Genome-wide differential expression profiling of long non-coding RNAs in FOXA2 knockout iPSC-derived pancreatic cells.

BACKGROUND: Our recent studies have demonstrated the crucial involvement of FOXA2 in the development of human pancreas. Reduction of FOXA2 expression during the differentiation of induced pluripotent stem cells (iPSCs) into pancreatic islets has been found to reduce α-and ß-cell masses. However, the extent to which such changes are linked to alterations in the expression profile of long non-coding RNAs (lncRNAs) remains unraveled. METHODS: Here, we employed our recently established FOXA2-deficient iPSCs (FOXA2-/- iPSCs) to investigate changes in lncRNA profiles and their correlation with dysregulated mRNAs during the pancreatic progenitor (PP) and pancreatic islet stages. Furthermore, we constructed co-expression networks linking significantly downregulated lncRNAs with differentially expressed pancreatic mRNAs. RESULTS: Our results showed that 442 lncRNAs were downregulated, and 114 lncRNAs were upregulated in PPs lacking FOXA2 compared to controls. Similarly, 177 lncRNAs were downregulated, and 59 lncRNAs were upregulated in islet cells lacking FOXA2 compared to controls. At both stages, we observed a strong correlation between lncRNAs and several crucial pancreatic genes and TFs during pancreatic differentiation. Correlation analysis revealed 12 DE-lncRNAs that strongly correlated with key downregulated pancreatic genes in both PPs and islet cell stages. Selected DE-lncRNAs were validated using RT-qPCR. CONCLUSIONS: Our data indicate that the observed defects in pancreatic islet development due to the FOXA2 loss is associated with significant alterations in the expression profile of lncRNAs. Therefore, our findings provide novel insights into the role of lncRNA and mRNA networks in regulating pancreatic islet development, which warrants further investigations. Video Abstract.

Expression profiling of N6-methyladenosine-modified mRNA in PC12 cells in response to unconjugated bilirubin.

BACKGROUND: Abnormal methylation of N6-methyladenosine (m6A) is reportedly associated with central nervous system disorders. However, the role of m6A mRNA methylation in unconjugated bilirubin (UCB) neurotoxicity requires further research. METHODS: Rat pheochromocytoma PC12 cells treated with UCB were used as in vitro models. After the PC12 cells were treated with UCB (0, 12, 18, and 24 µM) for 24 h, the total RNA m6A levels were measured using an m6A RNA methylation quantification kit. The expression of m6A demethylases and methyltransferases was detected through western blotting. We determined the m6A mRNA methylation profile in PC12 cells exposed to UCB (0 and 18 µM) for 24 h using methylated RNA immunoprecipitation sequencing (MeRIP-seq). RESULTS: Compared with the control group, UCB (18 and 24 µM) treatment decreased the expression of the m6A demethylase ALKBH5 and increased the expression of the methyltransferases METTL3 and METTL14, which resulted in an increase in the total m6A levels in PC12 cells. Furthermore, 1533 m6A peaks were significantly elevated and 1331 peaks were reduced in the UCB (18 µM)-treated groups compared with those in the control group. Genes with differential m6A peaks were mainly enriched in protein processing in the endoplasmic reticulum, ubiquitin-mediated proteolysis, cell cycle, and endocytosis. Through combined analysis of the MeRIP-seq and RNA sequencing data, 129 genes with differentially methylated m6A peaks and differentially expressed mRNA levels were identified. CONCLUSION: Our study suggests that the modulation of m6A methylation modifications plays a significant role in UCB neurotoxicity.

Protective effects of P2X7R antagonist in sepsis-induced acute lung injury in mice via regulation of circ_0001679 and circ_0001212 and downstream Pln, Cdh2, and Nprl3 expression.

