Ribonucleic Acid Levels Research Articles

Mechanism of metformin enhancing the sensitivity of human pancreatic cancer cells to gem-citabine by regulating the PI3K/Akt/mTOR signaling pathway.

To investigate the effect of metformin (MET) on enhancing the sensitivity of human pancreatic cancer cells to gemcitabine (GEM) by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. The GEM-resistant human pancreatic cancer PANC-1/GEM cell line was established, and the proliferation ability of PANC-1 and PANC-1/GEM cell lines was detected using the Cell Counting Kit-8 (CCK-8), which was then detected by flow cytometry after they were labeled by Ki67. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and Western blotting were adopted to detect the difference in the mTOR expression between PANC-1 and PANC-1/GEM cell lines. The proliferation ability of PANC-1/GEM/MET and PANC-1/GEM cell lines was determined using CCK-8 after drug-resistant cell lines were treated with 20 mmol/L MET combined with 0.4 μmol/L GEM or 0.4 μmol/L GEM alone for 48 h. Colony formation assay was applied to detect the proliferation ability of cells. The difference in the expression of mTOR/PI3K/Akt between PANC-1/GEM/MET and PANC-1/GEM cell lines was tested via qRT-PCR and Western blotting, respectively. Compared with PANC-1 cells, PANC-1/GEM cells had significantly enhanced proliferation ability (p<0.01). Flow cytometry results showed that the proliferation ability of PANC-1/GEM cells was notably enhanced (p<0.01). The expression level and phosphorylation level of mTOR in drug-resistant cell lines were increased (p<0.01). After the drug-resistant cell lines were treated with 20 mmol/L MET for 48 h, the proliferation ability of PANC-1/GEM/MET cells was evidently decreased compared with that of PANC-1/GEM cells (p<0.01). The messenger ribonucleic acid (mRNA) and protein expression levels of mTOR/PI3K/Akt were markedly down-regulated (p<0.01). MET can regulate the PI3K/Akt/mTOR signaling pathway to enhance the sensitivity of human pancreatic cancer cells to GEM.

Liver X Receptor Activation Impairs Neutrophil Functions and Aggravates Sepsis.

Liver X receptors (LXRs) are nuclear receptors activated by oxidized lipids and were previously implicated in several metabolic development and inflammatory disorders. Although neutrophils express both LXR-α and LXR-β, the consequences of their activation, particularly during sepsis, remain unknown. We used the model of cecal ligation and puncture (CLP) to investigate the role of LXR activation during sepsis. In this study, we verified that LXR activation reduces neutrophil chemotactic and killing abilities in vitro. Mice treated with LXR agonists showed higher sepsis-induced mortality, which could be associated with reduced neutrophil infiltration at the infectious foci, increased bacteremia, systemic inflammatory response, and multiorgan failure. In contrast, septic mice treated with LXR antagonist showed increased number of neutrophils in the peritoneal cavity, reduced bacterial load, and multiorgan dysfunction. More important, neutrophils from septic patients showed increased ABCA1 messenger ribonucleic acid levels (a marker of LXR activation) and impaired chemotactic response toward CXCL8 compared with cells from healthy individuals. Therefore, our findings suggest that LXR activation impairs neutrophil functions, which might contribute to poor sepsis outcome.

Mutation analysis of FBN1 gene in two Chinese families with congenital ectopia lentis in northern China.

To summarize the phenotypes and identify the underlying genetic cause of the fibrillin-1 (FBN1) gene responsible for congenital ectopia lentis (EL) in two Chinese families in northern China. A detailed family history and clinical data from all participants were collected by clinical examination. The candidate genes were captured and sequenced by targeted next-generation sequencing, and the results were confirmed by Sanger sequencing. Haplotyping was used to confirm the mutation sequence. Real-time PCR was used to determine the FBN1 messenger ribonucleic acid (mRNA) levels in patients with EL and in unaffected family members. The probands and other patients in the two families were affected with congenital isolated EL. A heterozygous FBN1 mutation in exon 21 (c.2420_IVS20-8 delTCTGAAACAinsCGAAAG) was identified in FAMILY-1. A heterozygous FBN1 mutation in exon 14 (c.1633C>T, p.R545C) was identified in FAMILY-2. Each mutation co-segregated with the affected individuals in the family and did not exist in unaffected family members and 200 unrelated normal controls. The insertion-deletion mutation (c.2420 IVS20-8delTCTGAAACA insCGAAAG) in the FBN1 gene is first identified in isolated EL. The mutation (c.1633C>T) in the FBN1 gene was a known mutation in EL patient. The variable phenotypes among the patients expand the phenotypic spectrum of EL in a different ethnic background.

