Protein Levels Of Biomarkers Research Articles

Po-Ge-Jiu-Xin decoction alleviate sepsis-induced cardiomyopathy via regulating phosphatase and tensin homolog-induced putative kinase 1 /parkin-mediated mitophagy

Ethnopharmacological relevanceSepsis is a life-threatening systemic syndrome usually accompanied by myocardial dysfunction. Po-Ge-Jiu-Xin decoction (PGJXD), a traditional Chinese prescription medicine, has been used clinically to treat cardiovascular disease including heart failure, sepsis-induced cardiomyopathy (SIC) and even septic shock. Previous clinical studies suggested PGJXD has shown promising results in improving cardiac function and treating heart failure in sepsis. However, more research is needed to elucidate the mechanisms underlying PGJXD's therapeutic effects in sepsis-induced cardiomyopathy. Materials and methodsInitially, we identified the major compounds of PGJXD through ultra-performance liquid chromatography-mass spectrometry technology analysis. We established in a SIC rat model using cecal ligation and puncture(CLP) and treated by PGJXD and levosimendan. We evaluated pathological damage by hematoxylin and eosin staining and measured serum myocardial injury biomarkers. Myocardial apoptosis was detected by Tunel staining and quantifying specific biomarker protein levels. Subsequently, we evaluated myocardium mitochondrial quality using Transmission electron microscope (TEM), antioxidant stress indexes and tissue adenosine triphosphate(ATP) content. We detected the expression of phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), parkin, LC3, and p62 using Western blotting and Quantitative real time polymerase chain reaction(qRT-PCR). (Lipopolysaccharides, LPS)-induced H9c2 cell model was established to further explore the mechanism of PGJXD on SIC. In addition to measuring cell viability, we measured mitochondrial membrane potential using JC-1 staining. Additionally, Parkin-siRNA transfected into H9c2 cells to validate whether PGJXD conducted protective effects against SIC through PINK1/Parkin-mediated mitophagy. ResultsIt has been demonstrated that PGJXD reduced mortality in septic rat, contributed to ameliorating myocardium injury, suppressed inflammatory response and ameliorated the myocardial apoptosis. PGJXD could also alleviate mitochondrial structural abnormality, mitigated oxidative stress injury and promoted energy synthesis in CLP models. Western blotting and qRT-PCR have further confirmed that PGJXD can activate PINK1/parkin pathway-mediated mitophagy, resulting in preserving mitochondrial quality in the myocardium. Furthermore, Parkin siRNA partially reversed the beneficial effect of PGJXD on mitochondrial fission/fusion and mitophagy in vitro. Therefore, the cardioprotective effect of PGJXD is achieved by inducing PINK1/Parkin-mediated mitophagy in maintaining mitochondrial homeostasis. ConclusionsThese results suggest that the potential therapeutic effect of PGJXD on cardiac dysfunction during sepsis and support its mechanism of targeted induction of PINK1-Parkin-mediated mitophagy.

Machine Learning-Enhanced Estimation of Cellular Protein Levels from Bright-Field Images.

In this study, we aimed to develop a novel method for non-invasively determining intracellular protein levels, which is essential for understanding cellular phenomena. This understanding hinges on insights into gene expression, cell morphology, dynamics, and intercellular interactions. Traditional cell analysis techniques, such as immunostaining, live imaging, next-generation sequencing, and single-cell analysis, despite rapid advancements, face challenges in comprehensively integrating gene and protein expression data with spatiotemporal information. Leveraging advances in machine learning for image analysis, we designed a new model to estimate cellular biomarker protein levels using a blend of phase-contrast and fluorescent immunostaining images of epidermal keratinocytes. By iterating this process across various proteins, our model can estimate multiple protein levels from a single phase-contrast image. Additionally, we developed a system for analyzing multiple protein expression levels alongside spatiotemporal data through live imaging and phase-contrast methods. Our study offers valuable tools for cell-based research and presents a new avenue for addressing molecular biological challenges.

Photodynamic Effects of Topical Photosensitizer, Photodithazine Using Micro-LED for Acne Bacteria Induced Inflammation.

