RT2 Profiler PCR Array Research Articles

Peritoneal Modulators of Endometriosis-Associated Ovarian Cancer.

Ovarian cancer is the 4th largest cause of cancer death in women. Approximately 10–15% of women of childbearing age suffer from endometriosis. Endometriosis is defined by the growth and presence of endometrial tissue (lesions) outside of the uterus. The women with endometriosis also have an increased presence of peritoneal fluid (PF) that comprises of inflammatory cells, growth factors, cytokines/chemokines, etc. Epidemiological studies have shown that >3% of women with endometriosis develop ovarian cancer (low-grade serous or endometrioid types). Our hypothesis is that the PF from women with endometriosis induces transformative changes in the ovarian cells, leading to ovarian cancer development. PF from women with and without endometriosis was collected after IRB approval and patient consent. IOSE (human normal ovarian epithelial cells) and TOV-21G cells (human ovarian clear cell carcinoma cell line) were treated with various volumes of PF (no endometriosis or endometriosis) for 48 or 96 h and proliferation measured. Expression levels of epigenetic regulators and FoxP3, an inflammatory tumor suppressor, were determined. A Human Cancer Inflammation and Immunity Crosstalk RT2 Profiler PCR array was used to measure changes in cancer related genes in treated cells. Results showed increased growth of TOV-21G cells treated with PF from women with endometriosis versus without endometriosis and compared to IOSE cells. Endo PF treatment induced EZH2, H3K27me3, and FoxP3. The RT2 PCR array of TOV-21G cells treated with endo PF showed upregulation of various inflammatory genes (TLRs, Myd88, etc.). These studies indicate that PF from women with endometriosis can both proliferate and transform ovarian cells and hence this microenvironment plays a major mechanistic role in the progression of endometriosis to ovarian cancer.

Glucocorticoid-induced Changes in the Transcriptional Activity of Genes of the Innate and Adaptive Immune System in the Blood of Patients with Acute Urticaria

Background: A number of the main effects of glucocorticoids (GCs) are their direct action on T cells, mainly through the transcriptional regulation: elevated expression of immune-regulatory proteins, inhibitory receptors, and reduced expression of pro-inflammatory cytokines, co-stimulatory molecules, and cell cycle mediators. But controversies arise due to the clinical effectiveness of GCs in the treatment of acute urticaria. Methods: In our research, we applied a pathway-specific PCR array (Human Innate & Adaptive Immune Responses RT2 Profiler PCR Array, QIAGEN, Germany) to detect and verify innate & adaptive immune responses pathway-focused genes expression in the blood of patients with acute urticaria who received treatment with glucocorticoids in addition to standard therapy. Results: Adding glucocorticoids to standard therapy did not notably affect the nature of the clinical presentation of acute urticaria, which was assessed according to the UAS scale (urticaria activity score). Analysis of the transcriptional profile of peripheral blood mononuclear cells in patients with acute urticaria against the background of glucocorticoid therapy showed the induction expression of the FOXP3 and IL10 genes against the background of repression of the transcriptional activity of the genes for chemokines and cytokines CCL5, CXCL8, IFNG, IL2, IL5, IL17A, IL1B, and TNF. Glucocorticoid-induced changes in the transcriptome also manifested by pronounced repression in genes of CD40 and CD80 (B7-1) co-stimulatory molecules, transcriptional regulators of Th1-cells differentiation - TBX21 and STAT1, Th17 cells - RORC, NLRP3-inflammasome genes, and the transcription factor NFKB1 compared with the control group. Conclusions: Adding glucocorticoids to the standard therapy of acute urticaria has a pronounced immunosuppressive potential at the transcriptome level of immune response genes in the blood; however, it does not have any noticeable clinical effect.

EMT-related genes are unlikely to be involved in extracapsular growth of lymph node metastases in gastric cancer

BackgroundIn gastric cancer (GC), extracapsular growth (ECG) pattern of lymph node metastases is associated with decreased overall survival rates compared to intracapsular lymph node metastases (ICG). Epithelial-to-mesenchymal transition (EMT) plays a pivotal role in hematogenous metastatic spread. Aim of the present study was to analyze if EMT related genes are involved in the growth pattern of lymph node metastases in GC. MethodsOut of our prospective database with 529 patients who underwent surgical resection for GC between 2002 and 2014 forty lymph node positive patients were identified (20 ECG, 20 ICG). The expression of 84 EMT-associated genes were analyzed by RT2 Profiler PCR Array (n = 20). Results were validated by Real-Time PCR (n = 20). ResultsGC with ECG showed differently expressed EMT related genes. GC leading to ECG showed an upregulation of three and downregulation of eleven genes. Those differences, however, could not be confirmed in PCR analysis. ConclusionsThis study demonstrates that EMT related genes are not responsible for the different growth patterns of lymph node metastases in GC. Further studies are required to evaluate the underlying mechanisms of ECG in GC as it might provide a potential therapeutic target for this subgroup of more aggressive tumors in the future.

Alterations of epigenetic regulators and P53 mutations in murine mesenchymal stem cell cultures: A possible mechanism of spontaneous transformation.

