TaqMan Gene Expression Assays Research Articles

Association of F11R polymorphisms and gene expression with primary Sjögren's syndrome patients.

F11R gene encodes junctional adhesion molecule-A protein (JAM-A), which is expressed in various types of cells and is involved in leukocyte extravasation during inflammation. Sjögren's syndrome (SS) is a chronic systemic inflammatory disease that involves lymphocytes invasion of exocrine glands. F11R has been studied in autoimmune diseases, but any association between F11R and SS has not yet been investigated. Therefore, experiments were undertaken to examine the relationships among F11R gene polymorphism, messenger RNA (mRNA) expression and SS patients. Three hundred and twenty-nine patients with SS, and 223 healthy controls were enrolled in their recruitment from the Kaohsiung Medical University Hospital. Genomic DNA was extracted from peripheral blood mononuclear cells and gene polymorphisms were genotyped by TaqMan real-time polymerase chain reaction (PCR). F11R mRNA expression was quantitated by quantitative real-time PCR with TaqMan Gene Expression Assay. Our study showed the genotype -688A/C (rs6695707) was not found in relation to SS patients. The odds ratio of -436A/G (rs12567886) genotype was notably associated with less susceptibility of SS in human leukocyte antigen (HLA)-DR2 negative and HLA-DR3 negative individuals. F11R mRNA expression was lower in SS patients than in the cells of healthy controls. The result indicated that G allele of -436A/G genotype has the potential protective effect against SS disease condition. F11R mRNA was expressed significantly lower in SS patients.

The Effect of Chronic Mild Stress and Escitalopram on the Expression and Methylation Levels of Genes Involved in the Oxidative and Nitrosative Stresses as Well as Tryptophan Catabolites Pathway in the Blood and Brain Structures

Previous studies suggest that depression may be associated with reactive oxygen species overproduction and disorders of the tryptophan catabolites pathway. Moreover, one-third of patients do not respond to conventional pharmacotherapy. Therefore, the study investigates the molecular effect of escitalopram on the expression of Cat, Gpx1/4, Nos1/2, Tph1/2, Ido1, Kmo, and Kynu and promoter methylation in the hippocampus, amygdala, cerebral cortex, and blood of rats exposed to CMS (chronic mild stress). The animals were exposed to CMS for two or seven weeks followed by escitalopram treatment for five weeks. The mRNA and protein expression of the genes were analysed using the TaqMan Gene Expression Assay and Western blotting, while the methylation was determined using methylation-sensitive high-resolution melting. The CMS caused an increase of Gpx1 and Nos1 mRNA expression in the hippocampus, which was normalised by escitalopram administration. Moreover, Tph1 and Tph2 mRNA expression in the cerebral cortex was increased in stressed rats after escitalopram therapy. The methylation status of the Cat promoter was decreased in the hippocampus and cerebral cortex of the rats after escitalopram therapy. The Gpx4 protein levels were decreased following escitalopram compared to the stressed/saline group. It appears that CMS and escitalopram influence the expression and methylation of the studied genes.

Exercise intervention differentially induces the expression of cystathionine beta synthase (CBS) in African American with MCI: Implication for endogenous H2S generation and component of aging pathway

AbstractBackgroundDecline in metabolic fitness, accumulation of undesirable metabolic changes and cellular senescence are the hallmarks of aging [1,2]. The incorporation of lifespan‐extending platforms such as exercise can promote metabolic fitness and delay the process of aging [3]. However, the mechanisms involved are unclear. We hypothesized that exercise intervention can drive beneficial outcome by increasing expression of two enzymes; CBS and CTH involved in the production of H2S, glutathione and taurine as well as splice factors; SRSF2 and HNRNPD [4,5] in African American with MCI.MethodWe used TaqMan gene expression assay to investigate mechanistic link between exercise and components of aging pathway by evaluating CBS and CTH, enzymes metabolizing sulfur containing amino acid. We also evaluated if CBS and CTH expression correlate with SRSF2 and HNRNPD splice factors by virtue of endogenous H2S production.ResultComparison of CBS and CTH at baseline, 3‐ and 6‐month exercise time points revealed that 3‐month exercise intervention doubled the expression of CBS. However, CTH showed a decreasing trend, suggesting differential expression. Interestingly, expression of CBS correlated with SRSF2 splice factor whereas expression of CTH correlated with HNRNPD at all time points tested.ConclusionIn this study, we provided mechanistic insight through which exercise drive beneficial outcome by investigating component of aging pathway at baseline and exercise intervention in African American with MCI. Our data suggest differential effect of exercise on the expression of CBS, CTH; and the splice factors linked to delay pace of cellular senescence and aging by way of H2S [4,5]. Thus, our study implicates exercise‐induced CBS expression and endogenous H2S as drivers of benefit of fitness adaptation on metabolic fitness and life span with potential for clinical translation.