Photodynamic therapy (PDT) using topical photosensitizers has been widely used worldwide as a therapeutic modality for acne. However, there are no published reports on photodithazine PDT for acne treatment. We investigated the effectiveness of PDT with photodithazine and micro-LED treatment for acne bacteria-induced inflammation. We established an acne bacteria-induced inflammation model by injecting of Cutibacterium acnes into the backs of HR-1 mice. The mouse models were divided into seven groups for control and comparison. Topical photosensitizer (photodithazine) was administered to the mice, and then their acne lesions were exposed to a micro-light-emitting diode (micro-LED) source. The effectiveness of the treatment on acne lesions was evaluated through clinical findings and measurements of acne inflammation biomarkers using semi-quantitative reverse transcription polymerase chain reaction, and Western blot. Additionally, hematoxylin and eosin staining and immunohistochemical staining were employed to evaluate the changes in inflammatory cells and biomarkers in skin tissues. Compared with the control groups treated with either LED or photosensitizer alone, the acne lesions were significantly reduced in severity and number after PDT. The mRNA and protein levels of biomarkers (interleukin [IL]-1α, IL-1β, tumor necrosis factor-α, Toll-like receptor 2, matrix metalloproteinase-2, and IL-8) exhibited variable decreases in the PDT group relative to the others. Moreover, there was a decline in inflammatory cells and biomarkers in skin specimens after PDT. This in vivo study demonstrated that PDT using photodithazine and micro-LED technology is effective against inflammation induced by acne bacteria.

Ultrasound-Guided Injections of HYADD4 for Knee Osteoarthritis Improves Pain and Functional Outcomes at 3, 6, and 12 Months without Changes in Measured Synovial Fluid, Serum Collagen Biomarkers, or Most Synovial Fluid Biomarker Proteins at 3 Months.

Prior studies have demonstrated improved efficacy when intra-articular (IA) therapeutics are injected using ultrasound (US) guidance. The aim of this study was to determine if clinical improvement in pain and function after IA hyaluronic acid injections using US is associated with changes in SF volumes and biomarker proteins at 3 months. 49 subjects with symptomatic knee OA, BMI < 40, and KL radiographic grade II or III participated. Subjects with adequate aspirated synovial fluid (SF) volumes received two US-guided IA-HA injections of HYADD4 (24 mg/3 mL) 7 days apart. Clinical evaluations at 3, 6, and 12 months included WOMAC, VAS, PCS scores, 6 MWD, and US-measured SF depth. SF and blood were collected at 3 months and analyzed for four serum OA biomarkers and fifteen SF proteins. Statistical differences were observed at 3, 6, and 12 months compared to baseline values, with improvements at 12 months for WOMAC scores (50%), VAS (54%), and PCS scores (24%). MMP10 levels were lower at 3 months without changes in SF volumes, serum levels of C2C, COMP, HA, CPII, or SF levels of IL-1 ra, IL-4, 6, 7, 8, 15, 18, ILGFBP-1, 3, and MMP 1, 2, 3, 8, 9. Baseline clinical features or SF biomarker protein levels did not predict responsiveness at 3 months. Clinical improvements were observed at 12 months using US needle guidance for IA HA, whereas only one SF protein biomarker protein was different at 3 months. Larger studies are needed to identify which SF biomarkers will predict which individual OA patients will receive the greatest benefit from IA therapeutics.

Development of a novel microneedle platform for biomarker assessment of atopic dermatitis patients.

Atopic dermatitis (AD) is a chronic inflammatory skin disease whose pathogenesis, cause, and treatment have been extensively studied. The association of AD with Th2 cytokines is well known; therefore, the analysis of this association is crucial for the diagnosis and treatment of AD. This study aimed to present a new method for measuring protein biomarkers in patients with AD, before and after treatment, using minimally invasive microneedles. First, hyaluronic acid-loaded microneedle patches (HA-MNs) for skin sample collection were fabricated. Next, after Institutional Review Board approval, 20 patients with AD were recruited and skin samples were taken before and after treatment using four different sampling techniques: (1) tape stripping, (2) hydrocolloid patches, (3) hollow microneedles, and (4) HA-MNs. Lastly, proteins were isolated from the collected samples, and AD-related biomarkers were analyzed by enzyme-linked immunosorbent assay. Proteins were successfully extracted from the skin samples collected by tape stripping, hydrocolloid patches, and HA-MNs, except hollow microneedles. Interleukin (IL)-4, IL-13, and interferon-γ were detected in the HA-MNs only. By comparing the biomarker level correlation before and after treatment and the improvement score of the patients, we observed a significant negative correlation between IL-4 and IL-13 with an improvement in AD symptoms. Overall, our results verified that HA-MNs can be used to effectively analyze protein levels of biomarkers from skin metabolites of patients with AD and can be applied to monitor the treatment progress of patients with AD in a minimally invasive manner.