BACKGROUND: Sepsis induces pulmonary P2X7 receptor (P2X7 R) expression and P2X7 R-knockout reduced lung inflammation in mice. The present study investigated the expression of circular RNA (circRNA) and mRNA in sepsis-induced acute lung injury (ALI) treated with a P2X7 R antagonist. METHODS: Sepsis was induced by tracheal administration of lipopolysaccharide (LPS), and the mice were then divided into two groups: without [sepsis + dimethyl sulfoxide (DMSO)] or with P2X7 R antagonist treatment (sepsis + P2X7 A). Sham mice were administrated sterile normal saline. Serum levels of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, pathological changes, cell apoptosis and P2X7 R expression in lung were assessed, followed by RNA sequencing (RNA-seq) and bioinformatics analyses. A quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to validate circRNAs and mRNAs. RESULTS: Compared to the sham group, LPS-induced sepsis produced obvious pathological changes in lung tissue, as well as increased apoptotic lung cells, serum TNF-α and IL-1ß levels, and P2X7 R expression; P2X7 R antagonism significantly ameliorated these changes. RNA-seq identified many dysregulated circRNAs and mRNAs during sepsis, whereas this changed with P2X7 R antagonism. RT-qPCR confirmed that Mus musculus (mmu)_circ_0001679, mmu_circ_0001212, phospholamban (Pln), cadherin-2 (Cdh2) and nitrogen permease regulator 3-like (Nprl3) expression were significantly increased in the sepsis + DMSO group compared to that in the sham group but were decreased in the sepsis + P2X7 A group compared to that in the sepsis + DMSO group. The circRNA-microRNA-mRNA coexpression network indicated that mmu_circ_0001679 may regulate Nprl3 and that mmu_circ_0001212 may similarly regulate Pln, Cdh2 and Nprl3 as a competing endogenous RNA. CONCLUSIONS: P2X7 R antagonism attenuates sepsis-induced ALI by inhibiting dysregulated expression of circRNA (circ_0001679, circ_0001212) and mRNA (Pln, Cdh2 and Nprl3).

Identification and verification of key genes in varicocele rats through high-throughput sequencing and bioinformatics analysis.

Varicocele (VC) is the most common treatable cause of infertility, but it is difficult to distinguish fertile from infertile VC populations because the pathogenesis is unclear. In order to study the related mechanism of VC causing male sterility, we made VC rat model by surgery, analysed the rat epididymal spermatozoa and used the transcriptome sequencing to compare all the mRNA expression differences in testicular tissue between VC rats and control rats. The differentially expressed genes (DEGs) of testicular tissue were also screened by the limma package in R software (version 3.6.1). The 273 DEGs were identified from the four profile data sets including 124 up-regulated genes and 149 down-regulated genes in the VC group compared to control group. We found that Sod1, Casp9, Atg7, Casp3 and Sirt1 in module 1 had higher degrees of connectivity in the first 10 hub genes. Gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis demonstrated that Sod1, Casp9, Atg7, Casp3 and Sirt1 are enriched in regulation of oxidative stress-induced cell death (GO:1,903,201) and Amyotrophic lateral sclerosis (KEGG:05,014). From the above evidence, we speculate that hypoxia plays an important role in the occurrence and development of VC, and it induced the abnormal expression of autophagy and apoptosis-related proteins may involve in the development of VC-associated infertility. Sod1, Casp9, Atg7, Casp3 and Sirt1 as well as their module are hub genes for VC, which will have attractive applications to provide new treatment targets for VC.

mRNA expression characteristics are different in irreversibly atrophic intrinsic muscles of the forepaw compared with reversibly atrophic biceps in a rat model of obstetric brachial plexus palsy (OBPP).

In obstetric brachial plexus palsy (OBPP), irreversible muscle atrophy occurs much faster in intrinsic muscles of the hand than in the biceps. To elucidate the mechanisms involved, mRNA expression profiles of denervated intrinsic muscles of the forepaw (IMF) and denervated biceps were determined by microarray using the rat model of OBPP where atrophy of IMF is irreversible while atrophy of biceps is reversible. Relative to contralateral control, 446 dysregulated mRNAs were detected in denervated IMF and mapped to 51 KEGG pathways, and 830 dysregulated mRNAs were detected in denervated biceps and mapped to 52 KEGG pathways. In denervated IMF, 10 of the pathways were related to muscle regulation; six with down-regulated and one with up-regulated mRNAs. The remaining three pathways had both up- and down-regulated mRNAs. In denervated biceps, 13 of the pathways were related to muscle regulation, six with up-regulated and seven with down-regulated mRNAs. Five of the pathways with up-regulated mRNAs were related to regrowth and differentiation of muscle cells. Among the 23 pathways with dysregulated mRNAs, 13 were involved in regulation of neuromuscular junctions. Our results demonstrated that mRNAs expression characteristics in irreversibly atrophic denervated IMF were different from those in reversibly atrophic denervated biceps; dysregulated mRNAs in IMF were associated with inactive pathways of muscle regulation, and in biceps they were associated with active pathways of regrowth and differentiation. Lack of self-repair potential in IMF may be a major reason why atrophy of IMF becomes irreversible much faster than atrophy of biceps after denervation.