Recent studies demonstrated the involvement of mesenchymal stem/stromal cells (MSCs) in carcinogenesis, but the molecular mechanism behind this transformation is still obscured. To screen both the expression levels of polycomb and trithorax epigenetic regulators and TrP53 mutations in early and late MSC culture passages in an attempt to decipher the mechanism of spontaneous transformation. The study was conducted on early and late passages of MSC culture model from C57BL/6J mice. The expression profile of 84 epigenetic regulators was examined using RT2 profiler PCR array. TrP53 mutations in the DNA binding domain was screened. Codons, amino acids positions and the corresponding human variants were detected in P53 sequences. Sixty-two epigenetic regulators were dysregulated. Abnormalities were detected starting the third passage. Nine regulators were dysregulated in all passages. (C>G) substitution P53 mutation was detected in passage3 resulting in Ser152Arg substitution. Passages 6, 9, 12 and the last passage showed T>C substitution resulting in Cys235Arg substitution. The last passage had T deletion and A insertion resulting in frame shift mutations changing the p.Phe286Ser and p.Asn103Lys respectively. In vitro expanded MSCs undergo transformation through alteration of epigenetic regulators which results in genomic instability and frequent P53 mutations.

Gene expression changes in vastus lateralis muscle after different strength training regimes during rehabilitation following anterior cruciate ligament reconstruction.

Impaired muscle regeneration has repeatedly been described after anterior cruciate ligament reconstruction (ACL-R). The results of recent studies provided some evidence for negative alterations in knee extensor muscles after ACL-R causing persisting strength deficits in spite of the regain of muscle mass. Accordingly, we observed that 12 weeks of concentric/eccentric quadriceps strength training with eccentric overload (CON/ECC+) induced a significantly greater hypertrophy of the atrophied quadriceps muscle after ACL-R than conventional concentric/eccentric quadriceps strength training (CON/ECC). However, strength deficits persisted and there was an unexpected increase in the proportion of slow type I fibers instead of the expected shift towards a faster muscle phenotype after CON/ECC+. In order to shed further light on muscle recovery after ACL-R, the steady-state levels of 84 marker mRNAs were analyzed in biopsies obtained from the vastus lateralis muscle of 31 subjects before and after 12 weeks of CON/ECC+ (n = 18) or CON/ECC strength training (n = 13) during rehabilitation after ACL-R using a custom RT2 Profiler PCR array. Significant (p < 0.05) changes were detected in the expression of 26 mRNAs, several of them involved in muscle wasting/atrophy. A different pattern with regard to the strength training mode was observed for 16 mRNAs, indicating an enhanced hypertrophic stimulus, mechanical sensing or fast contractility after CON/ECC+. The effects of the type of autograft (quadriceps, QUAD, n = 19, or semitendinosus tendon, SEMI, n = 12) were reflected in the lower expression of 6 mRNAs involved in skeletal muscle hypertrophy or contractility in QUAD. In conclusion, the greater hypertrophic stimulus and mechanical stress induced by CON/ECC+ and a beginning shift towards a faster muscle phenotype after CON/ECC+ might be indicated by significant gene expression changes as well as still ongoing muscle wasting processes and a negative impact of QUAD autograft.

Pro-atherogenic actions of signal transducer and activator of transcription 1 serine 727 phosphorylation in LDL receptor deficient mice via modulation of plaque inflammation.

Atherosclerosis is a chronic inflammatory disorder of the vasculature regulated by cytokines. We have previously shown that extracellular signal‐regulated kinase‐1/2 (ERK1/2) plays an important role in serine 727 phosphorylation of signal transducer and activator of transcription‐1 (STAT1) transactivation domain, which is required for maximal interferon‐γ signaling, and the regulation of modified LDL uptake by macrophages in vitro. Unfortunately, the roles of ERK1/2 and STAT1 serine 727 phosphorylation in atherosclerosis are poorly understood and were investigated using ERK1 deficient mice (ERK2 knockout mice die in utero) and STAT1 knock‐in mice (serine 727 replaced by alanine; STAT1 S727A). Mouse Atherosclerosis RT² Profiler PCR Array analysis showed that ERK1 deficiency and STAT1 S727A modification produced significant changes in the expression of 18 and 49 genes, respectively, in bone marrow‐derived macrophages, with 17 common regulated genes that included those that play key roles in inflammation and cell migration. Indeed, ERK1 deficiency and STAT1 S727A modification attenuated chemokine‐driven migration of macrophages with the former also impacting proliferation and the latter phagocytosis. In LDL receptor deficient mice fed a high fat diet, both ERK1 deficiency and STAT1 S727A modification produced significant reduction in plaque lipid content, albeit at different time points. The STAT1 S727A modification additionally caused a significant reduction in plaque content of macrophages and CD3 T cells and diet‐induced cardiac hypertrophy index. In addition, there was a significant increase in plasma IL‐2 levels and a trend toward increase in plasma IL‐5 levels. These studies demonstrate important roles of STAT1 S727 phosphorylation in particular in the regulation of atherosclerosis‐associated macrophage processes in vitro together with plaque lipid content and inflammation in vivo, and support further assessment of its therapeutical potential.