Combined Effects of Programmed Cell Death-1 Blockade and Endostar on Brain Metastases of Lung Cancer.

The blockade of programmed cell death-1 (PD-1) and recombinant human endostatin can be used for the treatment of non-small cell lung cancer (NSCLC) and its metastasis. This study aims to explore the therapeutically potential of PD-1 blockade plus Endostar in brain metastasis of NSCLC. The mouse brain metastases model was established using Lewis lung carcinoma luciferase (LLC-Luc) and PC-9-Luc cells. Tumor metastasis in the brain and tumor burden were analyzed by using bioluminescence imaging (BLI), qRT-PCR and ELISA which were used to determine the mRNA and protein levels of biomarkers in tumor tissues. Immunohistochemical staining was used to determine the expression and location of CD31 in tumor tissues in the brain. Treatment with anti-PD-1 and Endostar suppressed tumor metastasis in the brain and prolonged overall survival rate in LLC-Luc and PC-9-Luc brain metastases mouse model. In addition, treatment with anti-PD-1 and Endostar inhibited the expressions of CD31 and VEGF in tumor tissues in the brain. Furthermore, treatment with anti-PD- 1 and Endostar significantly suppressed the levels of IL1β, IFNγ, and TGFβ in the tumor tissues. The combination of PD-1 blockade and endostar suppressed brain metastases of NSCLC.

PSAT158 Dimeric [Cys25]PTH(1-34) and Monomeric PTH(1-34) Differentially Regulate the Expression of Genes Related to Calcium Homeostasis in Bone and Kidney

Abstract Osteoporosis and its resulting fractures threaten health and life of many patients worldwide; therefore, the development of effective and safe drugs for osteoporosis is an urgent need. In our previous report, dimeric [Cys25]PTH(1-34) peptide with arginine-to-cysteine mutation at position 25 (Dimer) showed a substantial osteo-anabolic effect similar or superior to that of PTH(1-34) in several mouse models. Surprisingly, cAMP was not elevated in serum or urine the Dimer-treated group unlike in the PTH(1-34)-treated group. From this, we hypothesized that the Dimer stimulates anabolic responses through a signaling pathway different from the cAMP-PKA pathway. To test this hypothesis, we assessed responses down-stream of the activated signal cascade from PTHR1 using GPCR Phospho Antibody Array in cells, as well as the expression levels of mRNAs and proteins related to calcium metabolism in bones and kidneys of mice. Cell lysates obtained from GP2.3 cells (treated with PTH(1-34) and Dimer with PBS as a control) were applied to the GPCR-MAPK Pathway Phosphorylation Antibody Array (Full Moon BioSystems, USA), containing 193 antibodies. Each of the antibodies has 6 replicates that are printed on standard-size coated glass microscope slides. The results of the Phospho-antibody array were further confirmed by Western Blot assay. Eighteen CD1 mice of 9 WO were divided into three groups: control, PTH(1-34)-treated, and Dimer-treated groups. 40 µg/kg of PTH(1-34) and 80 µg/kg of the Dimer were subcutaneously injected for each of the experimental groups, respectively. Then, two hours after the injection, the animals were sacrificed, and their kidneys and tibiae were harvested. Total mRNA samples from kidneys and tibiae were extracted using trizol reagent. Protein samples were extracted via protein extraction buffer. We estimated mRNA and protein levels of biomarkers related to calcium regulation via RT-PCR and ELISA with harvested bone and kidney tissues. Several changes of phosphorylation of signaling proteins in GP2.3 cells were detected between Dimer-treated group and PTH(1-34)-treated group. Expression of mRNA and protein levels of RANKL was lower in the Dimer-treated group compared to the PTH(1-34)-treated group. In contrast, expression of mRNA level of OPG in tibia samples was lower in the Dimer-treated group. In kidney, the mRNA and protein levels of 1α(OH)ase in the Dimer-treated group were lower than in the PTH(1-34)-treated group. However, mRNA level of 24(OH)ase was higher in the Dimer-treated group than in the PTH(1-34)-treated group. Other parameters were not significantly different between the Dimer-treated and PTH(1-34)-treated groups. We speculate that the osteoanabolic effect of Dimer uses a signaling pathway different from the cAMP-PKA pathway, regulating different expression parameters related to calcium metabolism in both the bone and kidney. Further studies are required to define exact signaling pathway that Dimer acts on. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Systematic exploration of the potential material basis and molecular mechanism of the Mongolian medicine Nutmeg-5 in improving cardiac remodeling after myocardial infarction