Integrative Transcriptomic Profiling of the Wilms Tumor.

Our study aimed to identify relevant transcriptomic biomarkers for the Wilms tumor, the most common pediatric kidney cancer, independent of the histological type and stage. Using next-generation sequencing, we analyzed the miRNA profiles of 74 kidney samples, which were divided into two independent groups: fresh frozen tissue and formalin-fixed paraffin-embedded tissue samples. Subsequent mRNA expression profiling and pathway analysis were performed to establish the interplay and potential involvement of miRNAs and mRNA in the Wilms tumor. Comparative analysis, irrespective of post-dissection tissue processing, revealed 41 differentially expressed miRNAs, with 27 miRNAs having decreased expression and 14 miRNAs having increased expression in the Wilms tumor tissue compared to healthy kidney tissue. Among global mRNA transcriptomic profile differences, cross-sectional analysis suggested a limited list of genes potentially regulated by differentially expressed miRNAs in the Wilms tumor. This study identified the comprehensive miRNA and mRNA profile of the Wilms tumor using next-generation sequencing and bioinformatics approach, providing better insights into the pathogenesis of the Wilms tumor. The identified Wilms tumor miRNAs have potential as biomarkers for the diagnosis and treatment of the Wilms tumor, regardless of histological subtype and disease stage.

Integrated Analysis of Transcriptome mRNA and miRNA Profiles Reveals Self-Protective Mechanism of Bovine MECs Induced by LPS.

Many studies have investigated the molecular crosstalk between mastitis-pathogens and cows by either miRNA or mRNA profiles. Here, we employed both miRNA and mRNA profiles to understand the mechanisms of the response of bovine mammary epithelial cells (bMECs) to lipopolysaccharide (LPS) by RNA-Seq. The total expression level of miRNAs increased while mRNAs reduced after LPS treatment. About 41 differentially expressed mRNAs and 45 differentially expressed miRNAs involved in inflammation were screened out. We found the NFκB-dependent chemokine, CXCL1, CXCL3, CXCL6, IL8, and CX3CL1 to be strongly induced. The anti-apoptosis was active because BCL2A1 and BIRC3 significantly increased with a higher expression. The effects of anti-microbe and inflammation were weakly activated because TNF, IL1, CCL20, CFB, S100A, MMP9, and NOS2A significantly increased but with a low expression, IL6 and ß-defensin decreased. These activities were supervised by the NFKBIA to avoid excessive damage to bMECs. The bta-let-7a-5p, bta-miR-30a-5p, bta-miR-125b, and bta-miR-100 were essential to regulate infection process in bMECs after LPS induction. Moreover, the lactation potential of bMECs was undermined due to significantly downregulated SOSTDC1, WNT7B, MSX1, and bta-miR-2425-5p. In summary, bMECs may not be good at going head-to-head with the pathogens; they seem to be mainly charged with sending out signals for help and anti-apoptosis for maintaining lives after LPS induction.

Calculating LRs for presence of body fluids from mRNA assay data in mixtures.

Messenger RNA (mRNA) profiling can identify body fluids present in a stain, yielding information on what activities could have taken place at a crime scene. To account for uncertainty in such identifications, recent work has focused on devising statistical models to allow for probabilistic statements on the presence of body fluids. A major hurdle for practical adoption is that evidentiary stains are likely to contain more than one body fluid and current models are ill-suited to analyse such mixtures. Here, we construct a likelihood ratio (LR) system that can handle mixtures, considering the hypotheses H1: the sample contains at least one of the body fluids of interest (and possibly other body fluids); H2: the sample contains none of the body fluids of interest (but possibly other body fluids). Thus, the LR-system outputs an LR-value for any combination of mRNA profile and set of body fluids of interest that are given as input. The calculation is based on an augmented dataset obtained by in silico mixing of real single body fluid mRNA profiles. These digital mixtures are used to construct a probabilistic classification method (a 'multi-label classifier'). The probabilities produced are subsequently used to calculate an LR, via calibration. We test a range of different classification methods from the field of machine learning, ways to preprocess the data and multi-label strategies for their performance on in silico mixed test data. Furthermore, we study their robustness to different assumptions on background levels of the body fluids. We find logistic regression works as well as more flexible classifiers, but shows higher robustness and better explainability. We test the system's performance on lab-generated mixture samples, and discuss practical usage in case work.