LXW7 attenuates inflammation via suppressing Akt/nuclear factor kappa B and mitogen-activated protein kinases signaling pathways in lipopolysaccharide-stimulated BV2 microglial cells

Microglia activation is closely linked to ischemia, various chronic neurodegenerative diseases (e.g., Alzheimer′s disease, Parkinson′s disease, multiple sclerosis, amyotrophic lateral sclerosis), and many other central nervous system diseases. Accumulating evidence suggests that depressing the microglial inflammatory response could be an effective treatment for inflammatory disorders. The integrin αvβ3 inhibitor LXW7 has a neuroprotective effect; however, its anti-inflammatory effects and underlying mechanism remain unclear. Thus, we examined whether LXW7 would inhibit inflammatory cytokines and mediators, and we evaluated the potential mechanisms of its neuroprotective effects. Nitrite analysis revealed LXW7 reduced the nitric oxide (NO) level. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) suggested that LXW7 suppressed the expression of proinflammatory genes for tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1β), inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and anti-inflammatory gene interleukin 10 (IL-10) at the messenger ribonucleic acid level. Enzyme-linked immunosorbent assay results demonstrated that LXW7 treatment reduced the expression of prostaglandin E2 (PGE2), TNF-α, IL-1β and IL-10 at the protein level. Western blotting was conducted to confirm the upregulation of inflammatory factors, including iNOS and COX-2 at the protein level. LXW7 inhibited major genes in the Akt/NF-κB and c-Jun NH2-terminal kinase/ mitogen-activated protein kinases (JNK/MAPK) signaling pathways. Immunofluorescence revealed that LXW7 inhibited the nuclear translocation of nuclear factor kappa B (NF-κB). Thus, LXW7 effectively alleviated LPS-induced inflammatory damage and had neuroprotective effects. The anti-inflammatory effects of LXW7 may be associated with the inhibition of microglial activation via Akt/NF-κB and JNK/MAPK signaling pathways by blocking integrin αvβ3 receptor. The present study′s findings suggest that LXW7 has a substantial therapeutic potential for treating inflammatory and neurodegenerative diseases.

CO2 Pneumoperitoneum Effects on Molecular Markers of Tumor Invasiveness in SH-SY5Y Neuroblastoma Cells.

CO2 pneumoperitoneum can influence the biological behavior of neuroblastoma (NB). Angiogenesis and genetic features are responsible for malignant phenotype of this tumor. We examined the CO2 effects on N-Myc, vascular endothelial growth factor (VEGF), and matrix metalloproteinase-2 (MMP-2) expression as critical biomarkers of tumor invasiveness, in NB cells without N-Myc amplification. SH-SY5Y cells were exposed to CO2 (100%) at 15 mm Hg pressure for 4 hours and then moved to normal condition for 24 hours. Control cells were incubated with 5% CO2 for the same time. In control and CO2-exposed cells, the messenger ribonucleic acid (mRNA) levels of hypoxia-inducible factor (HIF)-1α, HIF-2α, VEGF-A, and MMP-2 were quantified by real-time polymerase chain reaction. N-Myc expression was evaluated by Western blot analysis. The exposure to 15 mm Hg CO2 (100%) for 4 hours induced an increase in HIF-1α, but not in HIF-2α, mRNA levels. No differences were observed in N-Myc expression between exposed and control cells at each incubation time. Similarly, no significant differences were found for VEGF-A and MMP-2 transcript levels. In CO2 exposed cells, we observed only a slight increase in both VEGF-A and MMP-2 mRNA levels after 4 and 24 hours in comparison to controls. In our study, the hypoxic environment induced by CO2 exposure does not affect the expression of critical biomarkers of NB aggressiveness, such as N-Myc, VEGF, and MMP-2, in human SH-SY5Y NB cells without N-Myc amplification. These data suggest that CO2 pneumoperitoneum might not adversely impact NB cell invasiveness; however, it is necessary to evaluate these effects in others in vitro and in vivo models.