Ethnopharmacological relevanceNutmeg-5, which consists of Myristica fragrans Houtt., Aucklandia lappa Decne., Inula helenium L., Fructus Choerospondiatis and Piper longum L., is an ancient and classic formula in traditional Mongolian medicine that is widely used in the treatment of ischemic heart disease. However, its material basis and pharmacological mechanisms remain to be fully elucidated. Aim of the studyThe aim of this study was to explore the potential material basis and molecular mechanism of Nutmeg-5 in improving cardiac remodeling after myocardial infarction (MI). Materials and methodsThe constituents of Nutmeg-5 absorbed into the blood were identified by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). A mouse MI model was induced in male Kunming mice by permanent ligation of the left anterior descending coronary artery (LDA) ligation. Echocardiography was performed to assess cardiac function. The protective effect of Nutmeg-5 and compound Danshen dripping pills as positive control medicine on post-MI cardiac remodeling was evaluated by tissue histology and determination of the serum protein levels of biomarkers of myocardial injury. RNA sequencing analysis of mouse left ventricle tissue was performed to explore the molecular mechanism of Nutmeg-5 in cardiac remodeling after MI. ResultsA total of 27 constituents absorbed into blood were identified in rat plasma following gavage administration of Nutmeg-5 (0.54 g/kg) for 1 h. We found that ventricular remodeling after MI was significantly improved after Nutmeg-5 treatment in mice, which was demonstrated by decreased mortality, better cardiac function, decreased heart weight to body weight and heart weight to tibia length ratios, and attenuated cardiac fibrosis and myocardial injury. RNA sequencing revealed that the protective effect of Nutmeg-5 on cardiac remodeling after MI was associated with improved heart metabolism. Further study found that Nutmeg-5 treatment could preserve the ultrastructure of mitochondria and upregulate gene expression related to mitochondrial function and structure. HIF-1α (hypoxia inducible factor 1, alpha subunit) expression was significantly upregulated in the hearts of MI mice and significantly suppressed in the hearts of Nutmeg-5-treated mice. In addition, Nutmeg-5 treatment significantly activated the peroxisome proliferator-activated receptor alpha signaling pathway, which was inhibited in the hearts of MI mice. ConclusionsNutmeg-5 attenuates cardiac remodeling after MI by improving heart metabolism and preserving mitochondrial dysfunction by inhibiting HIF-1α expression in the mouse heart after MI.

Neuronal exosome proteins: novel biomarkers for predicting neonatal response to therapeutic hypothermia

ObjectiveCentral nervous system (CNS) derived exosomes can be purified from peripheral blood and have been used widely in adult neurological disease. Application to neonatal neurological disease deserves investigation in the...

Aspirin Improves Nonalcoholic Fatty Liver Disease and Atherosclerosis through Regulation of the PPARδ-AMPK-PGC-1α Pathway in Dyslipidemic Conditions.