Prospective Evaluation of a Circulating Tumor Cell Sensitivity Profile to Predict Response to Cisplatin Chemotherapy in Metastatic Breast Cancer Patients.

BACKGROUND: Cisplatin (cDDP) has regained interest for metastatic breast cancer (MBC) patients, given the platinum sensitivity in subtypes and better manageable toxicity. Here, the primary aim was to determine whether molecular characteristics of circulating tumor cells (CTCs) could identify patients responding to cDDP and to describe the outcomes to cDDP monotherapy in a large group of MBC patients pretreated with anthracycline- and taxane-based treatments. METHODS: Based on cell line data, a CTC-cDDP-sensitivity profile was generated. Applying an A'Herns single-stage phase II design, further investigation was considered worthwhile if 5/10 patients with a favorable profile responded to cDDP. Patients received 70mg/m2 cDDP every three weeks, CTCs were enumerated and the CTC-cDDP-sensitivity profile was determined. In total, 65 heavily pretreated MBC patients (77% received ≥2 lines of previous chemotherapy for MBC) were eligible for the per-protocol analysis. Primary endpoint was response rate, secondary endpoints included best observed response, progression-free survival (PFS) and overall survival (OS). RESULTS: The best observed response during cDDP therapy was a partial response in 7% and stable disease in 56% of the patients. None of the patients with a favorable CTC-cDDP-sensitivity profile had a response. The median baseline CTC count was 8 (range 0-3254). Patients with <5 CTCs had a better PFS and OS than patients with ≥5 CTCs (median PFS 4.5 months (95%CI 2.38-6.62) vs. 2.1 months [(95%CI 1.34-2.80)(p=0.009)] and median OS 13.1 months (95%CI 9.89-16.33) vs. 5.6 months [(95%CI 3.60-7.64)(p=0.003)]. No other factors than CTC count were associated with outcome to cDDP therapy, including triple-negative breast cancer versus ER-positive tumors. CONCLUSIONS: The CTC-cDDP-sensitivity profile was unable to select patients responding to cDDP monotherapy. In an unselected group of heavily pretreated MBC patients, cDDP yields outcomes comparable to other chemotherapeutic regimens for heavily pretreated MBC patients. CTC count was the only factor associated with outcome in these patients. CLINICAL TRIAL REGISTRATION: (https://www.trialregister.nl/trial/3885, identifier NTR4046).

mRNA Profiling for miR-124-mediated Repair in Spinal Cord Injury.

The miRNA miR-124 has been reported to be a promising target for the repair of spinal cord injury (SCI), which is a devastating neurological condition. This study aimed to investigate the underlying molecular mechanisms of miR-124-mediated SCI repair. We established miR-124 SCI model rats and further treated them with agomiR-124 for 14 days. After that, their spinal cords were sectioned, and levels of NeuN, GFAP, and NF-200 were measured via immunofluorescence or via immunohistochemistry. In addition, the spinal dorsal horns were collected for sequencing of total RNA. Differentially expressed (DE) mRNAs were then profiled and a number of these were further verified with qPCR. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to predict the potential functions of the DE mRNAs. AgomiR-124 was found to significantly inhibit the decrease of neurons and the activation of astrocytes, while promoting NF-200 expression in the dorsal horn. At fourteen days after agomiR-124 treatment, a total of 85 mRNAs were upregulated and 80 mRNAs were downregulated. We focused our analysis of the DE mRNAs on the top 20 most DE mRNAs, and found four upregulated genes (Nploc4, Yme1l1, LOC103693564, and Aspa) and four downregulated genes (Epb41l2, LOC100911685, LOC100910833, and Smarcc1), which are likely to be of interest to SCI researchers. In addition, we noted that Tal1 is a potential target gene of miR-124, and that a low level of this gene promoted the proliferation of neuronal precursor cells and inhibited their differentiation. In conclusion, miR-124 was able to mediate SCI repair by altering the expression of various mRNAs in rats. The miR-124/Tal1 axis may participate in the treatment of SCI by agomiR-124 by repopulating neural stem cells.

Ovary removal modifies liver message RNA profiles in single Comb White Leghorn chickens.

