Expression Of Signaling Pathway-related Genes Research Articles

DKK3 promotes adipogenic differentiation of stem cells by inhibiting Wnt/β-catenin signaling pathway related gene expression and mitochondrial autophagy function

Mesenchymal stem cells can differentiate into adipocyte precursor cells, and the balance of stem cell differentiation determines the quantity of adipocytes. Early-stage adipose tissue expression profiling revealed abnormal expression of DKK3 in the high-fat group. Moreover, DKK3 is enriched in the Wnt/β-catenin signaling pathway, and studies have shown that DKK3 can serve as a gene involved in early regulation of adipogenesis. Therefore, this study focuses on exploring how DKK3 regulates the molecular mechanism of adipocyte differentiation through the Wnt/β-catenin signaling pathway. In this experiment, the role of DKK3 in the differentiation of bone marrow mesenchymal stem cells into adipocyte precursors was validated using in vitro cultured chicken bone marrow mesenchymal stem cells. The results showed that overexpression of DKK3 led to a significant downregulation of Wnt/β-catenin signaling pathway-related marker gene expression (P < 0.05), a significant upregulation of adipogenic differentiation-related genes (P < 0.05), an increase in lipid droplet content, a significant increase in OD value (P < 0.05), a significant upregulation of mitochondrial oxidative respiratory-related marker gene expression (P < 0.05), and a significant downregulation of mitochondrial autophagy-related marker genes (P < 0.05). Conversely, the results were opposite after interfering with DKK3 gene expression. In this study, 4 SNP sites, including g.8419139, g.8419556, g.8419560, and g.8419598, were detected in the 7th exon of DKK3, among which the g.8419598 (C > T) site was significantly correlated with abdominal fat weight and abdominal fat rate in 100-day-old Ma Huang chickens (P < 0.001).

Differential Gene Expression Profile in DNA Damage Signaling Pathways, in De Novo Acute Myeloid Leukemia Patients before Induction Chemotherapy

Introduction: Acute Myeloid Leukemia (AML) is a life-threatening myeloid malignancy with poor prognosis. Response to induction treatment determines long-term prognosis; however, mechanisms underlying response have not been thoroughly investigated. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also important for refractoriness to treatment. Aim: The aim of this study is to investigate the possible alterations in the gene expression profile of DNA damage signaling pathways in two leukemic cell lines following their exposure to chemotherapeutic agents, namely, idarubicin and cytarabine, and to verify the findings in AML patient samples at diagnosis before the onset of chemotherapy. Methods: Cell lines Kasumi-1 with the t(8;21) and MV4-11 (biphenotypic B-myelomonocytic leukemia) were exposed either to idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Dead cells were eliminated from drug-treated cells using an appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2 Profiler, Qiagen) was performed in triplicate after RNA extraction from untreated, chemotherapy-treated and live (chemo resistant) cells following chemotherapy exposure. Human DNA damage signaling pathway-related gene expression and statistical analysis was performed through the RT2 Profiler PCR Array data analysis tool. Moreover, mononuclear cells were isolated from bone marrow of 11 lymphoma patients, used as controls, and from 64 AML patients at diagnosis who subsequently received chemotherapy with the 7+3 regimen. Response to first-line chemotherapy was recorded and patients were assigned in two groups: Responders (n=53 patients) and non-Responders (n=11 patients). Total RNA was isolated (RNeasy plus Mini Kit, Qiagen), quantified and cDNA was synthesized. Relative expression of genes of interest was quantified using SYBR® Greenbased real-time RT-PCR (QuantiTect® Primer Assay, Qiagen) based on the ddCt method (Housekeeping gene GADPH). All samples were obtained from patients after informed consent and treated in accordance with the Declaration of Helsinki. Statistical analysis was performed using R Statistical Software (version 4.2.1). Results: Five genes were found significantly [p < 0.05, Students t-test] up-regulated in live cells from either leukemic cell lines after treatment with both agents and had a FoldChange > 2. More specifically PPP1R15A, CDKN1A and GADD45G genes were up-regulated in both live cell lines, GADD45A in live MV4-11 cells and EXO1 in live Kasumi cells (Figure 1A). The aforementioned genes were selected and their expression levels were quantified in human bone marrow samples. CDKN1A and GADD45G were found down-regulated in Responders [p=0.032 and p=0.001 respectively; Welch t-test] while PPP1R15A was significantly up-regulated [over 8-fold change, p= 2.08E-09; Welch t-test] compared to controls. Likewise, GADD45G and PPP1R15A were also significantly down and up regulated respectively, in non-Responders [p=0.08 and p=1.51E-05, Welch t-test]. Interestingly, PPP1R15A presented a 1.7-fold overexpression trend in non-Responders compared to Responders [p=0.18, Welch t-test] (Figure 1B). Conclusion: The results of our study suggest a dysregulation of the DNA damage and repair pathways in AML. Upregulation of GADD45G is known to dramatically induce apoptosis, differentiation, and growth arrest while increasing sensitivity of AML cells to chemotherapeutic drugs (Guo, Dan et al., Blood 2021). CDKN1A is involved in p53/TP53 mediated inhibition of cellular proliferation in response to DNA damage. Both were down-regulated in AML patients and are likely to play an important role in the pathogenesis of AML. PPP1R15A participates in DNA damage response facilitating the recovery of cells from stress. The up-regulation of PPP1R15A both in live cells treated with Ara-C and Cytarabine, as well as the 1.7-fold overexpression trend in non-Responders compared to Responders, supports the notion that PPP1R15A could be associated with resistance to induction treatment, which may have significant prognostic and therapeutic implications. A greater sample of AML patients both responding and not responding to chemotherapy, would be essential to delineate the role of genes involved in DNA damage and repair pathways in resistance to induction treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PB1787: GADD45A IS OVER-EXPRESSED IN THE BONE MARROW OF PATIENTS WITH DE NOVO ACUTE MYELOID LEUKEMIA REFRACTORY TO INDUCTION CHEMOTHERAPY