This study is aimed at elucidating how aspirin could systemically and simultaneously normalize nonalcoholic fatty liver disease (NAFLD) and atherosclerosis through both in vitro and in vivo studies in hyperlipidemic conditions. We evaluated the effects and mechanism of aspirin on the levels of various biomarkers related to NAFLD, atherosclerosis, and oxidative phosphorylation in cells and animals of hyperlipidemic conditions. The protein levels of biomarkers (PPARδ, AMPK, and PGC-1α) involved in oxidative phosphorylation in both the vascular endothelial and liver cells were elevated by the aspirin in hyperlipidemic condition. Also in the stimulation pathway of oxidative phosphorylation by aspirin, PPARδ was a superior regulator than AMPK and PGC-1α in HepG2 cells. In the vascular endothelial cells, the phosphorylated endothelial nitric oxide synthase level was increased by the treatment. The protein levels of biomarkers related to lipid synthesis were decreased by the treatment in the liver cells. In rabbits administered with cholesterol diet, the levels of triglyceride, HDL-cholesterol, and alanine amino transferase in serums were ameliorated by the aspirin treatment, the levels of ATP and TNFα were increased or decreased, respectively, by the aspirin in liver and aorta tissues, and mannose receptor and C-C chemokine receptor type 2 levels were increased or decreased by the aspirin in spleen, respectively. The elevated levels of macrophage antigen, angiotensin II type1 receptor, and lipid accumulation were decreased in both the liver and aorta tissues in the aspirin-treated group. In conclusion, aspirin can systemically and simultaneously ameliorate NAFLD and atherosclerosis by inhibiting lipid biosynthesis and inflammation and by elevating catabolic metabolism through the activation of the PPARδ-AMPK-PGC-1α pathway. Furthermore, aspirin may normalize atherosclerosis and NAFLD by modulating the mannose receptor and CCR2 in macrophages.

MiR-17-5p knockdown inhibits proliferation, autophagy and promotes apoptosis in thyroid cancer via targeting PTEN.

Thyroid cancer is one common endocrine malignancy with various pathological types. MicroRNAs (miRNAs) play essential roles in development, prognosis and treatment of thyroid cancer. However, the role of miR-17-5p in thyroid cancer progression and its mechanism remain poorly understood. The expressions of miR-17-5p and phosphatase and tensin homolog (PTEN) were measured in thyroid cancer tissues and cells by quantitative real-time polymerase chain reaction or western blot. Cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and flow cytometry, respectively. The protein levels of biomarkers in autophagy or protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway were analyzed by western blot. The interaction between miR-17-5p and PTEN was probed by luciferase activity assay. We found that miR-17-5p expression was elevated and PTEN level was reduced in thyroid cancer tissues and cells compared with their corresponding controls. Knockdown of miR-17-5p or overexpression of PTEN suppressed cell proliferation and autophagy but promoted apoptosis in thyroid cancer cells. PTEN was indicated as a target of miR-17-5p and its interference reversed abrogation of miR-17-5p-mediated inhibition of proliferation and autophagy and increase of apoptosis. Moreover, downregulation of miR-17-5p impeded the activation of AKT/mTOR pathway in thyroid cancer cells, which was attenuated by silencing PTEN. Our data supported that knockdown of miR-17-5p upregulated PTEN expression, therefore leading to suppression of the malignancy of thyroid cancer and inactivation of AKT/mTOR pathway, providing a novel avenue for treatment of thyroid cancer.

Eicosapentaenoic Acid Enhances the Effects of Mesenchymal Stromal Cell Therapy in Experimental Allergic Asthma.

Asthma is characterized by chronic lung inflammation and airway hyperresponsiveness. Despite recent advances in the understanding of its pathophysiology, asthma remains a major public health problem and, at present, there are no effective interventions capable of reversing airway remodeling. Mesenchymal stromal cell (MSC)-based therapy mitigates lung inflammation in experimental allergic asthma; however, its ability to reduce airway remodeling is limited. We aimed to investigate whether pre-treatment with eicosapentaenoic acid (EPA) potentiates the therapeutic properties of MSCs in experimental allergic asthma. Seventy-two C57BL/6 mice were used. House dust mite (HDM) extract was intranasally administered to induce severe allergic asthma in mice. Unstimulated or EPA-stimulated MSCs were administered intratracheally 24 h after final HDM challenge. Lung mechanics, histology, protein levels of biomarkers, and cellularity in bronchoalveolar lavage fluid (BALF), thymus, lymph nodes, and bone marrow were analyzed. Furthermore, the effects of EPA on lipid body formation and secretion of resolvin-D1 (RvD1), prostaglandin E2 (PGE2), interleukin (IL)-10, and transforming growth factor (TGF)-β1 by MSCs were evaluated in vitro. EPA-stimulated MSCs, compared to unstimulated MSCs, yielded greater therapeutic effects by further reducing bronchoconstriction, alveolar collapse, total cell counts (in BALF, bone marrow, and lymph nodes), and collagen fiber content in airways, while increasing IL-10 levels in BALF and M2 macrophage counts in lungs. In conclusion, EPA potentiated MSC-based therapy in experimental allergic asthma, leading to increased secretion of pro-resolution and anti-inflammatory mediators (RvD1, PGE2, IL-10, and TGF-β), modulation of macrophages toward an anti-inflammatory phenotype, and reduction in the remodeling process. Taken together, these modifications may explain the greater improvement in lung mechanics obtained. This may be a promising novel strategy to potentiate MSCs effects.