Accelerated Intestinal Epithelial Cell Turnover Correlates with Stimulated BMP Signaling Cascade following Intestinal Ischemia-Reperfusion in a Rat.

Bone morphogenetic proteins (BMPs) are a family of proteins that regulate proliferation and differentiation of intestinal epithelial cells. The purpose of this study was to evaluate the role of BMP signaling following intestinal ischemia-reperfusion (IR) in a rat model. Male Sprague-Dawley rats were divided into four experimental groups: Sham-24 and Sham-48 rats underwent laparotomy and were sacrificed 24 or 48 hours later, respectively; IR-24 and IR-48 rats underwent occlusion of superior mesenteric artery and portal vein for 30 minutes followed by 24 or 48 hours of reperfusion, respectively. Enterocyte proliferation and apoptosis were determined at sacrifice. BMP-related genes and protein expression were determined using real-time polymerase chain reaction, Western blot, and immunohistochemistry for 48 hours followed by IR. IR rats demonstrated a significant increase in BMP2 (twofold increase, p < 0.05), BMP4 (sevenfold increase), STAT3 (70% increase), BMPR1 (70% increase) messenger ribonucleic acid levels in jejunum and was accompanied by a significant increase in BMP2 and BMP4 protein levels in jejunum (sixfold increase) (Western blot) and upward increase in the number of BMP-positive cells (by immunohistochemistry) in jejunal (48% increase) and ileal (56% increase) villi compared with Sham-48 animals. Elevation in BMP2 and BMP4 levels was associated with increased rates of cell proliferation and increased cell apoptosis. Forty-eight hours following intestinal IR in rats, BMP signaling pathway was stimulated. The increase in BMP signaling pathway activity correlates with accelerated cell turnover.

EXPLORE: A Prospective, Multinational, Natural History Study of Patients with Acute Hepatic Porphyria with Recurrent Attacks.

Background and AimsAcute hepatic porphyria comprises a group of rare genetic diseases caused by mutations in genes involved in heme biosynthesis. Patients can experience acute neurovisceral attacks, debilitating chronic symptoms, and long‐term complications. There is a lack of multinational, prospective data characterizing the disease and current treatment practices in severely affected patients.Approach and ResultsEXPLORE is a prospective, multinational, natural history study characterizing disease activity and clinical management in patients with acute hepatic porphyria who experience recurrent attacks. Eligible patients had a confirmed acute hepatic porphyria diagnosis and had experienced ≥3 attacks in the prior 12 months or were receiving prophylactic treatment. A total of 112 patients were enrolled and followed for at least 6 months. In the 12 months before the study, patients reported a median (range) of 6 (0‐52) acute attacks, with 52 (46%) patients receiving hemin prophylaxis. Chronic symptoms were reported by 73 (65%) patients, with 52 (46%) patients experiencing these daily. During the study, 98 (88%) patients experienced a total of 483 attacks, 77% of which required treatment at a health care facility and/or hemin administration (median [range] annualized attack rate 2.0 [0.0‐37.0]). Elevated levels of hepatic δ‐aminolevulinic acid synthase 1 messenger ribonucleic acid levels, δ‐aminolevulinic acid, and porphobilinogen compared with the upper limit of normal in healthy individuals were observed at baseline and increased further during attacks. Patients had impaired quality of life and increased health care utilization.ConclusionsPatients experienced attacks often requiring treatment in a health care facility and/or with hemin, as well as chronic symptoms that adversely influenced day‐to‐day functioning. In this patient group, the high disease burden and diminished quality of life highlight the need for novel therapies.

Correlations of the severity of diabetic retinopathy with EPO, Caspase-3 expression and oxidative stress.