GPR56 gene down-regulation in patients with Klinefelter syndrome: a candidate for infertility?

The etiology of azoospermia in patients with Klinefelter Syndrome (KS) is still unknown. The protein codified by the G protein-couple receptor 56 (GPR56) belongs to the adhesion family of G protein-coupled receptors (GPRs). Its mutations are involved in the pathogenesis of intellectual disability and, according to animal studies, infertility. As the expression of GPR56 in patients with KS has not been investigated so far, this study was undertaken with the purpose of evaluating its expression in peripheral blood mononuclear cells (PBMCs) of patients with KS and normal controls. This age-matched case-control study was performed in 10 patients with KS and 10 controls. Patients and controls underwent to blood sampling for next-generation sequencing (NGS) analysis, and differentially expressed mRNAs were identified using DESeq2 v.1.12. QRT-PCR was used to validate the results obtained by NGS analysis. TaqMan Gene Expression Assay primers were used to carry out the real-time PCR analysis for GPR56. GPR56 was down-regulated by -2081-fold (q-value <0.05) in PBMCs of patients with KS compared to controls. NGS data were confirmed by QRT-PCR. The possible contribution of the GPR56 gene down-regulation in the pathogenesis of spermatogenic failure in patients with KS is worthy to be further explored.

Supplementing chestnut tannins in the broiler diet mediates a metabolic phenotype of the ceca

As the demand for alternatives to antibiotic growth promoters (AGP) increases in food animal production, phytobiotic compounds gain popularity because of their ability to mimic the desirable bioactive properties of AGP. Chestnut tannins (ChT) are one of many phytobiotic compounds used as feed additives, particularly in South America, for broilers because of its favorable antimicrobial and growth promotion capabilities. Although studies have observed the microbiological and immunologic effects of ChT, there is a lack of studies evaluating the metabolic function of ChT. Therefore, the objective of this study was to characterize the cecal metabolic changes induced by ChT inclusion and how they relate to growth promotion. A total of 200 day-of-hatch broiler chicks were separated into 2 feed treatment groups: control and 1% ChT. The ceca from all the chicks in the treatment groups were collected on day 2, 4, 6, 8, and 10 after hatch. The cytokine mRNA quantitative RT-PCR was determined using TaqMan gene expression assays for IL-1B, IL-6, IL-8, IL-10, and interferon gamma quantification. The cytokine expression showed highly significant increased expressions of IL-6 and IL-10 on day 2 and 6, whereas the other proinflammatory cytokines did not have significantly increased expression. The results from the kinome array demonstrated that the ceca from birds fed with 1% ChT had significant (P < 0.05) metabolic alterations based on the number of peptides when compared with the control group across all day tested. The increased expression of IL-6 appeared to be strongly indicative of altered metabolism, whereas the increased expression of IL-10 indicated the regulatory effect against other proinflammatory cytokines other than IL-6. The ChT initiate a metabolic mechanism during the first 10 d in the broiler. For the first time, we show that a phytobiotic product initially modulates metabolism while also potentially supporting growth and feed efficiency downstream. In conclusion, a metabolic phenotype alteration in the ceca of chickens fed ChT may indicate the importance of enhanced broiler gut health.

The Impact of Chronic Mild Stress and Agomelatine Treatment on the Expression Level and Methylation Status of Genes Involved in Tryptophan Catabolic Pathway in PBMCs and Brain Structures.

