microRNA-142-3p inhibits apoptosis and inflammation induced by bleomycin through down-regulation of Cox-2 in MLE-12 cells.

microRNA (miR)-142-3p is implicated in malignancy and has been identified as a biomarker for aggressive and recurrent lung adenocarcinomas. This study aimed to evaluate the inhibitory effect of miR-142-3p on apoptosis and inflammation induced by bleomycin in MLE-12 cells. MLE-12 cells were first transfected either with miR-142-3p mimic or miR-142-3p inhibitor and then the cells were exposed to 50 µg/mL of bleomycin. Thereafter, cell viability, apoptosis and the expression of pro-inflammatory cytokines were assessed using CCK-8, flow cytometry, RT-PCR and western blot analyses. Cox-2, PI3K, AKT and mTOR expressions were detected by western blotting after bleomycin was administered together with NS-398 (an inhibitor of Cox-2). As a result, cell viability was significantly decreased, as well as apoptosis and the expression of IL-1 and TNF-α were remarkably increased after 50 and 100 µg/mL of bleomycin administration. miR-142-3p overexpression alleviated bleomycin-induced apoptosis and overproduction of these two pro-inflammatory cytokines, while miR-142-3p suppression exhibited completely opposite results. Up-regulation of Cox-2 and inactivation of PI3K/AKT/mTOR were found in bleomycin-pretreated cells, while these abnormal regulations were partially abolished by miR-142-3p overexpression and NS-398. In conclusion, this study demonstrated that miR-142-3p overexpression protected bleomycin-induced injury in lung epithelial MLE-12 cells, possibly via regulating Cox-2 expression and PI3K/AKT/mTOR signaling pathway. These findings provide evidence that miR-142-3p may be a therapeutic strategy for idiopathic pulmonary fibrosis (IPF) treatment.

microRNA-142-3p inhibits apoptosis and inflammation induced by bleomycin through down-regulation of Cox-2 in MLE-12 cells

microRNA (miR)-142-3p is implicated in malignancy and has been identified as a biomarker for aggressive and recurrent lung adenocarcinomas. This study aimed to evaluate the inhibitory effect of miR-142-3p on apoptosis and inflammation induced by bleomycin in MLE-12 cells. MLE-12 cells were first transfected either with miR-142-3p mimic or miR-142-3p inhibitor and then the cells were exposed to 50 μg/mL of bleomycin. Thereafter, cell viability, apoptosis and the expression of pro-inflammatory cytokines were assessed using CCK-8, flow cytometry, RT-PCR and western blot analyses. Cox-2, PI3K, AKT and mTOR expressions were detected by western blotting after bleomycin was administered together with NS-398 (an inhibitor of Cox-2). As a result, cell viability was significantly decreased, as well as apoptosis and the expression of IL-1 and TNF-α were remarkably increased after 50 and 100 μg/mL of bleomycin administration. miR-142-3p overexpression alleviated bleomycin-induced apoptosis and overproduction of these two pro-inflammatory cytokines, while miR-142-3p suppression exhibited completely opposite results. Up-regulation of Cox-2 and inactivation of PI3K/AKT/mTOR were found in bleomycin-pretreated cells, while these abnormal regulations were partially abolished by miR-142-3p overexpression and NS-398. In conclusion, this study demonstrated that miR-142-3p overexpression protected bleomycin-induced injury in lung epithelial MLE-12 cells, possibly via regulating Cox-2 expression and PI3K/AKT/mTOR signaling pathway. These findings provide evidence that miR-142-3p may be a therapeutic strategy for idiopathic pulmonary fibrosis (IPF) treatment.

Evaluation of the semen swim-up method for bovine sperm RNA extraction.