To investigate the relationships of the severity of diabetic retinopathy with erythropoietin (EPO), Caspase-3 expression, and oxidative stress. A total of 20 patients with non-proliferative diabetic retinopathy hospitalized from January 2017 to January 2018 were enrolled as observation group 1, 20 patients with proliferative diabetic retinopathy were chosen as observation group 2, and 20 patients with idiopathic macular hole were selected as control group. After admission, patients received all necessary examinations and underwent vitrectomy during which vitreous and retinal tissues were taken, and venous blood was collected. Then, the content of EPO, Caspase-3, nitric oxide (NO), and malondialdehyde (MDA) was detected through enzyme-linked immunosorbent assay (ELISA), the messenger ribonucleic acid (mRNA) levels of EPO, Caspase-3, NO, and MDA were measured via quantitative Polymerase Chain Reaction (qPCR), and the severity of diabetic retinopathy was evaluated by diabetic retinopathy grading score. Observation group 1 and 2 had significantly decreased the content of EPO (p<0.05) and overtly increased Caspase-3, NO, and MDA content (p<0.05) in comparison with control group. Compared with those in observation group 1, the EPO content was clearly lowered in observation group 2 (p<0.05), and the content of Caspase-3, NO, and MDA was evidently elevated (p<0.05). The diabetic retinopathy grading score was remarkably lower in control group than that in both observation group 1 and observation group 2 (p p<0.05), and it was significantly enhanced in observation group 2 compared with that in observation group 1 (p<0.05). Correlation analysis showed that the EPO content was negatively correlated with the severity of diabetic retinopathy, while the content of Caspase-3, NO, and MDA was positively related to the severity of diabetic retinopathy. The severity of diabetic retinopathy has a negative association with EPO and positive correlations with Caspase-3, NO, and MDA content.

Tim-3 Promotes Listeria monocytogenes Immune Evasion by Suppressing Major Histocompatibility Complex Class I.

T-cell immunoglobulin and mucin protein 3 (Tim-3) is an immune checkpoint inhibitor that has therapeutic implications for many tumors and infectious diseases. However, the mechanisms by which Tim-3 promotes immune evasion remain unclear. In this study, we demonstrated that Tim-3 inhibits the expression of major histocompatibility complex class I (MHC-I) in macrophages at both the messenger ribonucleic acid and protein levels by inhibiting the STAT1-NLRC5 signaling pathway. As a result, MHC-I-restricted antigen presentation by macrophages was inhibited by Tim-3 both in vitro and in a Listeria monocytogenes infection model in vivo. Systemic overexpression of Tim-3 or specific knockout of Tim-3 in macrophages significantly attenuated or enhanced CD8+ T-cell activation and infection damage in L monocytogenes-infected mice, respectively. Thus, we identified a new mechanism by which Tim-3 promotes L monocytogenes immune evasion. Further studies on this pathway might shed new light on the physio-pathological roles of Tim-3 and suggest new approaches for intervention.

Effect of Long-Term Polytrauma on Ventilator-Induced Diaphragmatic Dysfunction in a Piglet Model.

Mechanical ventilation is known to activate oxidative stress and proteolytic pathways in the diaphragm. Trauma by inducing inflammation and activating proteolytic pathways may potentiate the effects of mechanical ventilation on the diaphragm. In a blunt chest trauma with concomitant injuries we tested the hypothesis that trauma via inflammation further activates the proteolytic pathways and worsens atrophy in the diaphragm. Piglets were separated into two groups and underwent 72 h of mechanical ventilation. One group received a polytrauma (PT) by unilateral femur fracture, blunt chest trauma with lung contusion, laparotomy with standardized liver incision, and a predefined hemorrhagic shock. The second mechanically ventilated group (MV) did not receive any trauma. A non-ventilated group (Con) served as control.Diaphragmatic fiber dimensions, Western Blot analyses of proteolytic pathways, and lipid peroxidation and messenger ribonucleic acid (mRNA) levels of cytokines and nuclear factor kappa b subunit p65 were measured. Active Caspase-3 was significantly increased in MV (P = 0.019), and in PT (P = 0.02) compared with Con. Nuclear factor kappa b subunit p65, was upregulated in PT (P = 0.010) compared with Con. IL-6 mRNA increased significantly in PT compared with Con (P = 0.0024) but did not differ between Con and MV. Trauma and mechanical ventilation induced proteolysis and atrophy in the diaphragm, but only polytrauma induced an inflammatory response in the diaphragm. The additional traumatic inflammatory stimulus did not increase the levels of the prementioned variables. These data underline that inflammation is not a major contributor to ventilator-induced diaphragmatic dysfunction. AZ 84-02.04.2014.A265 (Landesamt für Natur-, Umwelt- und Verbraucherschutz, LANUV NRW, Germany).

