Decoding the key compounds and mechanism of Shashen Maidong decoction in the treatment of lung cancer.

BACKGROUND: Lung cancer is a malignant tumour with the fastest increase in morbidity and mortality around the world. The clinical treatments available have significant side effects, thus it is desirable to identify alternative modalities to treat lung cancer. Shashen Maidong decoction (SMD) is a commonly used traditional Chinese medicine (TCM) formula for treating lung cancer in the clinic. While the key functional components (KFC) and the underlying mechanisms of SMD treating lung cancer are still unclear. METHODS: We propose a new integrated pharmacology model, which combines a novel node-importance calculation method and the contribution decision rate (CDR) model, to identify the KFC of SMD and to deduce their mechanisms in the treatment of lung cancer. RESULTS: The enriched effective Gene Ontology (GO) terms selected from our proposed node importance detection method could cover 97.66% of enriched GO terms of reference targets. After calculating CDR of active components in key functional network, the first 82 components covered 90.25% of the network information, which were defined as KFC. 82 KFC were subjected to functional analysis and experimental validation. 5-40 µM protocatechuic acid, 100-400 µM paeonol or caffeic acid exerted significant inhibitory activity on the proliferation of A549 cells. The results show that KFC play an important therapeutic role in the treatment of lung cancer by targeting Ras, AKT, IKK, Raf1, MEK, and NF-κB in the PI3K-Akt, MAPK, SCLC, and NSCLC signaling pathways active in lung cancer. CONCLUSIONS: This study provides a methodological reference for the optimization and secondary development of TCM formulas. The strategy proposed in this study can be used to identify key compounds in the complex network and provides an operable test range for subsequent experimental verification, which greatly reduces the experimental workload.

Detecting Key Functional Components Group and Speculating the Potential Mechanism of Xiao-Xu-Ming Decoction in Treating Stroke.

Stroke is a cerebrovascular event with cerebral blood flow interruption which is caused by occlusion or bursting of cerebral vessels. At present, the main methods in treating stroke are surgical treatment, statins, and recombinant tissue-type plasminogen activator (rt-PA). Relatively, traditional Chinese medicine (TCM) has widely been used at clinical level in China and some countries in Asia. Xiao-Xu-Ming decoction (XXMD) is a classical and widely used prescription in treating stroke in China. However, the material basis of effect and the action principle of XXMD are still not clear. To solve this issue, we designed a new system pharmacology strategy that combined targets of XXMD and the pathogenetic genes of stroke to construct a functional response space (FRS). The effective proteins from this space were determined by using a novel node importance calculation method, and then the key functional components group (KFCG) that could mediate the effective proteins was selected based on the dynamic programming strategy. The results showed that enriched pathways of effective proteins selected from FRS could cover 99.10% of enriched pathways of reference targets, which were defined by overlapping of component targets and pathogenetic genes. Targets of optimized KFCG with 56 components can be enriched into 166 pathways that covered 80.43% of 138 pathways of 1,012 pathogenetic genes. A component potential effect score (PES) calculation model was constructed to calculate the comprehensive effective score of components in the components-targets-pathways (C-T-P) network of KFCGs, and showed that ferulic acid, zingerone, and vanillic acid had the highest PESs. Prediction and docking simulations show that these components can affect stroke synergistically through genes such as MEK, NFκB, and PI3K in PI3K-Akt, cAMP, and MAPK cascade signals. Finally, ferulic acid, zingerone, and vanillic acid were tested to be protective for PC12 cells and HT22 cells in increasing cell viabilities after oxygen and glucose deprivation (OGD). Our proposed strategy could improve the accuracy on decoding KFCGs of XXMD and provide a methodologic reference for the optimization, mechanism analysis, and secondary development of the formula in TCM.

Activin B-activated Cdc42 signaling plays a key role in regulating adipose-derived mesenchymal stem cells-mediated skin wound healing.