Cerebrospinal fluid biomarkers for prognosis of long-term cognitive treatment outcomes in patients with idiopathic normal pressure hydrocephalus

The prognosis of cognitive improvement after cerebrospinal fluid (CSF) shunting in idiopathic normal pressure hydrocephalus (iNPH) remains uncertain, with no reports on CSF biomarkers related to long-term cognitive prognosis. We performed a preliminary study of CSF biomarker protein levels for cognitive outcome prognostication of two-year outcomes after shunt treated iNPH in 36 patients (13 women) with a median age of 75years (IQR 69–78). CSF biomarkers included soluble amyloid precursor proteins (sAPP, sAPPα, sAPPβ), amyloid β (Aβ)1–38, Aβ1–42 and phosphorylated tau (p-tau), lipocalin-type prostaglandin D synthase (L-PGDS)/β-trace, and cystatin C. The results clearly showed that p-tau levels (sensitivity of 71.4%, specificity of 77.8%, cut-off value of 22.0pg/mL), Aβ1–38/Aβ1–42 ratio (77.8%, 81%, 3.58), and the Aβ1–42/p-tau ratio (76%, 72.7%, 14.6) in preoperative CSF have the potential to determine postoperative prognosis. Improved cognition may be associated with the improvement in CSF circulation after LPS, which likely induces cystatin C and L-PGDS and switches synthesis from Aβ1–42 to Aβ1–38.

Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity: comparison of HK-2, immortalized human proximal tubule epithelial, and primary cultures of human proximal tubular cells.

There has been intensive effort to identify in vivo biomarkers that can be used to monitor drug-induced kidney damage and identify injury before significant impairment occurs. Kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and human macrophage colony stimulating factor (M-CSF) have been validated as urinary and plasma clinical biomarkers predictive of acute and chronic kidney injury and disease. Similar validation of a high throughput in vitro assay predictive of nephrotoxicity could potentially be implemented early in drug discovery lead optimization to reduce attrition at later stages of drug development. To assess these known in vivo biomarkers for their potential for in vitro screening of drug-induced nephrotoxicity, we selected a panel of nephrotoxic agents and examined their effects on the overexpression of nephrotoxicity biomarkers in immortalized (HK-2) and primary (commercially available and freshly in-house produced) human renal proximal tubule epithelial cells. Traditional cytotoxicity was contrasted with expression levels of KIM-1, NGAL, and M-CSF assessed using ELISA and real-time quantitative reverse transcription PCR. Traditional cytotoxicity assays and biomarker assays using HK-2 cells were both unsuitable for prediction of nephrotoxicity. However, increases in protein levels of KIM-1 and NGAL in primary cells were well correlated with dose levels of known nephrotoxic compounds, with limited correlation seen in M-CSF protein and mRNA levels. These results suggest that profiling compounds against primary cells with monitoring of biomarker protein levels may have potential as in vitro predictive assays of drug-induced nephrotoxicity.

MiR-126-3p suppresses tumor metastasis and angiogenesis of hepatocellular carcinoma by targeting LRP6 and PIK3R2.