Ovaries produce sex hormones, and ovariectomized animals are often used as models for ovarian dysfunction. The liver is a vital organ involved in metabolism and immunity. In the present study, we conducted experiments to investigate the effects of ovariectomy on transcription and metabolic processes in the liver in chicken. Eight Single Comb White Leghorn (SCWL) female chickens were ovariectomized at 17 wk of age, and 8 intact SCWL females served as controls. At 100 wk of age, all chickens were euthanized. High-throughput transcriptome sequencing was performed on liver RNA obtained from ovariectomized and intact females. A total of 267 differentially expressed genes (DEG) were identified in our study. After analysis using DAVID functional annotation tool, one significant Kyoto Encyclopedia of Genes and Genomes pathway, the phosphatidylinositol signaling pathway, was clustered. Gene Ontology enrichment analysis yielded 46 significant Gene Ontology terms. Among terms describing biological processes, the glycerolipid metabolic and lipid localization processes were dominant. The anabolic genes, PEPCK and GK5, and the catabolic genes, VTG1; VTG2; PLD5; DGKQ; DGKE; and FABP3, were detected in ovariectomized chickens. Differentially expressed genes such as ENSGALG00000000162, IL-1Β, SVOPL, and CA12 implied that livers in ovariectomized chickens were subjected to strong inflammatory reactions, whereas defenses against endogenous materials were compromised. A comprehensive view of gene expression in the liver of ovariectomized chickens would advance our understanding of lipid metabolism, glycometabolism, and their relationships to pathologies induced by absence of the ovary. The identified DEG indicated that ovariectomy disturbed lipid metabolism in the liver and was accompanied by an increase in hepatic gluconeogenesis and reductions in phosphatidic acid synthesis and lipid carrier capacity.

Prediction of High-Grade Clear Cell Renal Cell Carcinoma Based on Plasma mRNA Profiles in Patients with Localized Pathologic T1N0M0 Stage Disease.

A high nuclear grade is crucial to predicting tumor recurrence and metastasis in clear cell renal cell carcinomas (ccRCCs). We aimed to compare the mRNA profiles of tumor tissues and preoperative plasma in patients with localized T1 stage ccRCCs, and to evaluate the potential of the plasma mRNA profile for predicting high-grade ccRCCs. Data from a prospective cohort (n = 140) were collected between November 2018 and November 2019. Frozen tumor tissues and plasma were used to measure PBRM1, BAP1, SET domain-containing 2 (SETD2), KDM5C, FOXC2, CLIP4, AQP1, DDX11, BAIAP2L1, and TMEM38B mRNA levels, and correlation with the Fuhrman grade was investigated. Multivariate logistic regression analysis revealed significant association between high-grade ccRCC and SETD2 and DDX11 mRNA levels in tissues (odds ratio (b) = 0.021, 95% confidence interval (CI): 0.001-0.466, p = 0.014; b = 6.116, 95% CI: 1.729-21.631, p = 0.005, respectively) and plasma (b = 0.028, 95% CI 0.007-0.119, p < 0.001; b = 1.496, 95% CI: 1.187-1.885, p = 0.001, respectively). High-grade ccRCC prediction models revealed areas under the curve of 0.997 and 0.971 and diagnostic accuracies of 97.86% and 92.86% for the frozen tissue and plasma, respectively. SETD2 and DDX11 mRNA can serve as non-invasive plasma biomarkers for predicting high-grade ccRCCs. Studies with long follow-ups are needed to validate the prognostic value of these biomarkers in ccRCCs.

Sub-pathway analysis for severe burns injury patients: Identification of potential key lncRNAs by analyzing lncRNA-mRNA profile.