Isolation of high-quality RNA is important for assessing sperm gene expression, and semen purification methods may affect the integrity of the isolated RNA. This study evaluated the effectiveness of the sperm swim-up method for seminal RNA isolation. Frozen semen samples in straws from three bulls of proven fertility were purified by the swim-up method. RNA extraction was carried out using the E.Z.N.A.(TM) Total RNA kit II, with non-swim-up sperm as a control. Total sperm RNA was analyzed by UV spectrophotometry, reverse transcription polymerase chain reaction (RT-PCR), and agarose gel electrophoresis, and expression of the sex-determining region on the Y chromosome (SRY), leptin (LEP), and ribosomal protein subunit 23 (RPS23) genes, were determined. 18S RNA was used as a positive control. Fewer somatic cells were found in sperm swim-up samples than in the non-swim-up counterparts (0 x 10(3) vs 17.33 ± 2.52 x 10(3) sperm, P < 0.05). In addition, high-quality RNA was obtained in about 2 h, with no significant difference between groups. Interestingly, the yields of RNA fragments containing ≥200 nucleotides were significantly reduced in sperm swim-up samples (0.92 ± 0.41 x 10(7) sperm) compared with the non-swim-up samples (1.36 ± 0.33 x 10(7) sperm, P < 0.05). After RT-PCR, clear bands representing SRY, LEP, and RPS23 in sperm cDNA were observed on agarose gel electrophoresis. Finally, no bands corresponding to 18S RNA were found in RNA samples from the sperm swim-up group. Our findings suggest that small amounts of sperm RNA can be efficiently extracted from frozen straw semen samples using the swim-up technique.

Identification of a novel mutation of the EDA gene in X-linked hypohidrotic ectodermal dysplasia.

This study aimed to identify the disease-causing mutation in the ectodysplasin A (EDA) gene in a Chinese family affected by X-linked hypohidrotic ectodermal dysplasia (XLHED). A family clinically diagnosed with XLHED was investigated. For mutation analysis, the coding region of EDA of 2 patients and 7 unaffected members of the family was sequenced. The detected mutation in EDA was investigated in 120 normal controls. A missense mutation (c.878T>G) in EDA was detected in 2 patients and 3 female carriers, but not in 4 unaffected members of the family. The mutation was not found in the 120 healthy controls and has not been reported previously. Our findings indicate that a novel mutation (c.878T>G) of EDA is associated with XLHED and adds to the repertoire of EDA mutations.

Establishment of a neuroblastoma mouse model by subcutaneous xenograft transplantation and its use to study metastatic neuroblastoma.

The aim of this study was to establish a metastatic human neuroblastoma (NB) mouse model by xenograft in order to study the metastatic mechanisms of NB. A human NB cell line was obtained from a 5-year-old patient and cultured in vitro. A suspension of these cells was subcutaneously inoculated into nude mice at the right flank next to the forelimb. The biological characteristics of the developed subcutaneous and metastatic tumors were analyzed by hematoxylin and eosin staining. The expression of the tumor marker neuron-specific enolase was determined by immunohistochemistry, and the invasive ability of metastatic tumors was examined by a Matrigel invasion assay. DNA microarray analyses were performed to examine the metastasis-related gene expression. Our results showed that tumors grew in 75% of the mice injected with NB cells and the rate of metastasis was 21%. The xenograft tumors retained the morphological and biological characteristics of the NB specimen from the pediatric patient. Neuron-specific enolase was highly expressed in both subcutaneous and metastatic tumors. The metastatic tumor cells possessed a higher invasive capability than the primary NB cells. The expression of 25 metastasis-related genes was found to be significantly altered in metastatic tumors compared to primary tumors, including RECK, MMP2, VEGF, MMP3, and CXCL12. In conclusion, we successfully established a human NB xenograft model with high tumor-bearing and metastatic rates in nude mice, providing an ideal animal model for the in vivo study of NB.

Elevated TrkA receptor expression is associated with all-trans retinoic acid-induced neuroblastoma differentiation.

Neuroblastoma is the most common and one of the deadliest among pediatric tumors; however, a subset of infants with neuroblastoma display spontaneous regression. The mechanism of spontaneous regression remains to be elucidated. TrkA plays an essential role in the differentiation and functionality of neurons; abundant TrkA expression is associated with favorable prognosis of neuroblastoma. All-trans retinoic acid (ATRA), a first-line drug for acute promyelocytic leukemia (APL) treatment, has been shown to induce differentiation and inhibit cell growth. Neuroblastoma tissues in our hospital inpatient were collected, primary cell culture was performed, and the cells were separated and purified to be cell line. Trypan blue exclusion was used to count the numbers of cells alive, morphological changes were observed under the phase-contrast microscope. RT-PCR was used to determine the expression level of TrkA. In this study, a human neuroblastoma cell line was successfully established; in addition, we demonstrated that ATRA induces growth arrest and promotes the differentiation of neuroblastoma cells. In addition, ATRA was shown to significantly increase the levels of TrkA mRNA expression. Therefore, we concluded that the elevated expression of the TrkA receptor is associated with ATRA-induced growth arrest and differentiation o neuroblastoma cells. The results of this study provide a theoretical basis for the clinical application of differentiation-inducing ATRA for neuroblastoma therapy.