Background: Acute myeloid leukemia (AML) is a life-threatening malignant myeloid disorder with poor prognosis. Response to induction treatment determines long-term prognosis; however, mechanisms underlying response have not been thoroughly investigated. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also very important for refractoriness to treatment. Aims: The aim of this study was to investigate the possible alterations in the gene expression profile of DNA damage signaling pathways in two leukemic cell lines following their exposure to chemotherapeutic agents, namely, idarubicin and cytarabine, and verify the findings in AML patient samples at diagnosis before the onset of chemotherapy. Methods: Cell lines Kasumi-1 with the t(8;21) and MV4-11 (biphenotypic B-myelomonocytic leukemia) were exposed either to idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Dead cells were eliminated from drug-treated cells using an appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2 Profiler, Qiagen) was performed in triplicate after RNA extraction from untreated, chemotherapy-treated and live cells following chemotherapy exposure. Human DNA damage signaling pathway-related gene expression was evaluated and analyzed through the RT2 Profiler PCR Array data analysis tool. Moreover, mononuclear cells were isolated from bone marrow of lymphoma patients, used as controls, and from AML patients at diagnosis who subsequently received chemotherapy with the 7 + 3 regiment. Response to first-line chemotherapy was recorded. Total RNA was isolated (RNeasy plus Mini Kit, Qiagen) and quantified and cDNA was synthesized. Relative expression of genes of interest was quantified using SYBR® Green-based real-time RT-PCR (QuantiTect® Primer Assay, Qiagen). Statistics were performed using linear regression. Results: The following genes were over two-fold up-regulated in live cells from leukemic cell lines after treatment with either agent: PPP1R15A and CDKN1A in both cell lines, GADD45A and GADD45G in live MV4-11 cells and EXO1 in live Kasumi cells. These genes were selected for relative quantification; 25 control samples and 38 AML samples (28 responders and 10 non-responders to treatment) were evaluated. GADD45A in AML compared to controls was found 2-fold up-regulated in treatment responders (ns) and 9-fold up-regulated in non-responders [p=0.01]. In addition, GADD45A was overexpressed in non-responders compared to responders [p=0.02]. PPP1R15A in AML compared to controls was 12-fold up-regulated in responders [p=0.001] and 16-fold up-regulated in non-responders [p=0.001]. (Figure 1) Image:Summary/Conclusion: GADD45A controls the balance between apoptosis and autophagy via the p38 and JNK/MAPK pathways and PPP1R15A participates in growth arrest and apoptosis in response to DNA damage by attenuating the translational elongation of key transcription factors. Both genes are likely to play an important role in the pathogenesis of AML. Furthermore, GADD45A overexpression appears to be related with resistance to induction treatment, which may have significant prognostic and therapeutic implications.

Protective Effect of Dandelion Leaf Water Extracts on APAP-Induced Liver Injury in Rats and Its Mechanism.