Depression is the serious mental disorder. Previous studies suggest that the development mechanism of depression may be associated with disorders of the tryptophan catabolic pathway (TRYCAT). Thus, this study investigates the effect of agomelatine treatment on the expression and methylation status of genes involved in TRYCAT in the brain and blood of rats exposed to a chronic mild stress (CMS). Separate groups of rats were exposed to CMS for two or seven weeks; the second group received vehicle or agomelatine for five weeks. After completion of both stress conditions and treatment, the expression levels of messenger RNA (mRNA) and protein, as well as the methylation status of promoters, were measured in peripheral blood mononuclear cells (PBMCs) and in brain structures with the use of TaqMan Gene Expression Assay, Western blot, and methylation-sensitive high-resolution melting techniques. In PBMCs, Kmo mRNA expression increased in the group after CMS, while this effect was normalized by agomelatine therapy. In brain, KatI and KatII expression changed following CMS exposure. Moreover, CMS decreased the methylation status of the second Tdo2 promoter in the amygdala. Protein expression of Tph1, Tph2, Ido1, and KatII changed in the group after CMS and agomelatine administration, most prominently in the basal ganglia, cerebral cortex, hippocampus, and amygdala. The results indicate that CMS and agomelatine affect the mRNA and protein expression, as well as the methylation of promoters of genes involved in the tryptophan catabolic pathway.

Upregulation of FOXO3 in New-Onset Type 1 Diabetes Mellitus.

Forkhead box O (FOXO) transcription factors have been implicated in the development and differentiation of the immune cells. FOXO3 plays a crucial role in physiologic and pathologic immune response. FOXO3, cooperatively with FOXO1, control the development and function of Foxp3+ regulatory T cells (Treg). Since the lack of Treg-mediated control has fundamental impact on type 1 diabetes mellitus (T1DM) development, we investigated FOXO3 expression in patients with T1DM. FOXO3 expression was estimated in peripheral blood mononuclear cells (PBMCs) from newly diagnosed T1DM pediatric patients (n = 28) and age-matched healthy donors (n = 27) by reahavel-time PCR and TaqMan gene expression assays. Expression analysis revealed significant upregulation of FOXO3 in T1DM (P = 0.0005). Stratification of the T1DM group according to the presence of initial diabetic ketoacidosis (DKA) did not indicate differences in FOXO3 expression in patients with DKA compared to a mild T1DM onset (P > 0.05). In conclusion, overexpression of FOXO3 is correlated with the ongoing islet autoimmune destruction and might suggest a potential role for this gene in the pathogenesis of type 1 diabetes mellitus.

ITGA4 gene methylation status in chronic lymphocytic leukemia.

Background:We aimed to investigate ITGA4 gene expression pattern and to explore its methylation heterogeneity in chronic lymphocytic leukemia (CLL).Patients & methods:Eighty one CLL patients and 75 healthy subjects were enrolled and prognostic evaluation of patients was assessed. ITGA4 q-realtime PCR was performed using Applied Biosystems, TaqMan gene expression assay. ITGA4 gene-specific CpG methylation was investigated in real time using pyrosequencing technology.Results:ITGA4 was differentially expressed in CLL patients. The CpG sites-1, 2 and 3 showed significantly higher mean levels than healthy controls (p = <0.001, 0.007 and 0.009). Significant association between CpG site-1 and CLL has been detected using age-adjusted logistic regression (p < 0.001).Conclusion:Hypermethylation at ITGA4 gene CpG sites (1,2,3) is a characteristic feature in CLL.

Intravenous Injection of Muse Cells as a Potential Therapeutic Approach for Epidermolysis Bullosa

Intravenous Injection of Muse Cells as a Potential Therapeutic Approach for Epidermolysis Bullosa

The Changes of Expression and Methylation of Genes Involved in Oxidative Stress in Course of Chronic Mild Stress and Antidepressant Therapy with Agomelatine.