BACKGROUND: In our previous study, activin B in combination with ADSCs enhances skin wound healing. However, the underlying molecular mechanisms are not well studied. Cdc42 is recognized to play a critical role in the regulation of stem cells. METHODS: Pull-down assay was performed to investigate the activity of Cdc42. The dominant-negative mutant of Cdc42 (Cdc42N17) was used to explore the role of Cdc42 in activin B-induced ADSCs migration, proliferation, and secretion in vitro. Cdc42N17-transfected ADSCs were injected into a full-thickness excisional wound model to explore their efficiency in wound healing in vivo. The wound healing efficacy was evaluated by the wound closure rates and histological examination. The neovascularization and wound contraction were detected by immunohistochemistry staining of CD31 and α-SMA. Finally, the underlying mechanisms were explored by RNA sequencing. RESULTS: Cdc42N17 inhibited ADSCs migration, proliferation, and secretion induced by activin B. Furthermore, Cdc42N17-transfected ADSCs inhibited the wound closure rate and suppressed the expression of CD31 and α-SMA induced by activin B in vivo. The RNA sequencing showed that the differentially expressed genes in Cdc42N17-transfected ADSCs versus ADSCs were associated with cell migration, proliferation, and adhesion. Further study revealed that the Cdc42-Erk-Srf pathway was required for activin B-induced proliferation in ADSCs. CONCLUSIONS: Our study indicates that Cdc42 plays a crucial role in ADSCs-mediated skin wound healing induced by activin B.

Continuous ZnO nanoparticle exposure induces melanoma-like skin lesions in epidermal barrier dysfunction model mice through anti-apoptotic effects mediated by the oxidative stress-activated NF-κB pathway.

BACKGROUND: Increasing interest in the hazardous properties of zinc oxide nanoparticles (ZnO NPs), commonly used as ultraviolet filters in sunscreen, has driven efforts to study the percutaneous application of ZnO NPs to diseased skin; however, in-depth studies of toxic effects on melanocytes under conditions of epidermal barrier dysfunction remain lacking. METHODS: Epidermal barrier dysfunction model mice were continuously exposed to a ZnO NP-containing suspension for 14 and 49 consecutive days in vivo. Melanoma-like change and molecular mechanisms were also verified in human epidermal melanocytes treated with 5.0 µg/ml ZnO NPs for 72 h in vitro. RESULTS: ZnO NP application for 14 and 49 consecutive days induced melanoma-like skin lesions, supported by pigmented appearance, markedly increased number of melanocytes in the epidermis and dermis, increased cells with irregular nuclei in the epidermis, recruited dendritic cells in the dermis and dysregulated expression of melanoma-associated gene Fkbp51, Trim63 and Tsp 1. ZnO NPs increased oxidative injury, inhibited apoptosis, and increased nuclear factor kappa B (NF-κB) p65 and Bcl-2 expression in melanocytes of skin with epidermal barrier dysfunction after continuously treated for 14 and 49 days. Exposure to 5.0 µg/ml ZnO NPs for 72 h increased cell viability, decreased apoptosis, and increased Fkbp51 expression in melanocytes, consistent with histological observations in vivo. The oxidative stress-mediated mechanism underlying the induction of anti-apoptotic effects was verified using the reactive oxygen species scavenger N-acetylcysteine. CONCLUSIONS: The entry of ZnO NPs into the stratum basale of skin with epidermal barrier dysfunction resulted in melanoma-like skin lesions and an anti-apoptotic effect induced by oxidative stress, activating the NF-κB pathway in melanocytes.

Corrigendum: Detecting key functional components group and speculating the potential mechanism of Xiao-Xu-Ming decoction in treating stroke.

[This corrects the article DOI: 10.3389/fcell.2022.753425.].

MicroRNA-200b/c-3p regulate epithelial plasticity and inhibit cutaneous wound healing by modulating TGF-ß-mediated RAC1 signaling.

Cutaneous wound healing is pivotal for human skin to regain barrier function against pathogens. MicroRNAs (miRNAs) have been found to play regulatory roles in wound healing. However, the mechanism of miRNA regulation remains largely unknown. In this study, we focused on microRNA-200b/c-3p (miR-200b/c-3p) whose expression was abundant in intact epidermis, but dramatically decreased in skin wounds. In silico prediction identified RAC1 as a potential miR-200b/c-3p target. Luciferase reporter assay confirmed that miR-200b/c-p repressed RAC1 by direct targeting to its mRNA 3'UTR. Consistently, miR-200b/c-3p expression was discordantly related to RAC1 protein level during wound healing. Forced miR-200b/c-3p expression repressed RAC1 and inhibited keratinocyte migration as well as re-epithelialization in a mouse back skin full-thickness wound healing model. Mechanistically, miR-200b/c-3p modulated RAC1 to inhibit cell migration by repressing lamellipodia formation and intercellular adhesion dissolution in keratinocytes. Furthermore, we found that TGF-ß1, which was highly expressed in skin wounds, contributed to the downregulation of miR-200b/c-3p in wound edge keratinocytes. Taken together, miR-200b/c-3p-mediated RAC1 repression inhibited keratinocyte migration to delay re-epithelialization. TGF-ß1 induction attenuated miR-200b/c-3p regulation of RAC1 signaling in cutaneous wounds and the repression of miR-200b/c-3p accelerated keratinocyte migration to promote wound healing. Our data provide new insight into how miR-200b/c-3p affects keratinocyte migration and highlight the potential of miR-200b/c-3p targeting for accelerating wound healing.