with the advent of a large number of drugs in recent years and the aggravation of human aging, drug-induced liver injury is increasing year by year. The protective effect of dandelion extract on acetaminophen (APAP) - induced drug-induced liver injury in rats and its specific mechanism was studied by in vitro cell culture. For this aim,twenty healthy SD rats with the same physiological status were divided into model group and normal group, with 10 rats in each group. The drug-induced liver injury model was made by intragastric administration of 1 g/kg APAP for 14 days. The liver function lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected to verify the success of the model. After that, the liver tissues were aseptically isolated from the normal group and APAP model group, and the primary hepatocytes were cultured. They were divided into control group (control), liver injury model group (model), medium-dose dandelion extract group (1mm dlwe) and high dose dandelion extract group (2mm dlwe). The cell proliferation activity was detected by CCK-8 cell proliferation activity kit. Cell samples were collected at 72 hours to detect the contents of AST and ALT in cell supernatant. The contents of oxidative stress-activated oxygen (ROS), reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) were detected by colorimetry, and the apoptosis was detected by flow cytometry. Inflammatory factors, key genes of liver injury, drug metabolic enzymes cytochrome P450 2E1 (CYP2E1), mitogen-activated protein kinase (MAPK) and nuclear transcription factors were detected by RT-PCR- κ B (NF- κ B) P65 signaling pathway-related gene expression level. Finally, the expression of CYP2E1, MAPK and NF-kB signaling pathwayswere analyzed by Western blot. Results showed thatSerum ast, ALT and LDH increased (P<0.05), suggesting that the liver injury model was successful. The hepatocytes in the normal group were oval, flat, evenly distributed and well adhered to the wall. The liver injury model group had more suspended cells, pseudopodia, polygonal and poor growth state. The cells in the medium-dose dandelion extract group (1mm dlwe) and high dose dandelion extract group (2mm dlwe) adhered well, mostly oval, similar to the normal group and grew well. CCK8 found that the cells in the model group decreased significantly, the proliferation activity decreased significantly, the ast, alt, LDH and ROS in the cell supernatant of the model group increased compared with other groups (P<0.05), and the contents of GSH and GSH PX decreased (P < 0.05). Apoptotic cells in the model group increased, and TNF in the model group-α, COX-2, CYP2E1, MAPK, JNK and NF KB p65 increased (P < 0.05). CYP2E1, MAPK and NF KB p65 increased in the model group (P < 0.05).

Neurotoxicity of Tris (1,3-dichloroisopropyl) phosphate in Caenorhabditis elegans

As a new type of flame retardant, Organic Phosphate Flame Retardant has been widely used worldwide. The purpose of our research is to determine the neurotoxicity of Tris (1,3-dichloroisopropyl) phosphate (TDCPP) to Caenorhabditis elegans and its mechanism. L1 larvae wild-type C. elegans were exposed to different concentrations of TDCPP, and the effects on motor behavior (head thrashes, body bends, pumping times, chemotaxis index), ROS levels, and p38MAPK signaling pathway-related gene expression levels were measured. Three transgenic nematode strains, BZ555, DA1240, and EG1285, were also used to study the effects of TDCPP on nematode dopamine neurons, glutamate neurons, and GABA neurons. The results showed that TDCPP can inhibit the head thrashes and body bends of the nematode, reduce dopamine production, increase the level of ROS in the body, and affect the expression of genes related to the p38MAPK signaling pathway. We next employed ROS production and motor behavior as toxicity assessment endpoints to determine the involvement of p38 MAPK signaling in the regulation of response to TDCPP. The results showed that the nematodes with low expression of pmk-1 were less sensitive to the TDCPP. It was suggested that TDCPP had neurotoxicity and regulated neurotoxicity to C. elegans by activating the p38-MAPK signaling pathway. The research in this article provides important information for revealing the environmental health risks of organophosphorus flame retardants and their toxic mechanism of action.

Enrichment of polystyrene microplastics induces histological damage, oxidative stress, Keap1-Nrf2 signaling pathway-related gene expression in loach juveniles (Paramisgurnus dabryanus)

Polystyrene microplastics (PS-MPs, particle size<5 mm) cause great harm to aquatic organisms. However, their precise effects are not completely understood. In China, placing plastic film at the pond bottom has become an important loach aquaculture mode. In this mode, MPs will affect loach health. This study investigated the enrichment of PS-MPs and its effects on the growth, liver histomorphology, antioxidant enzymes, and Keap1-Nrf2 signaling pathway-related gene expression in loach juveniles (Paramisgurnus dabryanus). The loach juveniles were raised at the concentration of 1000 μg/L fluorescent polystyrene microplastics (PS-MPs) with particle size of 0.5 µm or 5 µm for seven days, the results showed that fluorescent PS-MPs were found to be enriched in liver, intestine, and gill, and the enrichment amount was higher in liver than in gill and intestine (P < 0.05). Furthermore, the enrichment amount of different-sized PS-MPs was different in liver, gill, and intestine. The loach juveniles were cultured for 21 days in the water of the concentration of 100 or 1000 μg/L PS-MPs with particle size of 0.5 µm or 5 µm, the results showed that the survival rate, weight gain rate, and specific growth rate of loach juveniles were significantly reduced. The histological analysis revealed that PS-MPs caused liver damage. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), and acetylcholinesterase (AChE) were decreased with the extended exposure to PS-MPs. Generally, the expressions of Nrf2 and Keap1 showed the similar change trend. From 7–14 day, the expression trend of oxidative stressed-related genes was not completely consistent with that of Nrf2 gene, but on day 21, the gene expression trend of oxidative stress-related SOD, CAT, and GSH-PX in the downstream of Keap1-Nrf2 signaling pathway was roughly consistent with that of Nrf2 gene. Basically, the change trends of these three gene expression were similar to those of their corresponding enzyme activities. This study provides theoretical basis for the toxicological effects of PS-MPs on freshwater fish.