Preclinical studies conducted so far suggest that oxidative stress processes may be associated with the mechanism of depression development. This study shows the effects of chronic administration of agomelatine on expression and the methylation status of Sod1, Sod2, Gpx1, Gpx4, Cat, Nos1, and Nos2 in the brain stricture and blood in the chronic mild stress (CMS) animal model of depression. The animals were exposed to the CMS procedure and treatment with agomelatine (10 mg/kg/day, IP) for five weeks and then were sacrificed. TaqMan Gene Expression Assay, Western blot, and methylation-sensitive high-resolution melting techniques were used to evaluate mRNA and protein expression of the genes, and the methylation status of their promoters. Gpx1, Gpx4, and Sod2 expression in the PBMCs and Sod1 and Sod2 expression in the brain were reduced in the stressed group after agomelatine administration. CMS caused an increase in the methylation of the third Gpx4 promoter in peripheral blood mononuclear cells and Gpx1 promoter in the cerebral cortex. Additionally, stressed rats treated with agomelatine displayed a significantly lower Gpx4 level in the hypothalamus. The results confirm the hypothesis that the CMS procedure and agomelatine administration change the expression level and methylation status of the promoter region of genes involved in oxidative and nitrosative stress.

Selection of Endogenous Control Reference Genes for Studies on Type 1 or Type 2 Endometrial Cancer

A panel of 32 candidate reference genes was used to identify the most stable genes for gene normalisation in quantitative RT-PCR studies using endometrial biopsies obtained from women with endometrial cancer (type 1 or type 2) and without cancer (controls). RNA from the biopsies was isolated, examined for purity and quality, and then reverse transcribed into cDNA before being subjected to real-time qRT-PCR analysis in triplicate within the TaqMan gene Expression Assay kit. The most ‘stable’ endogenous control genes were then identified using the geNorm qbase + 2 and NormFinder software packages. PSMC4, PUM1 and IPO8 were identified as the best reference genes combination for type 1 endometrial cancer (grades 1, 2 and 3), whereas for type 2 endometrial cancer (serous and carcinosarcoma), UBC, MRPL19, PGK1 and PPIA were the best reference genes combination. We conclude that the use of these normaliser combinations should provide accurate interpretation of gene expression at the transcript level in endometrial cancer studies especially for types 1 and 2 cancers.

The Effect of Chronic Mild Stress and Venlafaxine on the Expression and Methylation Levels of Genes Involved in the Tryptophan Catabolites Pathway in the Blood and Brain Structures of Rats

A growing body of evidence suggests that depression may be associated with impairment of the tryptophan catabolites (TRYCATs) pathway. The present study investigated the effects of the chronic administration of venlafaxine on the expression and methylation status of Katl, Tph1/2, Ido1, Kmo and Kynu in the brain and blood of rats exposed to the CMS model of depression. The rats were subjected to the CMS procedure for 2 or 7 weeks and administered venlafaxine (10 mg/kg/day, IP) for 5 weeks. mRNA and protein expression and the methylation status of gene promoters in PBMCs and six brain structures were evaluated and analysed using the TaqMan Gene Expression Assay and Western blotting, and methylation-sensitive high-resolution melting (MS-HRM), respectively. We found that the CMS procedure increased KatI expression in the midbrain and KatII expression in the midbrain and the amygdala, while venlafaxine administration decreased KatII expression in the hypothalamus and the cerebral cortex. The methylation status of the Tph1 and Kmo promoters in peripheral blood mononuclear cells (PBMCs) was significantly increased in the stressed group after antidepressant therapy. The protein levels of Tph1 and Ido1 were decreased following venlafaxine administration. Our results confirmed that CMS and venlafaxine modulate the expression levels and methylation status of genes involved in the TRYCATs pathway.

Effects of venlafaxine on the expression level and methylation status of genes involved in oxidative stress in rats exposed to a chronic mild stress.

