlate with a severe impairment of the prepubertal spermatogenic wave, due to defective Sertoli cell maturation and incapacity to properly support meiosis and spermiogenesis. Importantly, we find several key genes known to be essential for Sertoli cell function to be significantly down-regulated in neonatal testes lacking Dicer in Sertoli cells. Overall, our results reveal novel essential roles played by the Dicer-dependent pathway in mammalian reproductive function, and thus pave the way for new insights into human infertility.

Birth of mice after intracytoplasmic injection of single purified sperm nuclei and detection of messenger RNAs and MicroRNAs in the sperm nuclei.

We have developed a method that effectively removes all of the perinuclear materials of a mouse sperm head, including the acrosome, plasma membrane, perinuclear theca, and nuclear envelope. By injection of a single purified sperm head into a metaphase II mouse oocyte followed by activation with strontium chloride, 93% of the zygotes developed into two-cell embryos. Although only approximately 17% of the transferred two-cell embryos were born alive, all live pups developed into adults, and they appeared to be normal in reproduction and behavior. We detected RNA species, including mRNAs and miRNAs from the purified sperm heads. Our data demonstrate that pure membrane-free sperm heads are sufficient to produce normal offspring through intracytoplasmic sperm injection and that at least part of the RNA molecules are deeply embedded in the sperm nucleus.

Cloning and expression profiling of testis-expressed microRNAs.

Using a new small RNA cloning method, we identified 141 miRNAs from the mouse testis, of which 29 were novel. The 141 miRNAs were mapped onto all chromosomes except the Y chromosome and 2/3 of these miRNA genes exist as clusters. approximately 70% of these miRNA genes were located in intronic or intergenic regions, whereas the remaining miRNAs were derived from exonic sequences. We further validated these cloned miRNAs by examining their expression in multiple mouse organs including developing testes and also in purified spermatogenic cells using semi-quantitative PCR analyses. Our expression profiling assays revealed that 60% of the testis-expressed miRNAs were ubiquitously expressed and the remaining are either preferentially (35%) or exclusively (5%) expressed in the testis. We also observed a lack of strand selection during testicular miRNA biogenesis, characterized by paired expression of both the 5' strands and 3' strands derived from the same precursor miRNAs. The present work identified numerous miRNAs preferentially or exclusively expressed in the testis, which would be interesting targets for further functional studies.

Cloning and expression profiling of small RNAs expressed in the mouse ovary.

Small noncoding RNAs have been suggested to play important roles in the regulation of gene expression across all species from plants to humans. To identify small RNAs expressed by the ovary, we generated mouse ovarian small RNA complementary DNA (srcDNA) libraries and sequenced 800 srcDNA clones. We identified 236 small RNAs including 122 microRNAs (miRNAs), 79 piwi-interacting RNAs (piRNAs), and 35 small nucleolar RNAs (snoRNAs). Among these small RNAs, 15 miRNAs, 74 piRNAs, and 21 snoRNAs are novel. Approximately 70% of the ovarian piRNAs are encoded by multicopy genes located within the repetitive regions, resembling previously identified repeat-associated small interference RNAs (rasiRNAs), whereas the remaining approximately 30% of piRNA genes are located in nonrepetitive regions of the genome with characteristics similar to the majority of piRNAs originally cloned from the testis. Since these two types of piRNAs display different structural features, we categorized them into two classes: repeat-associated piRNAs (rapiRNAs, equivalent of the rasiRNAs) and non-repeat-associated piRNAs (napiRNAs). Expression profiling analyses revealed that ovarian miRNAs were either ubiquitously expressed in multiple tissues or preferentially expressed in a few tissues including the ovary. Ovaries appear to express more rapiRNAs than napiRNAs, and sequence analyses support that both may be generated through the "ping-pong" mechanism. Unique expression and structural features of these ovarian small noncoding RNAs suggest that they may play important roles in the control of folliculogenesis and female fertility.

Tissue-dependent paired expression of miRNAs.

It is believed that depending on the thermodynamic stability of the 5'-strand and the 3'-strand in the stem-loop structure of a precursor microRNA (pre-miRNA), cells preferentially select the less stable one (called the miRNA or guide strand) and destroy the other one (called the miRNA* or passenger strand). However, our expression profiling analyses revealed that both strands could be co-accumulated as miRNA pairs in some tissues while being subjected to strand selection in other tissues. Our target prediction and validation assays demonstrated that both strands of a miRNA pair could target equal numbers of genes and that both were able to suppress the expression of their target genes. Our finding not only suggests that the numbers of miRNAs and their targets are much greater than what we previously thought, but also implies that novel mechanisms are involved in the tissue-dependent miRNA biogenesis and target selection process.