Genetic profiling of human bone marrow and adipose tissue-derived mesenchymal stem cells reveals differences in osteogenic signaling mediated by graphene

BackgroundIn the last decade, graphene surfaces have consistently supported osteoblast development of stem cells, holding promise as a therapeutic implant for degenerative bone diseases. However, until now no study has specifically examined the genetic changes when stem cells undergo osteogenic differentiation on graphene.ResultsIn this study, we provide a detailed overview of gene expressions when human mesenchymal stem cells (MSCs) derived from either adipose tissue (AD-MSCs) or bone marrow (BM-MSCs), are cultured on graphene. Genetic expressions were measured using osteogenic RT2 profiler PCR arrays and compared either over time (7 or 21 days) or between each cell source at each time point. Genes were categorized as either transcriptional regulation, osteoblast-related, extracellular matrix, cellular adhesion, BMP and SMAD signaling, growth factors, or angiogenic factors. Results showed that both MSC sources cultured on low oxygen graphene surfaces achieved osteogenesis by 21 days and expressed specific osteoblast markers. However, each MSC source cultured on graphene did have genetically different responses. When compared between each other, we found that genes of BM-MSCs were robustly expressed, and more noticeable after 7 days of culturing, suggesting BM-MSCs initiate osteogenesis at an earlier time point than AD-MSCs on graphene. Additionally, we found upregulated angiogenic markers in both MSCs sources, suggesting graphene could simultaneously attract the ingrowth of blood vessels in vivo. Finally, we identified several novel targets, including distal-less homeobox 5 (DLX5) and phosphate-regulating endopeptidase homolog, X-linked (PHEX).ConclusionsOverall, this study shows that graphene genetically supports differentiation of both AD-MSCs and BM-MSCs but may involve different signaling mechanisms to achieve osteogenesis. Data further demonstrates the lack of aberrant signaling due to cell-graphene interaction, strengthening the application of specific form and concentration of graphene nanoparticles in bone tissue engineering.Graphic abstract

Hepatic Glucose Regulations by Sago (Metroxylon sagu) Resistant Starch in Diabetic Goto Kakizaki Rat

Resistant starch (RS) Sago (Metroxylon sagu) intake has been linked with the improvement in postprandial hyperglycemia and diabetes management via several modes of action including delayed glucose absorption and inhibition of carbohydrate digestion in the gastrointestinal tract. However, to our knowledge, studies on local Malaysian sago RS associated with hepatic glucose production has not been reported elsewhere. Thus, this study was done to identify the underlying mechanisms of local Malaysian RS sago native and modified known as sago RS type 2 (sago RS2) and type 4 (sago RS4) respectively in glucose regulations by analyzing the targeted genes in hepatic glucose pathways. In this study, gene expression associated with Glucose and Glycogen Metabolism Pathways analysis in the liver of spontaneously type 2 diabetic rat, Goto kakizaki treated with water (control), Hi Maize (positive control), sago RS2 and RS4 was done using Rat Glucose Metabolism RT² Profiler PCR Array which consist of 84 genes. The results showed that several genes were significantly up- and down-regulated in the diabetic rats treated with Sago. Taldo1 was significantly upregulated whereas G6PC, Sdhb and Rplp1 genes were significantly downregulated in the rat liver treated with sago RS2. In the rat liver treated with sago RS4, Idh3g gene was significantly upregulated whereas G6pc, Pdk3, Eno3, Sdhb, Galm and Tkt genes were significantly downregulated. The gene expressions identified are associated in the blood glucose homeostasis involving the regulation and enzymatic pathways of glucose and glycogen metabolisms. In conclusion, the genes identified might be useful for therapeutic targets in glucose lowering effects by reducing hepatic glucose output indicating potential of our local sago in managing diabetes.

Transcriptomic Analysis of Short/Branched-Chain Acyl-Coenzyme a Dehydrogenase Knocked Out bMECs Revealed Its Regulatory Effect on Lipid Metabolism.

The acyl-CoA dehydrogenase family of enzymes includes short/branched-chain acyl-CoA dehydrogenase (ACADSB), which catalyzes the dehydrogenation of acyl-CoA derivatives in fatty acid metabolism. Our previous findings suggested that ACADSB was a critical candidate gene affecting milk fat synthesis by comparing the transcriptome in bovine mammary epithelial cells (bMECs) from Chinese Holstein dairy cows producing high-fat and low-fat milk as well as gene functional validation studies on the cellular level. In the present study, ACADSB in bMECs was knocked out (KO) using a CRISPR/Cas9 system, and mRNA transcriptome was further sequenced to verify the function of the ACADSB gene and analyze its correlation with lipid metabolism. The findings revealed that 15,693 genes were expressed, 1,548 genes were differentially expressed genes (DEGs), and 6,098 GO terms were enriched, of which 637 GO terms were greatly enhanced, such as phospholipid-translocation ATPase activity (GO:0004012), lipoprotein lipase activity (GO:0004465), acyl-CoA desaturase activity (GO:0016215), and so on. The analysis by KEGG showed that DEGs were distributed over 247 pathogens, of which 49 were significantly enriched, including the metabolism of fatty acids (PATH: 01212), metabolism of glycerolipid (PATH: 00561), and signaling of adipocytokines (PATH: 04920). The CHOL, TGs and FFA contents in bMECs were reduced when the ACADSB gene was knocked out. The RT2 Profiler PCR array also revealed that the loss of the ACADSB gene changed the expression levels of functional genes involved in lipid metabolism, including ACADL, ACOX2, ACAT2, and FABP3. In conclusion, the current findings show that ACADSB is a key regulator of lipid metabolism in bMECs. The ACADSB−/− bMECs could also be useful genetic material and tools for future research into gene functions related to lipid and fatty acid metabolism. It will be valuable for revealing the gene regulatory roles and molecular mechanisms in milk fat synthesis.