Recent human and animal studies indicate that oxidative and nitrosative stress may play a role in the aetiology and pathogenesis of depression. This study investigates the effect of chronic administration of the serotonin‐norepinephrine reuptake inhibitor, venlafaxine, on the expression and methylation status of SOD1, SOD2, GPx1, GPx4, CAT, NOS1 and NOS2 in the brain and blood of rats exposed to a chronic mild stress (CMS) model of depression. Separate groups of animals were exposed to CMS for 2 or 7 weeks; the second group received saline or venlafaxine (10 mg/kg/d, IP) for 5 weeks. After completion of both stress conditions and drug administration, the mRNA and protein expression of selected genes and the methylation status of their promoters were measured in peripheral mononuclear blood cells (PBMCs) and in brain structures (hippocampus, amygdala, hypothalamus, midbrain, cortex, basal ganglia) with the use of TaqMan Gene Expression Assay, Western blot and methylation‐sensitive high‐resolution melting techniques. CMS caused a decrease in sucrose consumption, and this effect was normalized by fluoxetine. In PBMCs, SOD1, SOD2 and NOS2 mRNA expression changed only after venlafaxine administration. In brain, CAT, Gpx1, Gpx4 and NOS1 gene expression changed following CMS or venlafaxine exposure, most prominently in the hippocampus, midbrain and basal ganglia. CMS increased the methylation of the Gpx1 promoter in PBMCs, the second Gpx4 promoter in midbrain and basal ganglia, and SOD1 and SOD2 in hippocampus. The CMS animals treated with venlafaxine displayed a significantly higher CAT level in midbrain and cerebral cortex. CMS caused an elevation of Gpx4 in the hippocampus, which was lowered in cerebral cortex by venlafaxine. The results indicate that CMS and venlafaxine administration affect the methylation of promoters of genes involved in oxidative and nitrosative stress. They also indicate that peripheral and central tissue differ in their response to stress or antidepressant treatments. It is possible that that apart from DNA methylation, a crucial role of expression level of genes may be played by other forms of epigenetic regulation, such as histone modification or microRNA interference.These findings provide strong evidence for thesis that analysis of the level of mRNA and protein expression as well as the status of promoter methylation can help in understanding the pathomechanisms of mental diseases, including depression, and the mechanisms of action of drugs effective in their therapy.

The Prognostic Impact of Epidermal Growth Factor Receptor (EGFR) in Patients with Acute Myeloid Leukaemia.

Expression of Epidermal Growth Factor Receptor (EGFR), an important proto-oncogene, regulates cell differentiation, proliferation, cell migration and survival in most of the cancer types. EGFR expression has been reported in Acute Myeloid Leukaemia (AML), however, many other reports nullified EGFR expression in AML. These contradictory data prompted us to reevaluate the expression of EGFR in AML and carry out a comparative survival analysis between EGFR expressing and non-expressing AML patients (Children and Acute Promyelocytic Leukaemia patients excluded). Bone marrow and/or peripheral blood samples were collected from 60 adult patients with AML with written informed consent. PCR, Real-Time Taqman gene expression assays were used for the detection of genetic alterations. Statistical analysis was conducted using SPSS software (IBM SPSS 20). In our study, EGFR expression was detected in 21 out of 60, in 35% (95% C.I. 23.45-48.48) AML patients. Overall survival was significantly shorter in patients with EGFR (p = < 0.01), with an average survival of 18.57months (95% C.I. 12.42-24.73months) compared with 31.27months (95% C.I. 28.19-34.33months) in patients without EGFR. EGFR expression was significantly higher in female patients compared to male (p = 0.037).This study confirms the presence of EGFR in AML and indicates that EGFR expression confers poor prognosis in AML. However, the underlying cause of this adverse prognostic effect has not been identified. Further clinical studies are warranted to determine the exact mechanism through which EGFR activity might contribute to AML progression and identify the potential therapeutic target for the reversal of resistance to conventional chemotherapeutics.

Specific gene expression in type 1 diabetic patients with and without cardiac autonomic neuropathy

We hypothesized that some molecular pathways might interact to initiate the process of nervous tissue destruction, promoting cardiac autonomic neuropathy (CAN) in the course of diabetes type 1 (T1D). The study group consisted of 60 T1D patients (58.33% women/41.67% men), on standard therapy. The control group consisted of twenty healthy volunteers recruited in accordance with age, gender and body weight. The presence of CAN was documented by the Ewing test method (ProSciCard apparatus). A microarray data analysis was performed using Gene Spring version 13. The microarray results for selected genes were confirmed by real-time PCR (qRT-PCR), using specific TaqMan Gene Expression Assays. Plasma IL‐6 content was measured by an enzyme-linked immunosorbent assay (ELISA). The p < 0.05 value was considered as statistically significant. The microarray analysis, confirmed by qRTPCR, showed significant up-regulation of autophagy, quantity of mitochondria, quality regulatory genes (mTOR, GABARAPL2) apoptosis, ER-stress and inflammation (NFKB1, IL1b, IL1R1, SOD1), in T1D when compared to the control group. A significantly higher IL-6 protein level was observed in T1D patients, in comparison to the control group. We concluded that the observed changes in gene expression and activation of intracellular pathways give a coherent picture of the important role of oxidative stress in inflammation and the activation of apoptosis in the pathomechanism of DM. The significance of the inflammatory process, confirmed by the increased level of the inflammation biomarker IL-6 in the pathomechanisms of CAN was shown even in patients with properly treated T1D.

