CITED1 Expression Research Articles

The Importance of Cited-1 and HIF-1α Immune Activity of Granulosa Cells in IVF Treatment

Aim: The aim of this study was to investigate the Cited-1 and HIF-1α immune activity in granulosa cells in follicular development in patients who underwent IVF for infertility. Materials and Methods: This study was conducted on 40 patients who were admitted to the assisted reproductive program with the complaint of infertility at the Gazi Yaşargil Training and Research Hospital Obstetrics and Gynecology Clinic IVF center between January 2022 and November 2022 and had primary or secondary infertility while starting the Ovum-Pick-Up (OPU) procedure. The fluid containing the granulosa cells was centrifuged at 3000 rpm for 10 min. The samples were fixed and processed for routine paraffine wax tissue embedding protocol. Sections were taken from paraffin blocks and immune stained with Cited-1 and HIF-1α. The preparations were examined under the microscope. Results: HIF-1α expression was positive in membrane of granulosa cells. The nuclei were apoptotic and pyknotic. Cited1 expression was positive in membrane of granulosa cells. the cells were pyknotic. Conclusion: The high level of HIF-1α immunopositivity and negative Cited1 immunoreactivity in the immunohistochemical staining after the granulosa cells around the oocytes collected from female patients admitted to the IVF clinic and diagnosed with infertility showed that granulosa cell viability may be important on oocyte quality.

CITED4 gene therapy protects against maladaptive cardiac remodeling after ischemia/reperfusion injury in mice

Cardiac signaling pathways functionally important in the heart's response to exercise often protect the heart against pathological stress, potentially providing novel therapeutic targets. However, it is important to determine which of these pathways can be feasibly targeted invivo. Transgenic overexpression of exercise-induced CITED4 has been shown to protect against adverse remodeling after ischemia/reperfusion injury (IRI). Here we investigated whether somatic gene transfer of CITED4 in a clinically relevant time frame could promote recovery after IRI. Cardiac CITED4 gene delivery via intravenous AAV9 injections in wild type mice led to an approximately 3-fold increase in cardiac CITED4 expression. After 4weeks, CITED4-treated animals developed physiological cardiac hypertrophy without adverse remodeling. In IRI, delivery of AAV9-CITED4 after reperfusion resulted in a 6-fold increase in CITED4 expression 1week after surgery, as well as decreased apoptosis, fibrosis, and inflammatory markers, culminating in a smaller scar and improved cardiac function 8weeks after IRI, compared with control mice receiving AAV9-GFP. Somatic gene transfer of CITED4 induced a phenotype suggestive of physiological cardiac growth and mitigated adverse remodeling after ischemic injury. These studies support the feasibility of CITED4 gene therapy delivered in a clinically relevant time frame to mitigate adverse ventricular remodeling after ischemic injury.

CITED1 expression in odontogenic cysts

BackgroundOriginating from odontogenic tissue, Odontogenic cysts are pathological cavities lined with epithelial cells and surrounded by fibrous connective tissue. This study investigated expression of CITED1 protein in different types of odontogenic cysts.Material and method40 keratocysts, 40 radicular cysts, and 40 dentigerous cysts were excised and processed for routine paraffin wax embedding protocol. Macroscopic and panoramic radiographies images were used for diagnosis. Demographical properties and dental parameters were recorded. Cystic tissues were stained with hematoxylin-eosin dye and CITED1 antibody. Semi-quantitative analysis was performed for immune staining. The protein-protein interaction network, hub gene detection and KEGG analysis were conducted using Cytoscape software.ResultOdontogenic keratocysts was imaged with 6–8 layered epithelial cells and fibrous cyst walls with inflammatory cells. Radicular cysts had stratified squamous epithelium with varying thickness, ciliated cells, and Rushton hyaline bodies. Dentigerous cysts presented hyperplastic non-keratinized epithelium, fibrous tissue, rete ridges, and inflammatory cells. CITED1 immunoexpression was highest in odontogenic keratocysts, followed by radicular cysts, and lowest in dentigerous cysts. Nuclear and cytoplasmic CITED1 expression was significantly elevated in odontogenic keratocysts compared to radicular and dentigerous cysts. The top five targets of CITED1 were identified, primarily showing enrichment in hormone and cancer related pathways.ConclusionsPositive CITED1 expression in all three types of odontogenic cysts suggest a potential role for CITED1 in the pathogenesis of odontogenic cysts, particularly in keratocysts. Further investigations are needed to elucidate the exact mechanisms underlying the differential expression of CITED1 and its implications for the development and progression of odontogenic cysts.