TOX3 Promotes Ovarian Estrogen Synthesis: An RNA-Sequencing and Network Study.

BackgroundWomen who undergo chronic exposure to excessive estrogen are at a high risk of developing breast cancer. TOX3 has been reported to be highly expressed in breast tumors and is closely related to estrogen receptors. However, the effect of TOX3 on estrogen synthesis remains poorly understood.MethodsUsing lentiviruses as a vector, we stably overexpressed TOX3 in the ovarian granulosa cell line KGN, the cells where estradiol is primarily produced, to investigate its role in estrogen production as well as cell viability and apoptosis. RNA-Sequencing was applied to uncover the global gene expression upon TOX3 overexpression.ResultsWe observed an increased level of cell viability and a reduced cell apoptosis rate after TOX3 overexpression, and the level of estradiol in the cell culture supernatant also increased significantly. Gene set enrichment analysis of the transcriptome showed that the ovarian steroidogenesis pathway was significantly enriched. Similarly, pathway mapping using the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses also showed that TOX3 overexpression affects the ovarian steroidogenesis pathway. Further experiments showed that upregulated FSHR, CYP19A1, and BMP6 accounted for the enhanced estrogen synthesis.ConclusionOur study demonstrated that TOX3 quantitatively and qualitatively stimulates estrogen synthesis by enhancing estrogen signaling pathway–related gene expression in ovarian granulosa cells. These findings suggest that TOX3 may play a vital role in the pathogenesis of breast cancer.

Functional Regulatory Mechanisms Underlying Bone Marrow Mesenchymal Stem Cell Senescence During Cell Passages.

Mesenchymal stem cell (MSC) transplantation is an effective periodontal regenerative therapy. MSCs are multipotent, have self-renewal ability, and can differentiate into periodontal cells. However, senescence is inevitable for MSCs. In vitro, cell senescence can be induced by long-term culture with/without cell passage. However, the regulatory mechanism of MSC senescence remains unclear. Undifferentiated MSC-specific transcription factors can regulate MSC function. Herein, we identified the regulatory transcription factors involved in MSC senescence and elucidated their mechanisms of action. We cultured human MSCs (hMSCs) with repetitive cell passages to induce cell senescence and evaluated the mRNA and protein expression of cell senescence-related genes. Additionally, we silenced the cell senescence-induced transcription factors, GATA binding protein 6 (GATA6) and SRY-box 11 (SOX11), and investigated senescence-related signaling pathways. With repeated passages, the number of senescent cells increased, while the cell proliferation capacity decreased; GATA6 mRNA expression was upregulated and that of SOX11 was downregulated. Repetitive cell passages decreased Wnt and bone morphogenetic protein (BMP) signaling pathway-related gene expression. Silencing of GATA6 and SOX11 regulated Wnt and BMP signaling pathway-related genes and affected cell senescence-related genes; moreover, SOX11 silencing regulated GATA6 expression. Hence, we identified them as pair of regulatory transcription factors for cell senescence in hMSCs via the Wnt and BMP signaling pathways.

MicroRNA-133a exerts tumor suppressive role in oral squamous cell carcinoma through the Notch signaling pathway via downregulation of CTBP2.

microRNAs (miRNAs) have been revealed to participate in some oral cancers and are proved to be effective. In the present study, we tried to explore the biological function of miR-133a in oral squamous cell carcinoma (OSCC) cells. The relationship that C-terminal-binding proteins 2 (CTBP2) was the putative target gene of miR-133a revealed from bioinformatics analysis was further was further validated by dual-luciferase reporter gene assay. In total, 40 patients with OSCC were enrolled for characterization of miR-133a, CTBP2, and Notch signaling pathway-related gene expression in clinical OSCC tissues. Low expression of miR-133a and high expression of CTBP2, Hes1, Notch-1, and Notch-3 were determined in OSCC tissues. OSCC cell lines were transfected with miR-133a inhibitor, miR-133a mimic, or shRNA targeting CTBP2, in response to which cell proliferation, migration, invasion, cell cycle, and apoptosis were evaluated. Transfection of miR-133a mimic induced apoptosis and inhibited OSCC cell proliferation, migration, and invasion and this was demonstrated to be attributable to decreased CTBP2 expression and suppression of the Notch signaling pathway. Taken together, we concluded that miR-133a acted as a tumor suppressor in OSCC through inhibition of the Notch signaling pathway via binding to CTBP2.