Potential roles of lncRNA-Cox2 and EGR1 in regulating epidural fibrosis following laminectomy.

Epidural fibrosis, one of the common complications after spinal surgery, seriously affects the surgical decompression effect. Effectively inhibiting the fibrous tissue hyperplasia is pivotal to reduce the scar adhesion. Previous studies showed that early growth response 1 (EGR1) is associated with the fibroblast reactivity induced by transforming growth factor-beta (TGF-β) and plays a vital regulatory role in scar formation; however, the upstream targets and mechanisms still remain unclear. In this work, it was found that the level of long non-coding ribonucleic acid (lncRNA)-cyclooxygenase-2 (COX2) was significantly negatively correlated with EGR1 expression and the severity of the scar. Therefore, it was conjectured that lncRNA-COX2 may decrease fibroplasia and scar formation by negatively regulating EGR1. TGF-β was used to activate the embryonic and adult rat fibroblasts. Rats underwent laminectomy to establish the epidural fibrosis model. The changes in the levels of fibroplasia-related genes were measured and analyzed through messenger RNA (mRNA), lncRNA, and micro RNA expression profile chips. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was applied to determine the levels of EGR1 and lncRNA-COX2, and Western blotting was adopted to detect the content of EGR1, collagen I (Col-1), Col-3, and alpha-smooth muscle actin (α-SMA). The scar formation was reflected by hematoxylin and eosin (HE) staining and Masson staining, and the expression level of α-SMA in the scar tissues was measured via immunohistochemistry. Finally, micro-magnetic resonance imaging (MRI) was utilized to examine the different degrees of epidural fibroplasia. It was found that the reactivity of embryonic rat fibroblasts to the TGF-β stimulation was different from that of adult rat fibroblasts. LncRNA-COX2 was highly expressed in the embryonic rat fibroblasts, but lowly expressed in the adult rat fibroblasts, which had negative correlations with the EGR1 level in embryonic and adult rat fibroblasts. In addition, it was revealed that the expression of EGR1 in the adult rat fibroblasts was remarkably higher than that in the embryonic rat fibroblasts after the activation with TGF-β. Meanwhile, the level of lncRNA-COX2 was lowered after the activation, especially in the adult rat fibroblasts. It was discovered in the in-vivo model that the degree of fibroplasia was positively associated with EGR1 level and negatively correlated with lncRNA-COX2 level. The results of this research elucidated that the down-regulation of lncRNA-COX2 is involved in the epidural scar formation and related to the elevated EGR1 level which regulates the activation of fibroblasts and secretion of massive extracellular matrixes, suggesting that lncRNA-COX2 may modulate the role of fibroblasts in scar formation as an upstream action target of EGR1.

A microfluidic chip-based assay outperforms common methods for quantification of cell-free DNA and RNA

Plasma and serum contain cell-free DNA and RNA and are common specimens used for pathology investigation. Circulating nucleic acids in plasma and serum (CNAPS) represent an important target for analyzing several conditions including prenatal diagnostics, tumors, infections, and inflammatory disorders. It is highly expected that plasma and serum contain potential biomarkers for the detection of cancers and other pathological conditions. The major challenges are that CNAPS are present in small quantities and are highly degraded, hampering their downstream analysis. For example, obtaining acceptable quality and quantity of CNAPS remains a bottleneck for advanced studies such as those using next-generation sequencing (NGS) platforms as the currently available platforms require nanogram to microgram levels of deoxyribonucleic acid or ribonucleic acid (DNA/RNA) input. This paper examines the use of various methods to quantify CNAPS, including quantitative real-time polymerase chain reaction (qRT-PCR), digital PCR (dPCR), fluorometric assay, gel electrophoresis, and Bioanalyzer. In particular, the results of CNAPS quantification using a fluorometer, a NanoDrop spectrophotometer, and a microfluidic chip-based assay were compared. It is asserted that spectrophotometry, fluorometric assay and gel electrophoresis lack sensitivity for detecting CNAPS; thus, highly sensitive methods such as qRT-PCR, dPCR, and Bioanalyzer are required.