Induced pluripotent stem cells-derived microvesicles accelerate deep second-degree burn wound healing in mice through miR-16-5p-mediated promotion of keratinocytes migration.

Background: Induced pluripotent stem cells (iPSCs) have emerged as a promising treatment paradigm for skin wounds. Extracellular vesicles are now recognized as key mediators of beneficial stem cells paracrine effects. In this study, we investigated the effect of iPSCs-derived microvesicles (iPSCs-MVs) on deep second-degree burn wound healing and explored the underlying mechanism. Methods: iPSCs-MVs were isolated and purified from conditioned medium of iPSCs and confirmed by electron micrograph and size distribution. In deep second-degree burn model, iPSCs-MVs were injected subcutaneously around wound sites and the efficacy was assessed by measuring wound closure areas, histological examination and immunohistochemistry staining. In vitro, CCK-8, EdU staining and scratch assays were used to assess the effects of iPSCs-MVs on proliferation and migration of keratinocytes. Next, we explored the underlying mechanisms by high-throughput microRNA sequencing. The roles of the miR-16-5p in regulation of keratinocytes function induced by iPSCs-MVs were assessed. Moreover, the target gene which mediated the biological effects of miR-16-5p in keratinocytes was also been detected. Finally, we examined the effect of local miR-16-5p treatment on deep second degree-burns wound healing in mice. Results: The local transplantation of iPSCs-MVs into the burn wound bed resulted in accelerated wound closure including the increased re-epithelialization. In vitro, iPSCs-MVs could promote the migration of keratinocytes. We also found that miR-16-5p is a critical factor in iPSCs-MVs-induced promotion of keratinocytes migration in vitro through activating p38/MARK pathway by targeting Desmoglein 3 (Dsg3). Finally, we confirmed that local miR-16-5p treatment could boost re-epithelialization during burn wound healing. Conclusion: Therefore, our results indicate that iPSCs-MVs-derived miR-16-5p may be a novel therapeutic approach for deep second-degree burn wound healing.

Effect of iPSCs-derived keratinocytes on healing of full-thickness skin wounds in mice.

Induced pluripotent stem cells (iPSCs) provide new approaches for the management of severe skin wound healing due to their infinite proliferative capacity, pluripotency into multiple lineages, and important ethical acceptability. In this study, we aimed to differentiate iPSCs into keratinocytes and to observe the therapeutic effects of transplanted iPSCs-derived keratinocytes on wound healing in mice. Here, mouse iPSCs had been successfully differentiated into keratinocytes. Next, iPSCs-derived keratinocytes labeled by CSFE were injected directly into the full-thickness skin wound. Hematoxylin & Eosin, Masson's trichrome, EdU staining and immunohistochemical staining were performed to assess the effects of iPSCs-derived keratinocytes on wound healing. Our results showed that transplantation of iPSCs-derived keratinocytes into full-thickness skin wound site accelerated re-epithelialization and reduced scar formation. In addition, we found that conditioned medium of iPSCs-derived keratinocytes reduced the expression of α-SMA and COL1 and increased the expression of MMP1 in fibroblasts in vitro. Further mechanism studies show the TNF-α-induced activation of NF-κB is involved in the effect of conditioned medium of iPSCs-derived keratinocytes on fibroblasts. In conclusion, this study has shown that iPSCs-derived keratinocytes decrease the healing time by increasing the epithelization rate and reduce scarring, suggesting a possible new treatment for skin wound healing.

Cdc42 Deficiency Leads To Epidermal Barrier Dysfunction by Regulating Intercellular Junctions and Keratinization of Epidermal Cells during Mouse Skin Development.