BackgroundThe deregulation of microRNAs has been reported to play a pivotal role in hepatocellular carcinoma (HCC). MiR-126-3p has been reported to be associated with poor prognosis in HCC. However the underlying mechanism of miR-126-3p in HCC remains unclear.MethodsThe expression levels of miR-126-3p in HCC tissues and cells were detected by RT-PCR. Transwell assay and capillary tube formation assay were applied to assess the metastasis and angiogenesis in vitro. Nude mice subcutaneous tumor model was used to perform in vivo study. Dual- luciferase reporter assay was conducted to confirm the direct binding of miR-126-3p and target genes. The changes of biomarker protein levels were examined by western blot and Immunohistochemistry.ResultsWe observed that the miR-126-3p expression levels in HCC tissues and cells were significantly down-regulated. Through gain- and loss- of function studies, we showed that miR-126-3p dramatically inhibited HCC cells from migrating and invading extracellular matrix gel and suppressed capillary tube formation of endothelial cells in vitro. Furthermore, overexpression of miR-126-3p significantly reduced the volume of tumor and microvessel density in vivo. LRP6 and PIK3R2 were identified as targets of miR-126-3p. Silencing LRP6 and PIK3R2 had similar effects of miR-126-3p restoration on metastasis and angiogenesis individually in HCC cells. Furthermore, the miR-126-3p level was inversely correlated with LRP6 and PIK3R2 in HCC tissues. In addition, the rescue experiments indicated that the metastasis and angiogenesis functions of miR-126-3p were mediated by LRP6 and PIK3R2.ConclusionOur results demonstrates that deregulation of miR-126-3p contributes to metastasis and angiogenesis in HCC. The restoration of miR-126-3p expression may be a promising strategy for HCC therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0259-1) contains supplementary material, which is available to authorized users.

High levels of circulating CD34+/VEGFR3+ lymphatic/vascular endothelial progenitor cells is correlated with lymph node metastasis in patients with epithelial ovarian cancer

Lymph node metastasis is one of the predictive factors associated with poor prognosis of epithelial ovarian cancer. To clarify the role of CD34 and vascular endothelial growth factor receptor-3-positive (CD34+/VEGFR3+) lymphatic/vascular endothelial progenitor cells (LVEPC) in patients with lymph node metastasis and epithelial ovarian cancer progression, the levels of circulating CD34+/VEGFR3+ LVEPC in epithelial ovarian cancer patients were detected. We also tested the plasma protein levels of VEGF and stromal cell-derived factor to find out their possible relationships with lymph node metastasis in our epithelial ovarian cancer cohort. Peripheral blood samples were collected from 54 patients diagnosed as epithelial ovarian cancer, and 31 normal samples as control. The circulating levels of LVEPC were carried out by flow cytometry, and blood protein levels of biomarkers were measured by enzyme-linked immunosorbent assay (ELISA). The level of circulating LVEPC was significantly higher in patients with ovarian cancer compared with that of healthy controls. There was also a statistically significant correlation between LVEPC levels and surgical staging of epithelial ovarian cancer (P < 0.01). The circulating levels of bone marrow-derived LVEPC are significantly increased in epithelial ovarian cancer patients and these levels correlate with lymph node metastasis too.

Abstract 2739: Claudin-3 expression is up-regulated in epithelial ovarian cancer tissues, including tumors with minimally elevated levels of CA125