The Influence of Tonality on Auditory Sensitivity in Different Types of Musical Texture

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, disrupts conservation programs of threatened species such as the white rhinoceros (Ceratotherium simum). Interferon gamma release assays have been developed for the diagnosis of M. bovis infection in rhinoceros, however, the discovery of additional diagnostic biomarkers might improve the accuracy of case detection. The aim of this pilot study was therefore to evaluate a novel unbiased approach to candidate biomarker discovery and preliminary validation. Whole blood samples from twelve white rhinoceros were incubated in Nil and TB antigen tubes of the QuantiFERON® TB Gold (In-Tube) system after which RNA was extracted and reverse transcribed. Using the equine RT2 profiler PCR array, relative gene expression analysis of samples from two immune sensitized rhinoceros identified CCL4, CCL8, IL23A, LTA, NODAL, TNF, CSF3, CXCL10 and GPI as upregulated in response to antigen stimulation. Novel gene expression assays (GEAs) were designed for selected candidates, i.e. CCL4, CXCL10 and IFNG, and analysis of QFT-processed samples showed the CXCL10 GEA could distinguish between five M. bovis-infected and five uninfected rhinoceros. These findings confirm the value of the equine RT2 profiler PCR array as a useful tool for screening biomarkers for the diagnosis of M. bovis infection in rhinoceros.

Antiproliferative effect of GTS-21 in glioblastoma cells.

Glioblastoma multiforme (GBM) is the most common malignant brain tumour in adults. The poor prognosis and short median overall survival of patients with GBM is associated with resistance to therapy after surgical and adjuvant treatment. The expression of various acetylcholine receptors (AChR) in GBM has been widely reported. The present study aimed to investigate the expression of cholinergic system-related genes in primary GBM and to explore the antiproliferative effect of 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21) in GBM cell lines. Therefore, the expression of 28 genes associated with the cholinergic system was detected using a customized RT2 Profiler PCR Array in 44 GBM and 5 healthy control brain tissue samples. In addition, the activity of GTS-21, an alpha 7 subunit nicotinic AChR (α7 nAChR) agonist, and that of α-bungarotoxin (α-BTX), an α7 nAChR antagonist, was determined in primary and established GBM cells. Therefore, the A172, U87 and G28 cell lines and primary GBM cells were treated with GTS-21, ACh or nicotine. Cell viability was evaluated using MTT assay at 24, 48 and 72 h following cell treatment with the corresponding compounds. The results revealed that the expression of cholinergic system-related components was notably downregulated, except that of cholinergic receptor nicotinic alpha 7 subunit (CHRNA7), in primary GBM and U87 cells. However, the dominant-negative duplicate form of CHRNA7 was also downregulated. Furthermore, A172 and G28 cells exhibited a heterogeneous gene expression pattern. Additionally, GTS-21 inhibited the proliferation of GBM cells in a dose- and time-dependent manner. Interestingly, treatment with α-BTX restored the proliferation of U87 cells, but not that of A172 and G28 cells. Collectively, the findings of the present study suggested that GTS-21 may inhibit the proliferation of GBM cells and may therefore serve as a novel therapeutic approach to the treatment of GBM, which warrants further investigation.

MiRNA-34c Regulates Bovine Sertoli Cell Proliferation, Gene Expression, and Apoptosis by Targeting the AXL Gene.

Simple SummaryFertility is one of the essential reproduction traits of bulls, and accurate prediction of fertility potential using a semen sample from a donor bull for artificial insemination is crucial to achieve consistently high reproductive efficiency. Somatic cells, such as Sertoli cells and Leydig cells, are important in testis formation and provide a nurturing and regulatory environment for spermatogenesis. Furthermore, it was suggested that non-coding RNAs, such as microRNAs, long non-coding RNAs, circular RNAs, and Piwi-interacting RNA, function as important regulators of gene expression at post-transcriptional level in spermatogenesis. In this study, microRNA-34c was verified to specifically regulate the AXL gene by targeting a sequence in the 3’ UTR; miRNA-34c can also influence the proliferation, apoptosis, and relative abundance of the transcript of male-reproduction-related genes. Therefore, microRNA-34c can be considered an essential regulator in the process of bull spermatogenesis. These results identify a key microRNA and functional genes in the process of cattle male reproduction, providing useful information for future marker-assisted selection of bulls with excellent sperm quality.MicroRNAs (miRNAs) play significant roles in mammalian spermatogenesis. Sertoli cells can provide a stable microenvironment and nutritional factors for germ cells, thus playing a vital role in spermatogenesis. However, few studies elucidate the regulation of bovine testicular Sertoli cells by miRNAs. Here, we have reported that miRNA-34c (miR-34c) regulates proliferation, apoptosis, and relative transcripts abundance gene in bovine Sertoli cells. In bovine Sertoli cells, overexpression of miR-34c inhibited proliferation and relative abundance of gene transcripts while promoting apoptosis of Sertoli cells, and the effects were the opposite when miR-34c was knocked down. Receptor tyrosine kinase (AXL) was identified as a direct target gene of miR-34c in Sertoli cells, validated by analysis of the relative abundance of AXL transcript and dual-luciferase reporter assay. The relative abundance of the transcript of genes related to male reproduction in Sertoli cells was changed after the AXL gene was overexpressed, as demonstrated by the RT2 Profiler PCR Array results. In summary, miR-34c specifically regulated the AXL gene by targeting a sequence in the 3′-UTR, which could influence proliferation, apoptosis, and relative abundance of the transcript of male reproduction-related genes. Therefore, miR-34c could be considered an essential regulator in the process of bull spermatogenesis.