Rationale: Cdc42 is a Rho GTPase that regulates diverse cellular functions. Here, we used genetic techniques to investigate the role of Cdc42 in epidermal development and epidermal barrier formation. Methods: Keratinocyte-restricted Cdc42 knockout mice were generated with the Cre-LoxP system under the keratin 14 (K14) promoter. The skin and other tissues were collected from mutant and wild-type mice, and their cellular, molecular, morphological, and physiological features were analyzed. Results: Loss of Cdc42 in the epidermis in vivo resulted in neonatal lethality and impairment of epidermal barrier formation. Cdc42 deficiency led to the loss of epidermal stem cells. The absence of Cdc42 led to increased thickening of the epidermis, which was associated with increased proliferation and reduced apoptosis of keratinocytes. In addition, Cdc42 deficiency damaged tight junctions, adherens junctions and desmosomes. RNA sequencing results showed that the most significantly altered genes were enriched by the terms of "keratinization" and "cornified envelope" (CE). Among the differentially expressed genes in the CE term, several members of the small proline-rich protein (SPRR) family were upregulated. Further study revealed that there may be a Cdc42-SPRR pathway, which may correlate with epidermal barrier function. Conclusions: Our study indicates that Cdc42 is essential for epidermal development and epidermal barrier formation. Defects in Cdc42-SPRR signaling may be associated with skin barrier dysfunction and a variety of skin diseases.

mDia1 and Cdc42 Regulate Activin B-Induced Migration of Bone Marrow-Derived Mesenchymal Stromal Cells.

In a previous study, we have shown that Activin B is a potent chemoattractant for bone marrow-derived mesenchymal stromal cells (BMSCs). As such, the combination of Activin B and BMSCs significantly accelerated rat skin wound healing. In another study, we showed that RhoA activation plays a key role in Activin B-induced BMSC migration. However, the role of the immediate downstream effectors of RhoA in this process is unclear. Here, we demonstrated that mammalian homolog of Drosophila diaphanous-1 (mDia1), a downstream effector of RhoA, exerts a crucial function in Activin B-induced BMSC migration by promoting membrane ruffling, microtubule morphology, and adhesion signaling dynamics. Furthermore, we showed that Activin B does not change Rac1 activity but increases Cdc42 activity in BMSCs. Inactivation of Cdc42 inhibited Activin B-stimulated Golgi reorientation and the cell migration of BMSCs. Furthermore, knockdown of mDia1 affected Activin B-induced BMSC-mediated wound healing in vivo. In conclusion, this study demonstrated that the RhoA-mDia1 and Cdc42 pathways regulate Activin B-induced BMSC migration. This study may help to optimize clinical MSC-based transplantation strategies to promote skin wound healing. Stem Cells 2019;37:150-162.

Inhibition of the JNK/MAPK signaling pathway by myogenesis-associated miRNAs is required for skeletal muscle development.

Skeletal muscle differentiation is controlled by multiple cell signaling pathways, however, the JNK/MAPK signaling pathway dominating this process has not been fully elucidated. Here, we report that the JNK/MAPK pathway was significantly downregulated in the late stages of myogenesis, and in contrast to P38/MAPK pathway, it negatively regulated skeletal muscle differentiation. Based on the PAR-CLIP-seq analysis, we identified six elevated miRNAs (miR-1a-3p, miR-133a-3p, miR-133b-3p, miR-206-3p, miR-128-3p, miR-351-5p), namely myogenesis-associated miRNAs (mamiRs), negatively controlled the JNK/MAPK pathway by repressing multiple factors for the phosphorylation of the JNK/MAPK pathway, including MEKK1, MEKK2, MKK7, and c-Jun but not JNK protein itself, and as a result, expression of transcriptional factor MyoD and mamiRs were further promoted. Our study revealed a novel double-negative feedback regulatory pattern of cell-specific miRNAs by targeting phosphorylation kinase signaling cascade responsible for skeletal muscle development.

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration.

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Antagonistic interaction between Wnt and Notch activity modulates the regenerative capacity of a zebrafish fibrotic liver model.