Investigation of the effects of dichlorvos poisoning on AMPK signaling pathway in chicken brain tissues

Dichlorvos is a common crop insecticide widely used by people which causes extensive and serious environmental pollution. However, it has been shown that organophosphorus poisoning causes energy metabolism and neural disorders. The overall purpose of this study was to investigate the damage to brain tissue and the changes in AMPK signaling pathway-related gene expression after dichlorvos poisoning in chickens. White-feathered broiler chickens, as the research subjects of this experiment, were divided into three groups: control group, low-dose group (77.5% dichlorvos at 1.13 mg/kg dose) and high-dose group (77.5% dichlorvos at 10.2 mg/kg dose). Clinical symptoms were observed after modeling, and an integrative analysis was conducted using HE staining microscopy, immune-histochemical microscopy, electron microscopy and PCR arrays. The results showed that the high-dose group had more obvious dyspnea, salivation, convulsion and other neurological phenomena. Pathological sections showed that nuclear disintegration of neurons was most obvious in the low-dose group, and apoptosis of brain cells was most obvious in the high-dose group, and the mitochondrial structure was destroyed in the two poisoned group, i.e. low-dose group and high-dose group. PCR arrays showed that AMPK signaling pathway was inhibited and the expressions of genes involved in energy metabolism (ACACA and PRKAA1) were significantly changed. Furthermore, genes associated with protein synthesis (EIF4EBP1) were significantly upregulated. FASN and HMGCR expressions were significantly increased. There were significant changes in the expressions of cell cycle-related genes (STK11, TP53 and FOXO3). Organophosphate poisoning can cause a lot of nuclear disintegration of brain neurons, increases cell apoptosis, disrupts the energy metabolism of mitochondrial structure, and inhibits the AMPK signaling pathway. These results provide a certain idea and basis for studying the mechanism of AMPK signaling after organophosphorus poisoning and provide a research basis for the prevention and treatment of organophosphorus poisoning.

Lactobacillus Plantarum HFY15 Helps Prevent Retinoic Acid-Induced Secondary Osteoporosis in Wistar Rats.

A rat model of secondary osteoporosis was constructed using retinoic acid as an inducer, and the genes, proteins, and bone mass of the rats were analyzed. qPCR detection of the Wnt/β-catenin and OPG/RANK/RANKL signaling pathway-related gene expression levels showed that Lactobacillus plantarum HFY15 played a positive role in regulating both pathways. HFY15 significantly increased β-catenin, Lrp5, Lrp6, Wnt10b, OPG, RANKL, and Runx2 expression and downregulated DKK1, RANK, CTSK, TRACP, and ALP expression. Enzyme-linked immunosorbent assays further confirmed the qPCR results. Tartrate-resistant acid phosphatase staining showed that HFY15 slowed retinoic acid-induced osteoclast formation. Microcomputed tomography showed that HFY15 reduced trabecular separation and increased the percent bone volume, trabecular numbers, trabecular thickness, and bone mineral density in the rats in vivo. These findings indicate that HFY15 may help prevent retinoic acid-induced secondary osteoporosis in vivo.

Effects of zearalenone-induced oxidative stress and Keap1–Nrf2 signaling pathway-related gene expression in the ileum and mesenteric lymph nodes of post-weaning gilts

Zearalenone (ZEA) contamination of feed affects animal husbandry and the human health. Currently, the molecular mechanism underlying small intestine-related diseases caused by ZEA-induced oxidative stress is not well understood. In this study, we aimed to identify the mechanisms involved in ZEA (0.5–1.5 mg/kg)-induced oxidative stress in the ileum and mesenteric lymph nodes (MLNs) and the role of the Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1)–nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in post-weaning gilts. Forty post-weaning gilts (Landrace × Yorkshire × Duroc) with an average body weight of 14.01 ± 0.86 kg were randomly allocated to four groups and fed a corn–soybean meal basal diet supplemented with < 0.1, 0.5, 1.0, or 1.5 mg/kg ZEA. The results showed that the activity of total superoxide dismutase and glutathione peroxidase decreased (p < 0.05) linearly and quadratically and that the content of malondialdehyde increased (p < 0.05) quadratically in the ileum and MLNs with increasing ZEA in the diet. Immunohistochemical analysis showed that the expression of Nrf2 and glutathione peroxidase 1 (Gpx1) immunoreactive proteins in the ileum and MLNs were significantly enhanced with increasing ZEA. The relative mRNA and protein expression of Nrf2, Gpx1, quinone oxidoreductase 1 (Nqo1), hemeoxygenase 1 (Ho1), modifier subunit of glutamate-cysteine ligase (Gclm), and catalytic subunit of glutamate-cysteine ligase (Gclc) increased (p < 0.05) linearly and quadratically, and the relative mRNA and protein expression of Keap1 decreased (p < 0.05) linearly and quadratically in the ileum with increasing ZEA concentrations in the diet. Further, the relative mRNA and protein expression of Nrf2 and Gpx1 increased (p < 0.05) linearly and quadratically, and the relative mRNA and protein expression of Nqo1, Ho1, and Gclm decreased (p < 0.05) quadratically in the MLNs as ZEA concentrations increased in the diet. Our results provide valuable genetic information on ZEA-induced oxidative stress in the ileum and MLNs of post-weaning gilts and have elucidated the key regulatory genes involved in the Keap1–Nrf2 signaling pathway. Results indicated that the Keap1–Nrf2 signaling pathway might be a key target to further prevent and treat ZEA-induced injury to the ileum in post-weaning gilts.