P–099 MYD88 dependent pathway through TLR 1,2 and 6 activation play a role in interaction of high DNA fragmented human sperm with fallopian tube epithelial cells

Abstract Study question How does sperm with high DFI disrupt the female reproductive system? Summary answer Sperm with damaged DNA could disrupt the female reproductive system by production of high inflammatory factors through activation of TLRs and MyD88-dependent pathway . What is known already Previous studies have also shown that the interaction between sperm and fallopian tube is a complex process and activates a number of immune mechanisms. Many TLRs, including TLR1, TLR2, and TLR6 are expressed in fallopian tube epithelial cells. TLRs have an important role in the immune interaction between fallopian tube and sperm. They are also involved in sperm capacitation, fertilization and pregnancy. Examining this interaction may lead us to valuable data. Study design, size, duration In this study, 10 Recurrent Implantation failure couples with high DFI ( &amp;gt;30% by SCSA evaluation) and 10 healthy donors men with low DFI (&amp;lt;30%) were considered as high DFI group and low DFI group respectively. After fresh semen preparation, sperm were co-cultured with human fallopian tube epithelial cell line( OE-E6/E7) for 24 hours. Participants/materials, setting, methods RNA were extracted from the cell line and PCR array for human innate and adaptive immune responses was performed by RT2 profiler PCR array. Main results and the role of chance Analysis of PCR array data showed that the expression of TLR–1, TLR–2, TLR–6, MYD88, TIRAP, IRAKS,TRAF6, MAPKS, NF-KB, G-CSF, GM-CSF, CXCL8, CXCL10, CCL2, IL–6, IL–1, TNFa in high DFI group was significantly higher than control group. All these factors are involved in the MyD88-dependent pathway. Our research suggests that the MyD88-dependent pathway through TLR1,2 and 6 activation may be the main inflammatory pathway activated by sperm with high DFI. The released DAMPs through damaged sperms were recognized by TLRs on the epithelial cells. Following the TLR1,2 and 6 signaling, and MYD88 activation, epithelial cells were producing the inflammatory cytokines which result in the neutrophils infiltration. In the lumen of the fallopian tube, the neutrophils were activated via TLR1/TLR2, complexes that lead to the neutrophils activation, phagocytosis, NET formation, and apoptosis. Limitations, reasons for caution Studying with a larger sample size and examining the final factors at the protein level will be better to detect the effect of sperm DNA fragmentation on fallopian tube. Wider implications of the findings: MyD88-dependent pathway could be one of the mechanisms that involved in interaction of high DNA fragmented sperm with female reproductive tract. Trial registration number Not applicable

P–446 Controlled ovarian stimulation protocols for oocyte vitrification induce differential gene expression profiles in primary tumours of breast cancer

Abstract Study question Could controlled ovarian stimulation (COS) protocols used in fertility preservation (FP) impact on malignant cell proliferation and tumour molecular profiling of breast cancer (BC) patients? Summary answer Letrozole supplementation during ovarian stimulation for oocyte vitrification could be considered as a safe procedure in estrogen-dependent BC patients undergoing FP. What is known already High estradiol levels associated to COS could promote changes in gene expression in estrogen-positive BC tumors. Estradiol levels reached during the ovarian stimulation could aggressively promote malignant cell proliferation and cell migration to adjacent organs. Aromatase inhibitors such as letrozole, are added to standard stimulation protocols to avoid this undesirable potential side effect. Despite the reassuring clinical results achieved by using letrozole for FP in BC patients, there is still a lack of evidence regarding its impact on malignant cell behaviour. For this reason, specific molecular studies to properly evaluate safety of letrozole in this specific population are still required. Study design, size, duration Experimental in vivo study. Thirty 5-week-old Nude-nu female mice were divided into three different groups: BC (n = 10), BC and FSH stimulation (BC-FSH, n = 10), or BC and letrozole stimulation (BC-LTZ, n = 10). BC was considered the control group, whereas BC-FSH and BC-LTZ represented distinct COS protocols. Hormone-dependent BC was induced in all mice. Animals were followed-up for 5 months and then euthanized to collect kidney, ovary, spleen, and liver tissues for gene expression and immunohistochemistry (IHC) analysis. Participants/materials, setting, methods One million of human MCF–7 BC cells were injected into the mouse left kidney capsule. Two days after xenograft, COS was induced by 10IU FSH or 1mg/ml letrozole + 10IU FSH, followed by ovarian triggering with 10IU hCG at 48h. Human BC RT2 Profiler PCR Arrays were performed to evaluate the impact of COS on tumour behaviour. BC biomarkers (Ki67, Erα, PR and HER–2) were also analyzed by IHC to validate gene expression results. Main results and the role of chance The differential gene expression was firstly assessed in kidney samples, as they represent the xenograft site, and different expression profiles were obtained depending on the COS protocol used. The BC-FSH group showed a global over-expression pattern of all genes of the array when compared to BC and BC-LTZ. Further gene ontology analysis revealed that cellular process, biological regulation, metabolic process, and proteases were the most over-represented biological terms, with a 20.5-fold over-expression for MMP2 compared to the other groups. On the other hand, BC-LTZ mice presented gene expression profiles similar to that of controls. When other tissues were analysed to detect malignant cell presence, our results revealed a significant up-regulation of matrix-proteases, cell cycle and proliferation related-genes, in liver samples from the BC-FSH group, but no amplification of any of the studied genes was detected in ovarian tissue or spleen. IHC findings confirmed the presence of human BC cells in 100% of samples from kidney tissue and in 30% of samples from liver tissue in the BC-FSH group. No human cells were detected by IHC in the BC and BC-LTZ groups. Limitations, reasons for caution Since this is an animal model of estrogen-dependent BC induced through a cell line, further validation with human tumour breast cancer samples would be required. Wider implications of the findings: Adjuvant letrozole in COS protocols prevents BC cell migration. The present study suggests that this protective effect could be mediated by interfering ER-pathway downstream genes involved in cell proliferation and matrix digestion. Altogether, letrozole could safely be used as a supplement during COS procedures for oocyte vitrification in BC women. Trial registration number Not applicable