Frequent Activation of Notch Signaling Pathway in Colorectal Cancers and Its Implication in Patient Survival Outcome.

Colorectal cancer is a major health concern as it ranks third in incidence and second major cause of cancer-related deaths worldwide. A leading cause of treatment failure has been attributed to cancer stem cells that can invariably resist existing chemotherapeutic regimens. Notch signaling pathway has been involved in the maintenance of stem cells besides being crucial in cell fate decision and embryonic development. This pathway has also been implicated in several human malignancies including colorectal cancer. We investigated mRNA expression of four Notch receptors (Notch1–4), five ligands (Jag1, Jag2, Dll1, Dll3, and Dll4), and four target genes (Hes1, Hes5, Hey1, and Hey2) using highly specific TaqMan gene expression assays in colorectal adenomas and cancers. Upregulated expression of Notch receptors ranged between 29 and 73% in colorectal cancers and between 11 and 56% in adenomas. Expression of Notch3 and Notch4 receptors was significantly higher in colorectal cancers compared to normal and adenoma tissues. The Jagged and Delta-like ligands were overexpressed between 25 and 52% in colorectal cancers, while in adenomas, it ranged between 0 and 33%. Combining the data for upregulation of receptors and ligands suggests that 86% colorectal cancers and 56% adenomas exhibited overexpression of Notch pathway genes in our cohort. Notch target genes were upregulated between 24 and 33% in colorectal cancers and between 11 and 22% in adenomas. Collating upregulation of Notch receptors and ligands with the target genes showed concordance in 58% colorectal tumors. Additionally, we evaluated expression of Notch receptors, ligands, and target genes with prognosis using the TCGA mRNA expression dataset. Patients overexpressing Notch3, Notch4, and Hey1 had significantly poorer overall survival relative to those having lower levels of these genes. Taken together, Notch signaling components are aberrantly overexpressed in colorectal tumors, and development of therapeutics targeting the Notch pathway may prove to be beneficial in the management of colorectal cancers.

MN1, FOXP1 and hsa-miR-181a-5p as prognostic markers in acute myeloid leukemia patients treated with intensive induction chemotherapy and autologous stem cell transplantation

BackgroundThe meningioma-1 (MN1) gene is expressed in hematopoietic CD34+ cells and down-regulated during myeloid differentiation. MN1 overexpression has been linked to shorter overall and disease free survival in AML patients treated with intensive induction chemotherapy. MN1 overexpression may still be an adverse prognostic marker in AML patients treated with autologous stem cell transplant (auto-SCT) after intensive induction chemotherapy. MethodsWe retrospectively analysed 54 peripheral blood mononuclear cell (PBMC) samples of AML patients who received auto-SCT at remission (CR1) after intensive induction chemotherapy. MN1 and putative MN1-associated mRNAs, as well as MN1-associated micro-RNAs were assessed at diagnosis in peripheral blood mononuclear cells using Taqman gene expression assays. ResultsAML patients with elevated MN1 or FoxP1 gene expression at diagnosis had a significantly shorter progression-free and overall survival after intensive induction chemo-therapy and auto-SCT. The presence of the favourable risk NPM1 mutation associated with reduced MN1 gene expression. In contrast to MN1 and FOXP1, elevated expression of the putative tumor suppressive micro-RNA hsa-miR-181a-5p was predictive for positive outcome. Correlation analysis of MN1 with myeloid gene expression levels revealed association of MN1 and BMI-1, CD34, FOXP1 and MDM2 expression. Analysis of non-coding RNAs revealed an inverse correlation of MN1 with hsa-miR-20a-5p and hsa-miR-181b-5p expression. ConclusionsMN1, FOXP1 and hsa-miR-181a-5p are prognostic markers in AML patients treated with intensive induction chemotherapy and auto-SCT. While MDM2 is a validated therapeutic target, the transcription factors MN1 and FOXP1, and the chromatin modulator BMI-1 are potential therapeutic targets in the treatment of AML. The tumor suppressor hsa-miR-181a-5p may be a candidate miRNA mimic for therapeutic use.