Lithium-calcium-silicate bioceramics stimulating cementogenic/osteogenic differentiation of periodontal ligament cells and periodontal regeneration

The ultimate goal of treatment of periodontal disease is not only to control inflammation and further to prevent development of the lesion, but also to achieve regeneration of the destroyed periodontal tissue. This study aimed to investigate the effect of lithium-calcium-silicate (Li2Ca4Si4O13, LCS) bioceramics on the cementogenic/osteogenic differentiation of human periodontal ligament cells (PDLCs) and regeneration of periodontal tissue as well as the corresponding mechanism. We prepared LCS bioceramics and explored the cementogenic/osteogenic differentiation of PDLCs after cultured with LCS extracts in vitro. Meanwhile, we further studied the involvement of the Wnt/β-catenin and ERK signaling pathway during this process. In the in vivo study, the regeneration of alveolar bone defect in New Zealand white rabbits and the periodontal tissue in beagle dogs were compared by applying LCS and β-tricalcium phosphate (β-TCP). Our results showed that a certain concentration of LCS extracts could significantly stimulate cementogenic/osteogenic differentiation of PDLCs, including alkaline phosphatase (ALP) activity, periodontal regeneration-related gene expression, Wnt/β-catenin and ERK signaling pathway related gene expression and protein expression of PDLCs. Moreover, it was found that LCS bioceramics could significantly activate Wnt/β-catenin and ERK signaling pathway in cementogenesis/osteogenesis of PDLCs. The in vivo results suggested that LCS significantly promoted the regeneration of bone defects and periodontal defects as compared with β-TCP in rabbit and beagle dog models, in which the LCS bioceramics possess the capacity to trigger the activation of the two signaling pathway, leading to significantly improved periodontal regeneration. Our study suggests that LCS is a promising bioactive material for periodontal regeneration, consequently offering a promising treatment strategy for the treatment of periodontitis-related disease.

Inhibition of Phospholipase D1 mRNA Expression Slows Down the Proliferation Rate of Prostate Cancer Cells That Have Transited to Androgen Independence.

To explore the role of phospholipase D1 (PLD1) mRNA in transition of prostate cancer (PCa) cells to androgen independence, we used Arraystar Human LncRNA Microarray V3.0 to detect and compare the differential expression of PLD1 and its signaling pathway-related gene in standard androgen dependence prostate cancer (ADPC) cell line LNCaP before and after the occurrence of androgen independence prostate cancer (AIPC) transition. In addition, we used the shRNA lentiviral vector to inhibit the PLD1 mRNA expression and observed its effect on LNCaP cell proliferation after AIPC transition by using MTS method. The results showed that the expression level of PLD1 mRNA was increased by 373-fold after AIPC transition (P<0.05); the PI3K/AKT signaling pathway-related gene expression was also elevated (P<0.05); the growth rate of LNCaP cells that had transited to androgen independence was reduced by about 30% when the PLD1 mRNA expression was inhibited by the shRNA lentivirus as compared with the negative control group (P<0.05). All these results suggest that PLD1 mRNA and the related PI3K/AKT signaling pathway may play an important role in AIPC. Down-regulating the expression of PLD1 mRNA could to some extent inhibit the proliferation rate of PCa cells after AIPC transition.

WITHDRAWN: Effect of LncRNA LINC00936 and its target NFKBIA on myocardial infarction through the NF-κB signaling pathway: an in vivo and in vitro study