O6 Analysis of angiogenic gene profiling after E-selectin + stem cell therapy in murine ischemic limb

Abstract Introduction There remains a paucity of novel therapeutics for limb salvage in patients with critical limb ischemia (CLI) for whom revascularization procedures have failed and amputation is imminent. We have shown that E-selectin+/Mesenchymal Stem Cell (MSC) injections into the ischemic limb tissue of a CLI mouse model improves revascularization and limb function. Thus, we sought to determine a mechanism of action for E-selectin+/MSC’s pro-angiogenic and tissue salvage properties. Methods MSC were extracted from donor mice bone marrow and subsequently engineered via viral transduction with E-selectin-ires-GFP/AAV and GFP/AAV (control) to create E-selectin-GFP+/MSC vs GFP+/MSC. Intramuscular injections of E-selectin-GFP+/MSC, GFP+/MSC, or PBS were performed in a mouse model of hindlimb ischemia. Laser doppler imaging (LDI), confocal laser microscopy, and treadmill exhaustion test were utilized to determine neovascularization and limb function. RNA extraction from engineered MSC (E-selectin-GFP+/MSC vs GFP+/MSC) and ischemic hindlimb tissues treated with E-selectin-GFP+/MSC vs GFP+/MSC was performed, followed by RT2 Profiler PCR Array analysis of 84 genes involved in angiogenesis. GFP+/MSC treated hindlimb tissue served as control. Student’s t-test or ANOVA was utilized to compare means and significance set at P &amp;lt; 0.05. Results Compared with GFP+/MSC and PBS, treatment with E-selectin-GFP+/MSC increased ischemic leg LDI reperfusion (54% vs. 39% vs. 22%, P &amp;lt; 0.001), treadmill distance traversed (162 m vs. 111 m vs. 110 m, P &amp;lt; 0.01) and ischemic mouse footpad vessel density (23% vs. 14% vs. 14%, P &amp;lt; 0.01). RT2 Profiler PCR Array demonstrated pro-angiogenic gene upregulation occurred in 7 genes (Csf3, Cxcl2, Cxcl5, Serpine1, F2, Lep, Tbx1, Table I.) in E-selectin-GFP+/MSC treated ischemic leg tissue while tumour necrosis factor (TNF) was found to be downregulated, when compared with GFP+/MSC treated tissues. Of these 7 upregulated genes, CXCl2, F2, Leptin and T-box1 (Table I.) are likely produced by E-selectin-GFP+/MSC, as analysis of cellular gene expression profiles of E-selectin-GFP+/MSC also revealed upregulation by 2-fold or more in these factors when compared to GFP+/MSC. Validation of gene functions in-vivo are under investigation. Conclusion Stem cell therapy using E-selectin-GFP+/MSC, in a murine model of CLI, confers both augmented postnatal neovascularization and increased limb function. The pro-angiogenic and pro-repair effects are likely mediated by upregulation of a panel of chemokines/cytokines and down-regulation of TNF in ischemic tissues treated with E-selectin-GFP+/MSC.

Linc8087 predicts favorable prognosis and inhibits cell migration and invasion in NSCLC

BackgroundNon-small cell lung cancer (NSCLC) is the most common cancer and has poor prognosis. Long non-coding RNA(LncRNA) plays important roles in the regulation of cell migration in various types of cancer. In this study, we aimed to demonstrate the function of linc8087 in regulating cell migration and invasion in NSCLC cells. MethodsA lncRNA microarray was used to identify differentially expressed lncRNAs between NSCLC tissues and normal tissues. RT-qPCR was used to confirm the expression of linc8087 in tumor tissues. The association between linc8087 expression and clinicopathological characteristics was analyzed. RNA fluorescence in situ hybridization (FISH) was performed to observe the subcellular localization of linc8087. We investigated the effects of linc8087 expression on cell migration and invasion by wound healing assay, Transwell and invasion assays. The Human Tumor Metastasis RT2 Profiler PCR Array was used to detect and analyze the mRNA levels of 84 genes involved in metastasis. ResultsWe found that linc8087 expression was obviously decreased in both NSCLC tissues and cell lines compared with paired normal tissues and a normal bronchial epithelium cell line. Low expression of linc8087 was significantly associated with poor survival. In addition, linc8087 was an independent risk factor for survival. Overexpressed linc8087 inhibited cell migration and invasion in A549 and PC9 cell lines. Knockdown of linc8087 promoted cell migration and invasion. The result of RT2 Profiler PCR Array showed that overexpressed linc8087 upregulated the expression of the COL4A2, CST7 and FAT1 genes and led to the downregulation of SERPINE1. ConclusionsThese results indicate that linc8087 plays a key role in the progression of NSCLC, and it may serve as a meaningful prognostic biomarker as well as a latent therapeutic target in NSCLC patients.