UNLABELLED: In chronic liver failure patients with sustained fibrosis, excessive accumulation of extracellular matrix proteins substantially dampens the regenerative capacity of the hepatocytes, resulting in poor prognosis and high mortality. Currently, the mechanisms and the strategies of inducing endogenous cellular sources such as hepatic progenitor cells (HPCs) to regenerate hepatocytes in various contexts of fibrogenic stimuli remain elusive. Here we aim to understand the molecular and cellular mechanisms that mediate the effects of sustained fibrosis on hepatocyte regeneration using the zebrafish as a model. In the ethanol-induced fibrotic zebrafish model, we identified a subset of HPCs, responsive to Notch signaling, that retains its capacity to regenerate as hepatocytes. Discrete levels of Notch signaling modulate distinct cellular outcomes of these Notch-responsive HPCs in hepatocyte regeneration. Lower levels of Notch signaling promote amplification and subsequent differentiation of these cells into hepatocytes, while high levels of Notch signaling suppress these processes. To identify small molecules facilitating hepatocyte regeneration in the fibrotic liver, we performed chemical screens and identified a number of Wnt agonists and Notch antagonists. Further analyses demonstrated that these Wnt agonists are capable of attenuating Notch signaling by inducing Numb, a membrane-associated protein that inhibits Notch signaling. This suggests that the antagonistic interplay between Wnt and Notch signaling crucially affects hepatocyte regeneration in the fibrotic liver. CONCLUSION: Our findings not only elucidate how signaling pathways and cell-cell communications direct the cellular response of HPCs to fibrogenic stimuli, but also identify novel potential therapeutic strategies for chronic liver disease.

Deciphering the transcriptional regulation of microRNA genes in humans with ACTLocater.

Understanding the transcriptional regulation of microRNAs (miRNAs) is extremely important for determining the specific roles they play in signaling cascades. However, precise identification of transcription factor binding sites (TFBSs) orchestrating the expressions of miRNAs remains a challenge. By combining accessible chromatin sequences of 12 cell types released by the ENCODE Project, we found that a significant fraction (~80%) of such integrated sequences, evolutionary conserved and in regions upstream of human miRNA genes that are independently transcribed, were preserved across cell types. Accordingly, we developed a computational method, Accessible and Conserved TFBSs Locater (ACTLocater), incorporating this chromatin feature and evolutionary conservation to identify the TFBSs associated with human miRNA genes. ACTLocater achieved high positive predictive values, as revealed by the experimental validation of FOXA1 predictions and by the comparison of its predictions of some other transcription factors (TFs) to empirical ChIP-seq data. Most notably, ACTLocater was widely applicable as indicated by the successful prediction of TF → miRNA interactions in cell types whose chromatin accessibility profiles were not incorporated. By applying ACTLocater to TFs with characterized binding specificities, we compiled a novel repository of putative TF → miRNA interactions and displayed it in ACTViewer, providing a promising foundation for future investigations to elucidate the regulatory mechanisms of miRNA transcription in humans.

MicroRNA-195 plays a tumor-suppressor role in human glioblastoma cells by targeting signaling pathways involved in cellular proliferation and invasion.

Accumulating evidence has implicated the deregluation of miRNAs in tumorigenesis. Previous studies have reported that microRNA-195 (miR-195) is markedly down-regulated in human glioblastoma cells, compared with normal brain tissue, but the biological role of miR-195 in glioblastoma development is currently unknown. In this study, we define a tumor-suppressor role for miR-195 in human glioblastoma cells. Over-expression of miR-195 in glioblastoma cell lines robustly arrested cell cycle progression and significantly repressed cellular invasion. We identified E2F3 and CCND3 as functional downstream targets of miR-195 in glioblastoma cells. Through knockdown studies, we demonstrated that E2F3 was the dominant effector of miR-195-mediated cell cycle arrest and that CCND3 was a key mediator of miR-195-induced inhibition of glioblastoma cell invasion. Furthermore, we showed that p27(Kip1) was an important regulator downstream of CCND3 and that the accumulation of p27(Kip1) in the cytoplasm might be responsible for the miR-195-mediated cell invasion inhibition in glioblastoma cells. This work provides evidence for the initial mechanism by which miR-195 negatively regulates both the proliferation and invasion of glioblastoma cells, suggesting that the down-regulation of miR-195 might contribute to the malignant transformation of glioblastoma cells and could be a molecular signature associated with glioblastoma progression.

Insulin-like growth factor-1 receptor is regulated by microRNA-133 during skeletal myogenesis.