Oncomir MicroRNA-346 Is Upregulated in Colons of Patients With Primary Sclerosing Cholangitis

INTRODUCTION:Primary sclerosing cholangitis (PSC) is a cholestatic liver disorder that is frequently associated with ulcerative colitis (UC). Patients with PSC and UC (PSC-UC) have a higher risk of colorectal neoplasia compared with patients with UC. The oncogenic properties of microRNA-346 (miR-346) have been recently reported. We investigated the expression of miR-346 and its 2 target genes, the receptor of vitamin D (VDR), and the tumor necrosis factor-α (TNF-α), which are known to modulate carcinogenesis.METHODS:Ascending and sigmoid colon biopsies were obtained from patients with PSC, PSC and UC (PSC-UC), UC, and healthy controls (n = 10 in each group). Expressions of VDR, TNF-α, 18S RNA, p27Kip1, miR-346, and reference microRNA, miR-191, were evaluated by real-time PCR using human TaqMan Gene Expression and TaqMan MicroRNA Assays. Functional studies with miR-346 mimic and inhibitor were conducted in HepG2 and Caco-2 cells. The effect of ursodeoxycholic acid on miR-346 expression was examined in Caco-2 cells.RESULTS:An increased expression of miR-346 in the ascending colon of PSC-UC was observed (P < 0.001 vs all groups). In patients with UC, an exceptionally low colonic expression of miRNA-346 was accompanied by the extensive upregulation of VDR and TNF-α genes. A functional in vitro analysis demonstrated that inhibition of miR-346 resulted in the upregulation of VDR and TNF-α, whereas the induction of miR-346 activity suppressed VDR, TNF-α, and p27Kip1.DISCUSSION:The upregulation of miRNA-346 in the colon of patients with PSC may be responsible for the disturbance of VDR and TNF-α signaling pathway, which could result in an inadequate suppression of neoplasia.

REGULATION OF MICRORNA EXPRESSION LEVEL BY CHOLERETIC THERAPY IN FUNCTIONAL DISORDERS OF THE GALLBLADDER AND ОDDI’S SPHINCTER IN CHILDREN

The alm: To study the effect of choleretic therapy on the level of microRNA expression in functional disorders of the gallbladder and Oddi's sphincter in children. Materials and methods: Fifty patients with functional disorders of the gallbladder and Oddi's sphincter who received standard therapy in combination with ursodeoxycholic acid, 20 patients - standard therapy without ursodeoxycholic acid, and 20 healthy children were examined. The level of expression of microRNA-378f, microRNA-4311, microRNA-4714- 3p in the blood serum by the method of real-time polymerase chain reaction with reverse transcription according to the protocol TaqMan Gene Expression Assays was performed. Results: It was demonstrated that the activity profile of microRNA-4714-3p was significantly lower in those examined with functional disorders of the gallbladder and Oddi's sphincter than in practically healthy children (p<0.05). After standard therapy combined with ursodeoxycholic acid in children with functional disorders of the gallbladder and Oddi's sphincter, the level of expression of microRNA-378f is significantly higher than before therapy (5.23±0.70 SU and 2.02±0.57 SU respectively) (p<0.05). Against the background of standard therapy with the addition of ursodeoxycholic acid or without it, the expression profile of microRNA-4714-3p in the blood serum in children with functional disorders of the gallbladder and Oddi's sphincter significantly decreased (1.93±0.58 SU and 1,14±0,53 SU respectively) (p<0.05). Conclusions: Ursodeoxycholic acid in children with functional disorders of the gallbladder and Oddi's sphincter affects the activity of generation of gene regulators of the cellular mechanisms of microRNA-378f and microRNA-4714-3p.

A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma

A Novel Recurrent Gain-of-Function Mutation of Rltpr Q575E in Adult T Cell Leukemia/Lymphoma