Inhibition of Secretin/Secretin Receptor Axis Ameliorates NAFLD Phenotypes.

Human NAFLD is characterized at early stages by hepatic steatosis, which may progress to NASH when the liver displays microvesicular steatosis, lobular inflammation, and pericellular fibrosis. The secretin (SCT)/secretin receptor (SCTR) axis promotes biliary senescence and liver fibrosis in cholestatic models through down-regulation of miR-125b signaling. We aim to evaluate the effect of disrupting biliary SCT/SCTR/miR-125b signaling on hepatic steatosis, biliary senescence, and liver fibrosis in NAFLD/NASH. In vivo, 4-week-old male wild-type, Sct-/- and Sctr-/- mice were fed a control diet or high-fat diet (HFD) for 16 weeks. The expression of SCT/SCTR/miR-125b axis was measured in human NAFLD/NASH liver samples and HFD mouse livers by immunohistochemistry and quantitative PCR. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were evaluated in mouse liver and human NAFLD/NASH liver samples. miR-125b target lipogenesis genes in hepatocytes were screened and validated by custom RT2 Profiler PCR array and luciferase assay. Biliary SCT/SCTR expression was increased in human NAFLD/NASH samples and in livers of HFD mice, whereas the expression of miR-125b was decreased. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were observed in human NAFLD/NASH samples as well as HFD mice, which were decreased in Sct-/- and Sctr-/- HFD mice. Elovl1 is a lipogenesis gene targeted by miR-125b, and its expression was also decreased in HFD mouse hepatocytes following Sct or Sctr knockout. Bile acid profile in fecal samples have the greatest changes between wild-type mice and Sct-/- /Sctr-/- mice. The biliary SCT/SCTR/miR-125b axis promotes liver steatosis by up-regulating lipid biosynthesis gene Elovl1. Targeting the biliary SCT/SCTR/miR-125b axis may be key for ameliorating phenotypes of human NAFLD/NASH.

Gene Expression Profile in Endothelial Cells: Effects of Defibrotide on Different Functional Pathways

Introduction.Sinusoidal obstruction syndrome/veno-occlusive disease (SOS/VOD) is a potentially life-threatening complication of hematopoietic stem cell transplantation (HSCT). It is characterized by endothelial cell (EC) damage and hepatocellular injury due to chemo- and radio-therapy toxicity, resulting in the local release of pro-inflammatory molecules, cell adhesion activation on endothelial cells and coagulative pathway activation. Fibrinolysis activation produces fibrosis of sinusoids followed by perivascular hepatocyte necrosis and venular occlusion. Defibrotide (DF) consists of a mixture of porcine derived single-stranded phosphodiester oligonucleotides and is efficaciously used in the treatment of SOS/VOD. Various in vivo and in vitro findings demonstrate that DF possesses only minimal anticoagulant activity, with potent antithrombotic and profibrinolytic effects antithrombotic properties, without significant systemic anticoagulant effects. Moreover, DF stimulates sinusoidal endothelium by increasing the availability of proangiogenic factors such as bFGF, protecting them from trypsin digestion. Since the mechanism of action of DF is not completely elucidated, we evaluated its effects on the gene expression of ECs.Methods.Endothelial colony forming cells (ECFCs) were obtained from umbilical cord blood according to the method of Ingram et al.(Blood 2004;104:2752-2760). Confluent ECFCs at passages III to IV were subjected to 6-h exposure to either Interferon γ (20 ng/ml; 3 experiments) or lipopolysaccharide (100 ng/ml, 4 experiments), with or without Defibrotide (100 ng/ml). Total RNA was extracted and reverted with a RT2 First Strand cDNA Kit (SABiociences, Qiagen). Genotype profile of control and treated cells was evaluated using the Human Endothelial Cell Biology“ (PAHS-015Z, SABiociences, Qiagen). Data were analyzed using the RT2 Profiler PCR array data analysis template v3.5 (SABiociences, Qiagen). Relative changes in gene expression were calculated using the Ct (cycle threshold) method.Results. The microarray included 84 genes involved in various pathways, including angiogenesis, cell adhesion, inflammatory response, coagulation and platelet activation, apoptosis, vasoconstriction/vasodilatation. We observed striking different effects of DF depending on the activator used. In particular, LPS treated cells displayed a significant upregulation of several genes, including CCL2, CCL5, CX3CL,TF, IL1B, IL6, endothelial selectin, TNF and VCAM1. DF exposure failed to revert the expression of LPS activated genes at levels comparable with unstimulated cells; nevertheless several genes scarcely expressed in LPS-treated cells were highly expressed in LPS+DF treated cells (Figure 1, upper panel). Similarly, CCL2, CCL5, CX3CL1, endothelial selectin, IL6 and VCAM1 were highly expressed in IFN-γ treated cells, although their expression was significantly lower in IFN-γ+DF treated cells (Figure 1, lower panel).Conclusions. These findings suggest that DF effects might be highly dependent on the specific activation setting. In particular, LPS and IFN-γ use different cell signaling pathways, though their downstream effects might partially overlap. According to our observations, DF effects could be mediated by the prevalent interference with specific signaling pathways more than by the direct modulation of target genes. This hypothesis agrees with experimental evidences indicating that DF is internalized by EC but dos not enter nuclei (Palomo et al. Blood 2016;127:1719-27). In alternative, the contemporary exposure of cells to DF and LPS, might result in their competition for modulating in opposite directions the same pathways, thus hampering the inhibitory effects of DF. This second hypothesis agrees with the evidence that DF attachment on cell membrane is sufficient to elicit its anti-inflammatory effect ((Palomo et al. Blood 2016;127:1719-27). [Display omitted] DisclosuresTeofili:Jazz Pharmaceutical: Research Funding. Orlando:Jazz Pharmaceutical: Research Funding.