The aim of the study was to identify key long non-coding RNAs (lncRNA) and related subpathways following severe burn injuries and research their functions. The miRNA-mRNA and lncRNA-miRNA interactions were downloaded from starBase v2.0 database. In addition, mRNA-miRNA interactions were obtained from TarBase, mirTarBase, mir2Disease, miRecords (V4.0) databases. The relationships of lncRNA-miRNA-mRNA were constructed. Genes of expression profiling were intersected with mRNA and lncRNA in lncRNA-mRNA interaction. Screened mRNAs were enriched into various pathways and screened lncRNAs were embedded into candidate pathways. Wallenius approximation methods were used to calculate the false discovery rate value of each sub-pathway. Based on the results of significant sub-pathways, the related lncRNA-mRNA network was constructed. A total of 18,081 genes were obtained. The lncRNA-mRNA intersections including 835 lncRNAs, 1,749 mRNAs and 7,693 interacting pairs were constructed. The enriched mRNAs were further enriched into various candidate pathways such as ribosome biogenesis in eukaryotes. Several sub-pathways were screened, including ribosome biogenesis in eukaryotes and MAPK signaling pathway. The network of pathway-lncRNA-mRNA was constructed. Hub-genes were identified, including C14orf169 and YLPM1. Several hub-lncRNAs were obtained, including PRKAG2 antisense RNA 1 and LEF1 antisense RNA 1. Several hub-lncRNAs including C14orf169, YLPM1, TTTY15, and PCBP1-AS1 were screened. The sub-pathways regulated by these lncRNAs were identified, and functions were predicted.

Alveolar macrophages from tuberculosis patients display an altered inflammatory gene expression profile.

Alveolar macrophages (AMs) are major targets of Mycobacterium tuberculosis (Mtb) infection, critical during the progression of active tuberculosis (TB). The complex immunopathology of TB generates diverse microenvironments in the lung, which shape immune responses by AMs. In the current study, we perform whole genome microarray transcriptional profiling on RNA isolated from AMs from TB patients (AMsTB) compared to AMs from control subjects (AMsCT) using bronchoalveolar lavage (BAL). Our hypothesis was that systemic effects on the local lung microenvironment during TB affect the transcriptional response of AMsTB. We found a unique gene expression profile of 51 genes, including up-regulated CHIT1, CHI3L1, CCL5, CCL22, CCL8, CXCL9, MMP9, MMP7 and MMP12, associated with a robust pro-inflammatory response, cell recruitment and tissue damage, and genes of the cyclin family (CCND1, CCND2, and CCNA1) associated with cell proliferation. These expression profiles may account for the inflammatory condition in the lungs of TB patients. CXCL5, IL1B, CAMP, and TGFB1 were down-regulated, suggesting an altered control of Mtb infection. Also, MARCO and COLEC12, affecting phagocytosis, and CES1, associated with an increase in free cholesterol, were down-regulated. The observed changes in mRNA expression profiles may partially account for the inability of AMsTB to effectively control Mtb infection, suggesting that a balanced control of pro- and anti-inflammatory immune responses is crucial for infection control.

Cell cycle and histone modification genes were decreased in placenta tissue from unexplained early miscarriage.

Genetic defect is a major cause of early miscarriage, but still in many cases the etiology are not fully understood. Recent studies have shown that dysregulation of genes in placenta tissue are participated in the pathogenesis of unexplained early miscarriage. The aim of our study is to explore mRNA expression profile in placental chorionic villi and to reveal the underlying mechanism of unexplained early miscarriage. Chorionic villous were isolated and extracted from early miscarriage (n=3) and control pregnancy (n=3) placenta with normal chromosome karyotype using MLPA assay, and then mRNA expression profiles were determined by microarray. For verification the reproducibility of the microarray, three up-regulated genes and six down-regulated genes were chosen and examined by real-time PCR (n=30). A total of 81 genes were up-regulated and 231 genes were down-regulated when compared to the control group, and the differences were reached statistically significances (P<0.05). After Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, we found that almost down-regulation genes are associated with cell cycle and histone modification, and these genes are participated in several important physiological processes, such as cell proliferation, nuclear division, chromatic assembly, DNA packing and modification. These results indicated that cell cycle and histone modification genes, and related signaling pathway maybe contribute to the genesis and development of unexplained early miscarriage. Further studies and validations are necessary to elucidate the exact roles of these genes in miscarriage pathogenesis, which can develop tools for early detection and management.

Microcystin-LR induces dysfunction of insulin secretion in rat insulinoma (INS-1) cells: Implications for diabetes mellitus.