Association between the rs4753426 polymorphism in MTNR1B with fasting plasma glucose level and pancreatic ß-cell function in gestational diabetes mellitus.

We investigated the association between rs4753426 single nucleotide polymorphisms in the melatonin receptor 1B (MTNR1B) gene and the risk of developing gestational diabetes mellitus (GDM). A total of 516 gravidas (186 with GDM and 330 non-diabetic controls) were enrolled in the study. Genotype and allele frequencies of rs4753426 in the MTNR1B gene were detected by DNA sequencing. Fasting plasma glucose and fasting insulin levels were measured to calculate the homeostasis model assessment for insulin resistance (HOMA-IR) and for ß-cell function. Three genotypes (CC, CT, and TT) were found in both groups. The frequencies of CC, CT, and TT genotypes for the GDM group were 70.97, 22.58, and 6.45% vs 53.03, 39.70, and 7.27% in the control group, respectively. Significant differences were observed in genotype frequencies between groups (P < 0.05). T and C allele frequencies in the GDM group were 17.74 and 82.26%, respectively, and in the control group were 27.12 and 72.88%, respectively. Significant differences in T and C allele frequencies were found between groups (P < 0.05). In the GDM group, the C allele was associated with increased fasting plasma glucose level and reduced pancreatic ß-cell function (P < 0.05). There were no significant differences in total cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein concentration, or HOMA-IR between groups (P > 0.05). The single nucleotide polymorphism rs4753426 in MTNR1B may be a susceptibility gene locus for GDM, and the C allele may contribute to the increased fasting plasma glucose level and reduced pancreatic ß-cell function.

MicroRNAs in avian influenza virus H9N2-infected and non-infected chicken embryo fibroblasts.

There is limited information about microRNAs (miR-NAs) in H9N2 subtype influenza virus-infected chicken cells or tissues. In this study, 10,487,469 and 13,119,795 reads were obtained from in-fected and non-infected chicken embryo fibroblasts, respectively. Seven hundred and thirty-six and 1004 miRNAs, including mature miRNAs and precursors, were obtained from the infected and non-infected fibro-blasts, respectively. Of those miRNAs, 48 were expressed differently between the groups: 37 had a low expression level in the infected chick-en embryo fibroblast, and the remaining 11 had a higher expression level. Every miRNA was predicted to target immune response-related genes. It has been found that some of the miRNAs, such as gga-miR-146c, gga-miR-181a, gga-miR-181b, gga-miR-30b, gga-miR-30c, gga-miR-30e, and gga-miR-455, are expressed differently in other types of influenza-infected chicken cells or tissues.

Isolation of microsatellite loci for Rhodiola alsia (Crassulaceae), an important ethno-medicinal herb endemic to the Qinghai-Tibetan plateau.

Rhodiola alsia, which has been used widely in traditional Chinese medicine for a considerable time, grows on moist habitats at high altitude near the snow line. Microsatellite loci were developed for R. alsia to investigate its population genetics. In total, 17 polymorphic microsatellites were developed based on ESTs from the Illumina HiSeq(TM) 2000 platform. The microsatellite loci were checked for variability using 80 individuals of R. alsia sampled from four locations on the Qinghai-Tibet Plateau. The total number of alleles per locus ranged from 10 to 20, and the observed heterozygosity ranged from 0.000 to 1.000. The null allele frequency ranged from 0.000 to 0.324. These microsatellites are expected to be helpful in future studies of population genetics in R. alsia and related species.

Association between macrophage migration inhibitory factor rs1007888 and GDM.