In Vivo Cigarette Smoke Exposure to Examine the Expression of Genes Involved in the Inflammatory Response in the Mouse Uterus.

Cigarette smoke may impair uterine function, but the underlying mechanisms are poorly characterized. In this article, we describe the methodology for whole-body exposure to cigarette smoke together with assessment of the impact of this exposure on the expression of a panel of genes related to stress and toxicity pathways in mouse uteri using an in vivo model. C57BL/6 mice are whole-body-exposed to three cigarettes daily, 7 days/week, for 2 months using a specific rodent ventilator. Uteri are then collected and subjected to qRT-PCR analysis using the Stress & Toxicity PathwayFinder RT2 Profiler PCR Array (Qiagen). Cigarette smoke was found to be associated with an upregulation (≥2-fold) of C-reactive protein (Crp; 2.65-fold, p-value = 0.02), growth arrest and DNA-damage-inducible45γ (Gadd45γ; 2.11-fold, p-value = 0.04), interferon γ (Ifnγ; 2.05-fold, p-value = 0.01), and interleukin1α (Il1α; 7.74-fold, p-value = 0.003) and downregulation of matrix metallopeptidase-9 (Mmp9; -2.42-fold, p-value = 0.01). The protocol used in this study may represent a new experimental model of mouse in vivo mainstream exposure to cigarette smoke. In addition, the resulting overexpression of pro-inflammatory cytokines and genes involved in cell cycle proliferation, together with the downregulation of extracellular matrix metallopeptidases, may represent a toxicological response to cigarette smoke exposure, with potential repercussion for the processes of uterine remodeling and growth that are essential for uterine receptiveness. A recommendation to expand upon this research area is made. © 2021 Wiley Periodicals LLC.

P066 Exploration of critical molecules for optimizing treatment by analyzing in flammatory cytokines in the intestinal mucosa of patients with Inflammatory Bowel Disease

Abstract Background Inflammatory cytokines initiate and sustain the perpetuation of processes leading to chronic inflammatory conditions such as inflammatory bowel diseases (IBD). Research into the cytokine biology of IBD has nurtured the dogmatic view that distinct immunotypes characterize different IBD subtypes. Recently, many biologics have emerged as the treatment options for IBD patients. Still, it is challenging to establish biomarkers for predicting their effectiveness because the cytokines that determine the IBD clinical phenotype vary in individual patients. Therefore, this study aims to explore critical molecules for optimizing treatment by analyzing inflammatory cytokines in the intestinal mucosa of IBD patients. Methods From September 2019, to February 2020, patients with ulcerative colitis (UC) and Crohn’s disease (CD) who underwent colonoscopy or flexible sigmoidoscopy, and the control group, patients who received colorectal polypectomy at our institution, were enrolled in the study. We took biopsies from inflamed ileal or colonic mucosa. We analyzed the gene expression of various inflammatory cytokines by real-time PCR using RT2 Profiler PCR Array (96-Well Format) Human Inflammatory Cytokines &amp; Receptors (QIAGEN). Cytokines not included in this array were subjected to real-time PCR separately. Based on these data, we investigated the relationship between mucosal cytokines and clinical characteristics. Results The total number of patients was 23 (control 5, UC 12, CD 6) cases, the median age was 51 years (18–78), genders were fifteen males and eight females. Disease phenotype was divided into extensive colitis/ left-sided colitis/proctitis (10/1/1) in UC patients, and A1/A2/A3 (2/3/1), L1/L3 (2/4), B1/B1+p/B2/B3 (3/1/1/1) in CD patients. Seven patients were steroid-dependent, and six patients were refractory to the anti-TNFα agent. The higher gene expressions of CCR2/CXCL1/CXCL3/CXCL13, IL-8, IL-33, and SPP1 in inflamed mucosa of UC compared to those of CD (p&amp;lt;0.05). The positive correlation between Mayo score and IL-1R1 expression in intestinal mucosa was observed (r=0.68, p=0.028). The expression of IL-1β, IL-6, IL-8, and oncostatin M (OSM) was higher in steroid-dependent UC patients, and the primary non-responders to anti-TNFα therapies had higher expressions of IL-7 in colonic mucosa than the secondary non-responders in UC. Conclusion Our results suggest that UC exhibits different cytokine patterns in the intestinal mucosa from CD as reported previously. Exploration of cytokine expressions in the intestinal mucosa may lead to predicting refractory IBD. The identification of molecules that can predict therapeutic effectiveness helps to optimize IBD treatment in the future.