// Xian-Zhao Zhang 1 , Gui-Ling Sun 1 , Xi-Dong Li 1 , Jing-Fang Li 1 and Qing-Rong Ji 1 1 Department of Cardiology, Linyi People&rsquo;s Hospital, Linyi, P.R. China Correspondence to: Qing-Rong Ji, email: qingrong_jqr@163.com Keywords : long noncoding RNA; long intergenic non-protein coding RNA 936; NF-kappa-B inhibitor alpha; nuclear factor-kappa-B signaling pathway; myocardial infarction Received: June 07, 2017&nbsp;&nbsp;&nbsp;&nbsp; Accepted: October 28, 2017&nbsp;&nbsp;&nbsp;&nbsp; Epub: December 30, 2017 Abstract Objective: The study aimed to explore the protective effect of LINC00936 against myocardial infarction (MI) by targeting NFKBIA through the NF-&kappa;B signaling pathway. Methods: Gene Expression Omnibus and Multi Experiment Matrix website were employed for Bioinformatics prediction. MI mouse models were established. Ultrasonic cardiogram assessments were conducted to quantify cardiac function. TTC and TUNEL staining were conducted to observe infarct size and apoptosis of myocardial tissues. Myocardial cells were assigned into blank, NC, LINC00936 vector, NFKBIA vector, siRNA-LINC00936, siRNA-NFKBIA, LINC00936 vector + NFKBIA vector and siRNA-LINC00936 + siRNA-NFKBIA groups. RT-qPCR and Western blotting were performed to measure expression of LINC00936, NFKBIA, NF-&kappa;Bp50, NF-&kappa;Bp65 and IKK&beta;. MTT assay, flow cytometry and ELLSA were conducted to measure cell viability, cell cycle, apoptosis and inflammatory cytokine expression. Results: LINC00936 showed high expression in MI. NFKBIA was confirmed as the target gene of LINC00936 by dual-luciferase reporter gene assay and RNA-IP assay. Compared with the sham group, the MI group exhibited larger infarct sizes, increased apoptotic myocardial cells, over-expressions of LINC00936, NF-&kappa;Bp50, NF-&kappa;Bp65 and IKK&beta; as well as reductions in expressions of NFKBIA. LINC00936 inhibited the activation of the NF-&kappa;B signaling pathway by suppressing NFKBIA expression. Overexpression of NFKBIA reversed the up-regulation of the NF-&kappa;B signaling pathway-related gene expression, inhibition of myocardial cell growth, apoptosis of myocardial cells and increased expressions of inflammatory cytokines due to high LINC00936 expression. Conclusion: Our study provided evidence suggesting that LINC00936-targeted overexpression of NFKBIA exhibits protective ability against MI through the blockade of the NF-&kappa;B pathway in mice.

Eluted zinc ions stimulate osteoblast differentiation and mineralization in human dental pulp stem cells for bone tissue engineering

ObjectiveZinc is an essential element for proliferation, differentiation and survival in various cell types. In a previous study, we found that zinc ions released from zinc-modified titanium surfaces (eluted zinc ions; EZ) stimulate cell viability, osteoblast marker gene expression and calcium deposition in human bone marrow-derived mesenchymal cells (hBMCs). The aim of the present study was to investigate the effects of EZ on osteoblast differentiation among dental pulp stem cells (DPSCs) in vitro. MethodIn this study, we evaluated the effects of EZ on osteogenesis in DPSCs. Osteoblast and osteoclast marker gene expression was evaluated by real-time PCR. We also evaluated alkaline phosphatase (ALP) staining and calcium deposition. ResultsWe found that EZ stimulated osteoblast marker gene (type I collagen, alkaline phosphatase (ALP), osteocalcin (OCN) and Runx2) expression, vascular endothelial growth factor A (VEGF-A), and TGF-beta signaling pathway-related gene expression after 7days of incubation. Osteoclastogenesis occurs in a receptor for activated nuclear-factor kappa B ligand (RANKL)/osteoprotegerin (OPG)-independent manner. Real-time PCR analysis revealed that EZ did not affect RANKL or OPG mRNA expression. It was also revealed that EZ induced alkaline phosphatase (ALP) staining and calcium deposition in DPSCs. Collectively, these results demonstrate the potential for clinical application to prospective treatment of bone diseases.

Effects of transforming growth factor-β1 on the expression of Wnt signaling pathway related genes in the differentiation of bone marrow stromal cells

Objective To observe the transforming growth factor-β1 (TGF-β1) on the expression of Wnt signaling pathway related genes during the chondrogenesis of bone marrow stromal cells (BMSCs) in vitro.Methods BMSCs cultured in TGF-β1 medium were observed by inverted phase contrast microscope and identified by toluidine blue staining.The expression levels of glycosaminoglycan (GAG) and target genes including Wnt1,Wnt3a,β-catenin,Sox9 and collagen Ⅱ mRNA under different concentrations of TGF-β1 were detected by using enzyme linked immunosorbent assay (ELISA) and real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) respectively.Results At day 21 after TGF-β1 induction,toluidine blue staining showed that more than 95％ cells differentiated into chondrocytes; the amount of GAG in the cell cultured medium in 2 μg/L TGF-β1-induced group was (69.16 ± 1.18) mg/L,significantly higher than other groups (P ＜ 0.05).FQ-PCR results showed that Wnt1,Wnt3a and β-catenin mRNA expression in 2 μg/L TGF-β1-induced group was inhibited [0.0266 ±0.0047,0.0154 ±0.0033,0.0187 ±0.0021,(P ＜0.05)],but in contrast Sox9 and collagen Ⅱ mRNA expression levels were increased [0.0682 ± 0.0024,0.1134 ± 0.0048 (P ＜ 0.05)] as compared with the control group.Conclusion TGF-β1 could promote BMSCs chondrogenic differentiation,and the expression of Wnt/β-catenin signaling pathway related genes Wnt1,Wnt3a and β-catenin mRNA was inhibited and the expression of chondrocyte specific genes Sox9 and collagen Ⅱ mRNA was increased. Key words: Transforming growth factor-beta1; Bone marrow stromal cells; Wnt signaling pathway; Glycosaminoglycan