We investigated the association between macrophage migration inhibitory factor (MIF) rs1007888 single-nucleotide polymorphisms and the genetic susceptibility to gestational diabetes mellitus (GDM). A total of 240 GDM pregnant women (GDM group) and 330 healthy pregnant women (control group) were included in the study. Differences in the MIF rs1007888 genotype and allele frequencies and differences between fasting blood glucose, fasting insulin, homeostatic model assessment (HOMA)-insulin resistance, and HOMA-b levels of pregnant women with different genotypes were compared. MIF genotype distributions were significantly different in the GDM group compared to the control group (P < 0.05). No significant difference was observed in the allele distributions of MIF rs1007888 between the GDM group and control group (P > 0.05). GDM patients had higher fasting blood glucose, fasting insulin, and HOMA-insulin resistance levels, but lower HOMA-b levels than normal gestational women (P < 0.05). Fasting blood glucose, fasting insulin, and HOMA-insulin resistance in pregnant women with the GG genotype were significantly higher than those with GA and AA genotypes, while HOMA-b in pregnant women with the GG genotype was lower (all P < 0.05). Our findings demonstrated the associations among MIF polymorphism rs1007888, insulin resistance, and pancreatic ß cell functions in GDM patients. The GG genotype of MIF rs1007888 may be a genetic susceptibility factor in the pathogenesis of GDM.

New support vector machine-based method for microRNA target prediction.

MicroRNA (miRNA) plays important roles in cell differentiation, proliferation, growth, mobility, and apoptosis. An accurate list of precise target genes is necessary in order to fully understand the importance of miRNAs in animal development and disease. Several computational methods have been proposed for miRNA target-gene identification. However, these methods still have limitations with respect to their sensitivity and accuracy. Thus, we developed a new miRNA target-prediction method based on the support vector machine (SVM) model. The model supplies information of two binding sites (primary and secondary) for a radial basis function kernel as a similarity measure for SVM features. The information is categorized based on structural, thermodynamic, and sequence conservation. Using high-confidence datasets selected from public miRNA target databases, we obtained a human miRNA target SVM classifier model with high performance and provided an efficient tool for human miRNA target gene identification. Experiments have shown that our method is a reliable tool for miRNA target-gene prediction, and a successful application of an SVM classifier. Compared with other methods, the method proposed here improves the sensitivity and accuracy of miRNA prediction. Its performance can be further improved by providing more training examples.

New roles of filopodia and podosomes in the differentiation and fusion process of osteoclasts.

The cytoskeleton mediates various cellular processes such as differentiation and fusion, including in the filopodia and podosomes. However, apart from cell migration and formation of the sealing zone, little is known regarding the changes and related regulatory mechanisms of the cytoskeleton and additional roles of the filopodia and podosomes during the differentiation and fusion of osteoclasts. The cytomorphology and cytoskeleton of osteoclasts in the differentiation process were evaluated using tartrate-resistant acid phosphatase staining and immunofluorescence staining. Moreover, the expression levels of Rho GTPases and enzymes related to osteoclast differentiation and bone resorption were detected by quantitative reverse transcription-polymerase chain reaction. We detected 3 types of filopodia in osteoclast precursors and only 1 type of filopodia in undifferentiated cells. Mature osteoclasts were completely devoid of filopodia. Interestingly, cell fusion was highly specific, and the fusion initially occurred to the filopodia. Confocal images revealed that F-actin and microtubules significantly differed among fused cells. These results suggest that filopodia and podosomes not only play important roles in cell migration and the formation of sealing zones but also in the pre-fusion selectivity of 2 cells and the movement direction of the cell nucleus and cytoplasm during the fusion process. In addition, cdc42v1, RhoU, and RhoF regulate the formation of 3 types of filopodia during various stages of differentiation, while Rac1, Rac2, and filament A may be associated with cell selectivity during the fusion process.

Molecular characterization and expression analysis of the Lrh-1 gene in Chinese Hu sheep.