Microcystins (MCs) are the most frequent cyanobacterial toxins observed in freshwater systems. Accumulating evidence suggests that MCs pose a serious threat to public health. However, the contributions of the exposure of MCs to the occurrence of human diseases remain largely unknown. This study provides the evidence of the effects of MC-LR on pancreatic ß-cell function through the exposure of rat insulinoma (INS-1) cells to 0, 10, 20, or 40µM MC-LR for 72h and explores the underlying molecular mechanisms. Our results demonstrate that exposure to MC-LR for 72h suppresses cell viability, disturbs glucose-stimulated insulin secretion (GSIS), and decreases the expression of insulin protein. Moreover, MC-LR disrupts the cell cycle distribution and increases cell apoptosis at 20 or 40µM for 72h, respectively, indicating that the ß-cell mass would be decreased by MC-LR exposure. A transcriptomic analysis revealed several key genes (e.g., Pdx-1, Neurod1, and Abcc8) involved in insulin secretion are significantly differentially expressed in INS-1 cells in response to MC-LR exposure. In addition, several signal transduction pathways associated with diabetes (e.g., type 1 and 2 diabetes) were also identified compared with the control cells. We recommend that MC be considered as a new environmental factor that promotes diabetes development. The identified key genes or pathways may potentially contribute to the future therapies in the environmental contaminants induced ß-cell damage.

Integrated analysis miRNA and mRNA profiling in patients with severe oligozoospermia reveals miR-34c-3p downregulates PLCXD3 expression.

Our previous research suggested that an integrated analysis of microRNA (miRNA) and messenger RNA (mRNA) expression is helpful to explore miRNA-mRNA interactions and to uncover the molecular mechanisms of male infertility. In this study, microarrays were used to compare the differences in the miRNA and mRNA expression profiles in the testicular tissues of severe oligozoospermia (SO) patients with obstructive azoospermia (OA) controls with normal spermatogenesis. Four miRNAs (miR-1246, miR-375, miR-410, and miR-758) and six mRNAs (SLC1A3, PRKAR2B, HYDIN, WDR65, PRDX1, and ADATMS5) were selected to validate the microarray data using quantitative real-time PCR. Using statistical calculations and bioinformatics predictions, we identified 33 differentially expressed miRNAs and 1,239 differentially expressed mRNAs, among which one potential miRNA-target gene pair, miR-34c-3p and PLCXD3 (Phosphatidylinositol-Specific Phospholipase C, X Domain Containing 3), was identified. Immunohistochemical analysis indicated that PLCXD3 was located within the germ cells of the mouse and human testis. Moreover, we found that miR-34c-3p was able to decrease PLCXD3 expression in mouse (GC-1 and TM4) and human (NCM460) cell lines, presumably indicating the possibility that miR-34c-3p acts as an intracellular mediator in germinal lineage differentiation. Notably, we reported the expression of the PLCXD3 protein in a man with normal spermatogenesis and the lack of the PLCXD3 protein in a man with SO. Therefore, the identified miRNA and mRNA may represent a potentially novel molecular regulatory network and therapeutic targets for the study or treatment of SO, which might provide a better understanding of the molecular basis of spermatogenesis dysfunction.

Transcriptome of normal lung distinguishes mouse lines with differentsusceptibility to inflammation and to lung tumorigenesis

AIRmax and AIRmin mouse lines show a differential lung inflammatory response and differential lung tumor susceptibility after urethane treatment. The transcript profile of 24,000 known genes was analyzed in normal lung tissue of untreated and urethane-treatedAIRmax and AIRmin mice. In lungs of untreated mice, inflammation-associated genes involved in pathways such as ‘‘leukocyte transendothelial migration”, ‘‘cell adhesion” and ‘‘tight junctions” were differentially expressed. Moreover, gene expression levels differed significantly in urethane-treated mice; in AIRmin mice, modulation of expression of genes involved in pathways associated with inflammatory response paralleled the previously observed persistent infiltration of inflammatory cells in the lung of these mice.

Serial Analysis of Gene Expression in Parasitological Research / 中国寄生虫学与寄生虫病杂志

Serial analysis of gene expression (SAGE) is a powerful high-throughput experimental technique that allows rapid, quantitative analysis of global gene expression in eukaryotic organisms. A short sequence taq (10~14 bp),which is defined by an anchoring enzyme site at a fixed distance from polyA tail, contains sufficient information to iden-tify mRNA transcript from which it originates. The taqs are ligated to obtain concatemers that are cloned into a plasmid vector for sequencing. The identification and abundance of mRNA can be observed through bioinformatics and statistical analysis of a given tag. SAGE is not only applied in obtaining global profile of gene expression in a given cell or tissue, but also help identify a set of specific genes to the cellular conditions by comparing the profiles constructed for a pair of cells that are kept at different conditions. This review covers a general introduction of SAGE, its protocol, meth-odological evolution and applications in parasite biology.