BACKGROUND: The insulin-like growth factor (IGF) signaling pathway has long been established as playing critical roles in skeletal muscle development. However, the underlying regulatory mechanism is poorly understood. Recently, a large family of small RNAs, named microRNAs (miRNAs), has been identified as key regulators for many developmental processes. Because miRNAs participate in the regulation of various signaling pathways, we hypothesized that miRNAs may be involved in the regulation of IGF signaling in skeletal myogenesis. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we determined that the cell-surface receptor IGF-1R is directly regulated by a muscle-specific miRNA, microRNA-133 (miR-133). A conserved and functional binding site for miR-133 was identified in the 3'untranslated region (3'UTR) of IGF-1R. During differentiation of C2C12 myoblasts, IGF-1R protein, but not messenger RNA (mRNA) expression, was gradually reduced, concurrent with the upregulation of miR-133. Overexpression of miR-133 in C2C12 cells significantly suppressed IGF-1R expression at the posttranscriptional level. We also demonstrated that both overexpression of miR-133 and knockdown of IGF-1R downregulated the phosphorylation of Akt, the central mediator of the PI3K/Akt signaling pathway. Furthermore, upregulation of miR-133 during C2C12 differentiation was significantly accelerated by the addition of IGF-1. Mechanistically, we found that the expression of myogenin, a myogenic transcription factor reported to transactivate miR-133, was increased by IGF-1 stimulation. CONCLUSION/SIGNIFICANCE: Our results elucidate a negative feedback circuit in which IGF-1-stimulated miR-133 in turn represses IGF-1R expression to modulate the IGF-1R signaling pathway during skeletal myogenesis. These findings also suggest that miR-133 may be a potential therapeutic target in muscle diseases.

High glucose down-regulates miR-29a to increase collagen IV production in HK-2 cells.

Deposition of collagen IV in proximal tubule cells (PTCs) plays an important role during diabetic nephropathy, but the mechanism underlying excessive production of collagen IV remains poorly understood. In this study, we examined the miRNA profile of HK-2 cells and found that high glucose/TGF-beta1 induced significant down-regulation of miR-29a. We then showed that miR-29a negatively regulated collagen IV by directly targeting the 3'UTRs of col4a1 and col4a2. These results suggest that miR-29a acts as a repressor to fine-tune collagen expression and that the reduction of miR-29a caused by high glucose may increase the risk of excess collagen deposition in PTCs.

Identification of novel chicken microRNAs and analysis of their genomic organization.

MicroRNAs (miRNAs) represent a family of small noncoding RNAs with important regulatory roles in diverse biological processes ranging from cell differentiation to organism development. In chickens, the full set of miRNAs and the expression patterns of miRNAs during development are still poorly understood when compared to the other vertebrates. In this study, we identified 29 novel miRNAs and 140 potential miRNA loci in the chicken genome by combining the experimental and computational analyses. Detailed expression patterns of 49 miRNAs were first characterized by Northern blotting and indicated the cooperativity of the miRNA expression with their function in embryogenesis and organogenesis. Twenty-seven miRNA clusters were systematically evaluated in the chicken genome and diverse expression patterns for closely linked miRNAs were observed. Our results significantly expand the set of known miRNAs in the chicken and provide the basis for understanding the structural and functional evolution of miRNA genes in vertebrates.

snoSeeker: an advanced computational package for screening of guide and orphan snoRNA genes in the human genome.

Small nucleolar RNAs (snoRNAs) represent an abundant group of non-coding RNAs in eukaryotes. They can be divided into guide and orphan snoRNAs according to the presence or absence of antisense sequence to rRNAs or snRNAs. Current snoRNA-searching programs, which are essentially based on sequence complementarity to rRNAs or snRNAs, exist only for the screening of guide snoRNAs. In this study, we have developed an advanced computational package, snoSeeker, which includes CDseeker and ACAseeker programs, for the highly efficient and specific screening of both guide and orphan snoRNA genes in mammalian genomes. By using these programs, we have systematically scanned four human-mammal whole-genome alignment (WGA) sequences and identified 54 novel candidates including 26 orphan candidates as well as 266 known snoRNA genes. Eighteen novel snoRNAs were further experimentally confirmed with four snoRNAs exhibiting a tissue-specific or restricted expression pattern. The results of this study provide the most comprehensive listing of two families of snoRNA genes in the human genome till date.