Increased expression of pentraxin 3 in placental tissues from patients with unexplained recurrent pregnancy loss

Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a evolutionarily conserved multimeric pattern recognition receptor involved in the humoral component of the innate immune system. Pentraxin 3 is released when tissue is stressed or damaged, and interacts with many different ligands. Pentraxin 3 exerts a pivotal role both as a regulator and as an indicator of inflammatory response in the pathogenesis of many diseases such as sepsis, vasculitis and preeclampsia. Uncontrolled inflammatory response is considered a major cause of unexplained recurrent pregnancy loss (URPL). We determined the PTX3 messenger ribonucleic acid (mRNA) and protein expression levels in placentai tissues from 50 women with URPL, and made comparison with those in 50 age-matched control subjects. In quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry analyses, PTX3 mRNA and protein levels, respectively, were significantly increased in URPL patients compared with their respective controls (p = 0.0001). Although no significant correlations were identified between PTX3 expression levels and clinical parameters such as maternal age, numbers of previous pregnancy losses, and gestational age at miscarriage, PTX3 mRNA expression was significantly higher in patients with no live births than in women with previous live births (p = 0.0001). Our study suggests that tissue-specific expression of PTX3 is associated with URPL. Further larger studies are required to determine whether PTX3 expression can be used as a biomarker to manage URPL in routine clinical practice.

A Bioinformatics Toolkit: In Silico Tools and Online Resources for Investigating Genetic Variation.

With the advent of large-scale next-generation sequencing initiatives, there is an increasing importance to interpret and understand the potential phenotypic influence of identified genetic variation and its significance in the human genome. Bioinformatics analyses can provide useful information to assist with variant interpretation. This review provides an overview of tools/resources currently available, and how they can help predict the impact of genetic variation at the deoxyribonucleic acid, ribonucleic acid, and protein level.

LINC01503 promotes cell proliferation, invasion and EMT process in cholangio-carcinoma.

This work aimed to analyze the relative expression level of long intergenic non-protein coding ribonucleic acid 1503 (LINC01503) in cholangiocarcinoma tissues and cells, and to explore the effects of LINC01503 on cell proliferation, migration and invasion. Logarithmic growth phase cholangiocarcinoma cells were selected and transfected with Lipofectamine 2000 (si-LINC01503, si-NC). The expression of LINC01503 was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The proliferation of cells was observed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to detect cell apoptosis. Transwell assay was used to observe the cell migration and invasion ability. The expression of LINC01503 was significantly increased in cholangiocarcinoma tissues compared with adjacent tissues (p<0.05), and the up-regulated expression of LINC01503 was associated with lymph node metastasis. Compared with normal bile duct cells (HIBEC), cholangiocarcinoma cells (RBE, QBC939) have higher expression of LINC01503, and si-LINC01503 transfection can effectively reduce the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of cholangiocarcinoma cells. LINC01503 is highly expressed in cholangiocarcinoma and can effectively promote the proliferation, migration, invasion and EMT process of cancer cells, and LINC01503 is expected to be a potential biomarker for cholangiocarcinoma.

Correlation analysis between COX-2 gene polymorphism and eclampsia.

The aim of this study was to investigate the expression of cyclooxygenase-2 (COX-2) in eclampsia patients, and to explore the correlation between COX-2 polymorphism and incidence of eclampsia. From January 2016 to January 2018, a total of 280 pregnant women diagnosed in the Obstetrics and Gynecology Department of our hospital were selected for this study. All patients were divided into two groups, including normal pregnancy control group (n=120) and eclampsia group (n=160). The expression of COX-2 in placenta and umbilical cord tissues of eclampsia group and normal group was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Western blotting and immunohistochemical staining. The single-nucleotide polymorphisms (rs1526172, rs1245231 and rs2198532) in the promoter region of the COX-2 gene were typed via conformation difference gel electrophoresis. Whether the distribution frequency of COX-2 genotypes met Hardy-Weinberg equilibrium law was detected via the Chi-square test. Meanwhile, the correlation between COX-2 alleles and gene polymorphisms and the incidence of eclampsia was analyzed. The messenger ribonucleic acid (mRNA) and protein expression levels of COX-2 in the eclampsia group were significantly higher than those of the normal group (p<0.05). According to the analysis, three polymorphisms of COX-2 gene were all in line with Hardy-Weinberg equilibrium distribution (p>0.05). Gene association analysis revealed that only polymorphisms (rs1526172 and rs1245231) and alleles were correlated with the incidence of eclampsia (p<0.05). However, polymorphism rs2198532 and alleles were not correlated with the incidence of eclampsia (p>0.05). Rs1526172 and rs1245231 in the promoter region of COX-2 are correlated with the incidence of eclampsia, while rs2198532 has no correlation with eclampsia.