Liver receptor homologue 1 (Lrh-1) is a member of the nuclear receptor belonging to the second subfamily of the nuclear receptor family 5A (NR5A), also named NR5A2, which is important for lipid homeostasis, embryogenesis, and regulation of aromatics. The present study aimed to understand the sequence of ovine Lrh-1 and the expression traits in reproductive organ tissues. Initially, we cloned Lrh-1 from the liver of Hu sheep through degenerate primer of reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. Characteristic functional domains of DNA binding and ligand binding, conserved among transcription factors of the nuclear receptor superfamily, were identified in Lrh-1 of Hu sheep. The Lrh-1 protein levels in the tissues detected by Western blotting correlated significantly with the transcript levels measured by quantitative real-time polymerase chain reaction (qRT-PCR). To understand the Lrh-1 expression change in the hypothalamus and hypophysis during the estrous cycle, we analyzed the expression pattern of Lrh-1 mRNA and protein by qRT-PCR and Western blotting, respectively. This analysis revealed that Lrh-1 expression in the hypothalamus was highest during the metestrus phase, while the Lrh-1 level was similar during other phases. In the hypophysis, the expression was significantly different during the 4 phases of the estrous cycle but highest during the estrus phase, significantly correlating with FSH concentration. These results indicate that Lrh-1 expression is correlated with gonadotropic hormone secretion, influencing follicular formation in the ovary.

Expression of the luteinizing hormone receptor (LHR) gene in ovine non-gonadal tissues during estrous cycle.

Luteinizing hormone (LH) is an important glycoprotein hormone that regulates gonadal function in mammals and in turn regulates physiological status changes during the estrous cycle. The function of LH is mediated by luteinizing hormone receptor (LHR). In order to examine the expression patterns of the LHR gene in non-gonadal tissues during the 4 phases of the ovine estrous cycle, tissues from healthy non-pregnant adult Hu sheep were examined according to the estrous cycle for normal ovaries using real-time fluorescence quantitative PCR and ELISA methods with GAPDH as the reference gene. LHR mRNA expression levels were significantly correlated with protein concentrations and the LHR gene was abundantly expressed in olfactory bulb, hypothalamus, rumen, small intestine, kidney, and uterine tissues. When comparing the expression levels of LHR during the 4 estrous phases in particular tissues, the results showed that LHR expression levels were significantly different and relatively lower at the estrous stage in a number of non-gonadal tissues. The trends of change in LHR expression levels were highly significantly correlated between hypothalamus and rectum, hypophysis and oviduct, ileum and uterus, and among jejunum, olfactory bulb, and kidney (P < 0.01), and there was also significant correlation between duodenum and oviduct, hypothalamus and medulla oblongata, jejunum and uterus, omasum and abomasum, and reticulum and colon (P < 0.05). These results indicate that the ovine LHR gene (or LH) might control important mechanisms in non-gonadal tissues and that the level of LH activity in some tissues may be influenced by hormonal status during the estrous cycle.

A novel single-base deletion mutation of the RUNX2 gene in a Chinese family with cleidocranial dysplasia.

We identified a disease-causing mutation of the RUNX2 gene in a four-generation Chinese family affected with cleidocranial dysplasia (CCD). For mutation analysis, the coding region of RUNX2 was sequenced with DNA from two patients and three unaffected family members. The RUNX2 mutation was investigated in 50 normal controls by denaturing high pressure liquid chromatography. A heterozygous single-base deletion (c.549delC) of RUNX2, which predicts a termination site at the 185th codon and leads to a stop in the runt domain of RUNX2 protein, was detected in both patients but not in the three unaffected members of the family. This mutation was also not found in 50 controls and has not been reported previously. We demonstrated that a novel mutation (c.549delC) of RUNX2 is associated with CCD in a Chinese family, adding to the repertoire of RUNX2 mutations related to CCD.

Studies of beta-thalassemia mutations in families living in three provinces in southern China.

beta-Thalassemia is a common disease in Southern China and 10 different mutations or frameshifts are responsible for most types of beta-thalassemia in this area. We studied 126 chromosomes of 80 beta-thalassemia patients from the Guangxi, Guangdong, and Sichuan Provinces using the polymerase chain reaction followed by dot-blot hybridization with specific oligonucleotide probes. The most common mutation in the three provinces is the frameshift at codons 41-42, followed by the A----T mutation at codon 17. The A----G mutation at nt -29 of the promoter is common in Sichuan but not in the other two provinces. Three mutations (T----C at nt -30; G----T at IVS-I-1, and G----C at IVS-I-5) were not observed. These data were used to initiate a prenatal diagnosis program using the same techniques for identification. Eleven fetuses at risk for beta-thalassemia have been diagnosed.