Abstract Background: Epithelial ovarian cancer (EOC) is a leading cause of death from gynecological malignancies in the US. The high mortality rate associated with EOC is largely due to the late disease stage at which the majority of women present clinical symptoms. CA125 (MUC16) is a serum marker used for detection of ovarian cancer. Serum CA125 is elevated in the majority of women with ovarian cancer, but its clinical usefulness is limited in part by low sensitivity for detection of early stage disease. Therefore, biomarkers that specify early stage ovarian cancer are needed. Objective: To identify markers that improve upon the performance of CA125, we searched for genes that were increased in tumors expressing only low levels of CA125 mRNA. We reasoned that such biomarkers may have utility in detecting the early stage ovarian cancers that are missed by screening for serum CA125 alone. Methods: The expression levels of CA125 and additional candidate biomarkers were measured using real-time quantitative RT-PCR across a panel of normal ovary tissue specimens and epithelial ovarian tumors. Biomarker protein levels in normal ovary and ovarian tumor tissue extracts were assessed by immunoblotting. Results: CA125 mRNA levels were characterized across a panel of 27 normal ovary specimens and 96 epithelial ovarian tumors. In 78% of the ovarian tumor tissues, levels of CA125 transcript were elevated greater than 10-fold relative to the average expression measured in the normal ovary specimens. In the remaining 21 EOC specimens, CA125 mRNA levels were much more modestly elevated, with increased expression measuring less than 10-fold relative to the normal ovarian tissues. Claudin-3 (CLDN3) was identified as a biomarker whose transcript level was elevated greater than ten-fold in 90% of EOC tissues including the subset of ovarian tumors expressing only modestly elevated levels of CA125 mRNA. In 17 of the 21 tumor specimens exhibiting only modestly elevated CA125 message (81%), CLDN3 transcript was up-regulated greater than 10-fold. CLDN3 message was up-regulated over 100-fold in 11 of these 21 specimens (52%). Eleven of the 21 tumors with low CA125 mRNA and high CLDN3 mRNA were diagnosed at an early stage (stage 1 + 2). This increased expression of CLDN3 mRNA in ovarian tumor tissues displaying only slightly elevated CA125 transcript was confirmed by immunoblotting tumor tissue lysates with a monoclonal antibody specific for CLDN3. Conclusion: The elevated expression of CLDN3 mRNA and protein in epithelial ovarian cancer tissues exhibiting only modest up-regulation of CA125 suggests that CLDN3 may complement CA125 as a biomarker for detection and management of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2739.

Histopathological and molecular changes during apoptosis produced by 7H-dibenzo[c,g]-carbazole in mouse liver.

The topical administration of 7H-dibenzo[c,g]carbazole (7H-DBC) at very low but repeated doses causes genotoxic effects such as DNA adduct formation and produces hepatocellular apoptosis in mouse liver. The purpose of this work was to investigate the alterations in gene expression and protein levels of biomarkers associated with the p53 pathway in mouse liver after exposure to cumulative low doses of 7H-DBC by skin paint applications. The compound was administered topically at the dose of 13.35 microg per animal every 2 days to give either 6, 8, 10, or 12 applications. Animals were sacrificed 48 hours after the different treatments. The apoptotic index increased with the number of applications, with a major proportion of apoptotic cells in the periportal areas. A significant increase of Bax mRNA and protein expression was observed after the 8th application whereas the expression of mRNA levels of Fas and p53 did not show significant differences between treated and control animals. Nuclear staining of p53 was detected in hepatocyte nuclei showing the activation of this protein. Later in the apoptosis process we observed the up-regulation of TGF-beta1 in parenchymal cells. In addition to the induction of the p53 apoptosis pathway in vivo by 7H-DBC, we have observed molecular changes related to cell proliferation such as the overexpression of the antiapoptotic gene Bcl-2.

Measurement of inflammatory biomarkers in synovial tissue extracts by enzyme-linked immunosorbent assay.

We developed methods for measuring inflammatory biomarkers (cytokines, chemokines, and metalloproteinases) in synovial biopsy specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Soluble extracts of synovial fragments were prepared with mild detergent and analyzed by enzyme-linked immunosorbent assay (ELISA) for interleukin 1beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), and matrix metalloproteinase 3. The optimal detergent was 0.1% Igepal CA-630, which interfered minimally with ELISA detection but extracted 80% of IL-6 from synovial tissue. Upon spiking, 81 to 107% of added biomarkers could be recovered. To determine within-tissue variability, multiple biopsy specimens from each RA synovial extract were analyzed individually. A resulting coefficient of variation of 35 to 62% indicated that six biopsy specimens per synovial extract would result in a sampling error of < or = 25%. Preliminary power analysis suggested that 8 to 15 patients per group would suffice to observe a threefold difference before and after treatment in a serial biopsy clinical study. The previously described significant differences in IL-1beta, IL-6, IL-8, and TNF-alpha levels between RA and OA could be detected, thereby validating the use of synovial extracts for biomarker analysis in arthritis. These methods allow monitoring of biomarker protein levels in synovial tissue and could potentially be applied to early-phase clinical trials to provide a preliminary estimate of drug efficacy.