Vascular endothelial growth factor in tumor cells in platinum-containing chemotherapy of advanced ovarian cancer

Aim. Study of the relationship of vascular endothelial growth factor expression in tumor cells of ascites and primary tumor in patients with ascitic form of ovarian cancer undergoing neoadjuvant chemotherapy according to the AP scheme (doxorubicin + cisplatin).&#x0D; Methods. The level of vascular endothelial growth factor messenger ribonucleic acid (mRNA) was assessed using PCR-RT in epithelial cells of ascites and primary tumor tissue obtained intraoperatively after 2–4 courses of neoadjuvant chemotherapy according to the AP scheme from 25 patients with a verified diagnosis of ovarian cancer (serous adenocarcinoma) at stage III–IV according to the International Federation of Gynecology and Obstetrics. Processing of the results was performed using the software package Statistica 10.&#x0D; Results. An increase in the expression of vascular endothelial growth factor mRNA in epithelial cells of ascites in 60% of samples was established. In the intraoperatively obtained tumor tissue after 2–4 courses of neoadjuvant chemotherapy according to the AP scheme, vascular endothelial growth factor expression was increased in 73% of the samples. At the same time, the increase of relative expression in the tumor after neoadjuvant chemotherapy did not depend on the degree of therapeutic pathomorphosis but was related to early relapse and increased level of cancer antigen-125 (CA-125). Median of relapse-free survival in vascular endothelial growth factor-positive ovarian cancer was less than 5 months.&#x0D; Conclusion. Neoadjuvant chemotherapy according to the AP scheme increases the expression of vascular endothelial growth factor in the tumor tissue in advanced ovarian cancer; the degree of vascular endothelial growth factor overexpression in ovarian tumor is related to the presence of early relapse and an increase in the serum CA-125 level.

Fibulin-2 inhibits development of gastric cancer by downregulating β-catenin.

The aim of the present study was to investigate the expression of fibulin-2 and β-catenin in gastric cancer tissues and the association to mutual regulation. Forty-nine cases of gastric cancer specimens obtained via surgical resection in the Pathology Department of Heping Hospital Affiliated to Changzhi Medical College from March 2013 to August 2017 were collected. The expression levels of fibulin-2 and β-catenin in 49 cases of gastric cancer and para-carcinoma tissues were detected via quantitative polymerase chain reaction and immunohistochemistry. The correlation of expression of fibulin-2 and β-catenin proteins in gastric cancer was detected via Spearman's analysis. The correlation of fibulin-2 and β-catenin protein expression with clinicopathological indexes of patients was also analyzed. Moreover, the fibulin-2 overexpression plasmid was constructed and transfected into human gastric cancer AGS and SGC-790 cell lines, so as to observe changes in β-catenin and its downstream indexes. Fibulin-2 messenger ribonucleic acid (mRNA) level in gastric cancer tissues was significantly lower than that in para-carcinoma tissues, while β-catenin mRNA level was significantly increased (P<0.05). The positive rate of fibulin-2 protein was 73.47% (36/49) in gastric cancer tissues and 16.33% (8/49) in para-carcinoma tissues, while that of β-catenin was 77.55% (38/49) in gastric cancer tissues and 28.57% (14/49) in para-carcinoma tissues, indicating that fibulin-2 protein is significantly deleted in gastric cancer tissues, and β-catenin protein is significantly upregulated (P<0.001). Fibulin-2 and β-catenin had a negative correlation (r=−0.361, P=0.003), but was closely correlated with the tumor size and lymph node metastasis (P<0.05). After overexpression of fibulin-2, expression of β-catenin, cyclin D1 and c-Myc protein was obviously downregulated (P<0.05). The tumor suppressor gene, fibulin-2, is significantly deleted in gastric cancer tissues, while β-catenin is remarkably enriched. Overexpression of fibulin-2 can inhibit the development of gastric cancer by downregulating β-catenin.