CTNNB1, AXIN1 and APC expression analysis of different medulloblastoma variants

OBJECTIVES:We investigated four components of the Wnt signaling pathway in medulloblastomas. Medulloblastoma is the most common type of malignant pediatric brain tumor, and the Wnt signaling pathway has been shown to be activated in this type of tumor.METHODS:Sixty-one medulloblastoma cases were analyzed for β-catenin gene (CTNNB1) mutations, β-catenin protein expression via immunostaining and Wnt signaling pathway-related gene expression. All data were correlated with histological subtypes and patient clinical information.RESULTS:CTNNB1 sequencing analysis revealed that 11 out of 61 medulloblastomas harbored missense mutations in residues 32, 33, 34 and 37, which are located in exon 3. These mutations alter the glycogen synthase kinase-3β phosphorylation sites, which participate in β-catenin degradation. No significant differences were observed between mutation status and histological medulloblastoma type, patient age and overall or progression-free survival times. Nuclear β-catenin accumulation, which was observed in 27.9% of the cases, was not associated with the histological type, CTNNB1 mutation status or tumor cell dissemination. The relative expression levels of genes that code for proteins involved in the Wnt signaling pathway (CTNNB1, APC, AXIN1 and WNT1) were also analyzed, but no significant correlations were found. In addition, large-cell variant medulloblastomas presented lower relative CTNNB1 expression as compared to the other tumor variants.CONCLUSIONS:A small subset of medulloblastomas carry CTNNB1 mutations with consequent nuclear accumulation of β-catenin. The Wnt signaling pathway plays a role in classic, desmoplastic and extensive nodularity medulloblastoma variants but not in large-cell medulloblastomas.

Analysis of the role of the integrin signaling pathway in hepatocytes during rat liver regeneration.

To explore the role of the integrin signaling pathway in hepatocytes during rat liver regeneration, the integrin signaling pathway-related gene expression profile in hepatocytes of regenerative liver was detected using Rat Genome 230 2.0 array. The chip data showed that 265 genes of the integrin signaling pathway were included by Rat Genome 230 2.0 array and 132 genes showed significant expression changes in hepatocytes of regenerative liver. The numbers of up-, down- and up/down-regulated genes were 110, 15 and 7 respectively. In addition, bioinformatics and systems biology methods were used to analyze the role of the integrin signaling pathway in hepatocytes. The analysis of gene synergy value indicated that paths 1, 8, 12, and 15 promoted hepatocyte proliferation at the priming phase of liver regeneration; paths 1, 3, 8, and 12–15 enhanced hepatocyte proliferation at the progressing phase; paths 11 and 14 promoted hepatocyte proliferation, while paths 12 and 13 reduced hepatocyte proliferation at the terminal phase. Additionally, the other 8 paths (2, 4, 5–7, 9–10, and 16) were not found to be related to liver regeneration. In conclusion, 132 genes and 8 cascades of the integrin signaling pathway participated in regulating hepatocyte proliferation during rat liver regeneration.

Signaling pathway-related gene expression in neuroimmunoregulation

An important question in the field of psychoneuroimmuto 4 h, following ACTH treatment. TNFa expression also nology involves the intracellular mechanisms activated by neuropeptide receptors to modulate immune system functioning. It is reasonable to expect that cross talk occurs or the pathways converge when two different signals such as a hormone or a cytokine mediate a common effect such as inhibition of a cytokine. The NF-nB transcription factor pathway, because it enhances the expression of many immune and viral-related genes might be a convergence point for different receptors to have similar immune affects. In fact, one means by which corticosteroids mediate part of their anti-inflammatory effect is by inhibiting NF-nB activity through the induction of the endogenous inhibitor InBa (Auphan et al., 1995; Scheinman et al., 1995). The aim of this study was to determine if selected neuropeptides, corticotropin (ACTH) and corticotropin releasing hormone (CRH), which are respectively immunosuppressive (Smith 1994) and immunoenhancing (Auphan et al., 1995; McGillis et al., 1989) act through alteration of NF-nB activity and related gene expression. Our initial studies investigated the effect of ACTH on NF-nB-related gene expression. A NF-nB pathway specific macro-gene array system (GEArray) was used to identify potential mechanisms for ACTH inhibition of NF-nB. Human H9 T-lymphoma cells were stimulated with tumor necrosis factor alpha (TNFa) for 24 h to induce NF-nB, then the cultures were treated with ACTH. Total RNA was then extracted for the macro-array assay. Expression was normalized to the housekeeping gene, GAPDH and in this assay a two-fold or greater increase over control was considered significant. Enhanced expression was seen in multiple genes related to NF-nB signaling (Table 1). InBa and c-Rel were elevated within 45 min and still evident out