Systematic expression analysis of the mitochondrial respiratory chain protein subunits identifies COX5B as a prognostic marker in clear cell renal cell carcinoma.

To analyze the expression of mitochondrial respiratory chain protein subunits in clear cell renal cell carcinoma. Possible prognostic candidates were determined using The Cancer Genome Atlas database (n=605). The database provided valid messenger ribonucleic acid expression data for 93 genes encoding for the subunits. Selected subunits were further investigated at the messenger ribonucleic acid and protein level by real-time polymerase chain reaction, western blot and immunohistochemistry with the cohorts of the University Hospital Bonn. The Cancer Genome Atlas messenger ribonucleic acid expression data indicated univariate and multivariate prognostic impact for seven subunits (NDUFS8, NDUFS7, COX5B, COX6B1, SDHD, COX15 and COX19). Using real-time polymerase chain reaction, significant downregulation (P<0.05, n=74) could be shown for COX5B, COX6B1, NDUFS7 and NDUF8 in clear cell renal cell carcinoma tissue. Survival analysis of polymerase chain reaction data showed a non-significant relationship (P=0.067) of high COX5B expression and poor overall survival. Western blot (n=8) and immunohistochemistry analysis (n=167) confirmed significant COX5B downregulation on the protein level. Immunohistochemistry analysis identified COX5B as a prognostic marker for overall (P=0.017) and cancer-specific survival (P=0.042). The present study findings suggest downregulation of additional subunits of mitochondrial respiratory chain proteins in clear cell renal cell carcinoma. Remarkably, COX5B, a subunit of the respiratory chain complexIV, can be identified as a novel prognostic marker.

Effects of Achyranthes bidentata alcohol on proliferation capacity of osteoblasts and miRNA in Runx2.

Achyranthes bidentata is a herbal plant commonly used in the treatment of osteoporosis and bone nonunion with traditional Chinese medicine. Achyranthes bidentata alcohol is a major component extracted from Achyranthes bidentata, which has been proved to be able to exert a variety of pharmacological effects, such as anti-inflammation, antipyresis, anti-rheumatism, diuresis and anti-osteoporosis. Thirty male Sprague-Dawley rats aged 4 weeks were used in the experiment. All primary rat osteoblasts were cultured and amplified for further experiments. The osteoblasts were divided into six groups (5 rats in each group): the culture medium control group, the 25 µg/ml achyranthol group, the 50 µg/ml achyranthol group, the 100 µg/ml achyranthol group, 200 µg/ml achyranthol group, and the 25 µM PD98059+200 µg/ml achyranthol group. In this study, the effect of Achyranthes bidentata alcohol on the proliferation of osteoblasts was detected via methyl thiazolyl tetrazolium (MTT) assay. The effect of Achyranthes bidentata alcohol on the alkaline phosphatase (ALP) activity in osteoblasts was analyzed via ALP assay. The effect of Achyranthes bidentata alcohol on the expression of osteoblast marker gene, Runt-related transcription factor 2 (Runx2), was detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Moreover, the phosphorylation or activation of extracellular signal-regulated kinase (ERK) in osteoblasts induced by Achyranthes bidentata alcohol was analyzed using western blotting. Achyranthes bidentata alcohol increased cell proliferation in a dose-dependent manner, increased the micro ribonucleic acid (miRNA) level in Runx2, enhanced the ALP activity in osteoblasts, and stimulated the activation of ERK (P<0.05). The expression of Runx2 with the inhibitor PD98059 was decreased significantly compared with that in the Achyranthes bidentata alcohol group (P<0.01). Immunohistochemical results manifested that the percentage of Runx2 positive cells in treated tissues was obviously higher than that in untreated tissues (P<0.01). Therefore, Achyranthes bidentata alcohol promotes the proliferation capacity of osteoblasts in a dose-dependent manner, enhances the expression of miRNA in Runx2, and stimulates the osteogenic differentiation of osteoblasts through activating the ERK signal transduction pathway.