MTHFR as a Novel Candidate Marker for Litter Size in Rabbits.

Litter size is a significant economic trait during animal reproduction. This current study attempted to decipher whether MTHFR promotes the apoptosis of granulosa cells (GCs) and inhibits their proliferation by investigating the effects of the MTHFR gene using flow cytometry and a Cell Counting Kit-8 (CCK-8) assay. MTHFR is linked with ovarian follicle development in the reproductive performance of 104 female New Zealand rabbits. We observed that MTHFR could regulate the mRNA of follicular development-related genes (TIMP1, CITED1, FSHR, GHR, HSD17B1, and STAR) with a qRT-PCR, and we observed the protein expression of CITED1 and GHR using a western blot (WB) analysis. The dual luciferase activity assays helped identify the core promoter region of the MTHFR gene, and the polymorphism of the MTHFR promoter region was studied using Sanger sequencing. The results indicated four single nucleotide polymorphisms (SNPs) within the core promoter region, among which the g.-680C>A locus was significantly associated with both the total and alive litter sizes. Additionally, the CC genotype was associated with the largest total and alive litter sizes, compared to the CA and AA genotypes (p < 0.05). In conclusion, this study investigated the effects of MTHFR on ovarian granulosa cells and its association with selected reproductive parameters in rabbits. The results provide a theoretical foundation for the use of MTHFR as a molecular marker in rabbits.

Dysregulation of CITED2 in abnormal lung development in the nitrofen rat model.

CITED2 both modulates lung, heart and diaphragm development. The role of CITED2 in the pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. We aimed to study CITED2 during abnormal lung development in the nitrofen model. Timed-pregnant rats were given nitrofen on embryonic day (E) 9 to induce CDH. Fetal lungs were harvested on E15, 18 and 21. We performed RT-qPCR, RNAscope™ in situ hybridization and immunofluorescence staining for CITED2. We observed no difference in RT-qPCR (control: 1.09 ± 0.22 and nitrofen: 0.95 ± 0.18, p = 0.64) and in situ hybridization (1.03 ± 0.03; 1.04 ± 0.03, p = 0.97) for CITED2 expression in E15 nitrofen and control pups. At E18, CITED2 expression was reduced in in situ hybridization of nitrofen lungs (1.47 ± 0.05; 1.14 ± 0.07, p = 0.0006), but not altered in RT-qPCR (1.04 ± 0.16; 0.81 ± 0.13, p = 0.33). In E21 nitrofen lungs, CITED2 RNA expression was increased in RT-qPCR (1.04 ± 0.11; 1.52 ± 0.17, p = 0.03) and in situ hybridization (1.08 ± 0.07, 1.29 ± 0.04, p = 0.02). CITED2 protein abundance was higher in immunofluorescence staining of E21 nitrofen lungs (2.96 × 109 ± 0.13 × 109; 4.82 × 109 ± 0.25 × 109, p < 0.0001). Our data suggest that dysregulation of CITED2 contributes to abnormal lung development of CDH, as demonstrated by the distinct spatial-temporal distribution in nitrofen-induced lungs.

CITED2 Attenuates Ischemia Reperfusion-Induced Pyroptosis and Injury in Cardiomyocyte.

To examine the role of CITED2 in myocardial ischemia/reperfusion injury (MIRI) in a cell model and uncover the mechanism, hypoxia/reoxygenation (H/R) -stimulated H9C2 cell model was utilized as a MIRI cell model. Quantitative polymerase chain reaction (qPCR) as well as immunoblot assays were carried out to determine the expression of CITED2 in the MIRI cell model. MTT as well as lactate dehydrogenase assays were employed to detect the survival of H/R-stimulated H9C2 cells. Immunoblot, flow cytometry, qPCR, and enzyme-linked immunosorbent assay were carried out to assess the pyroptosis and inflammation in H9C2 cells. Immunoblot assays were used to confirm the mechanism. The expression of CITED2 was low in H/R-stimulated H9C2 cells. CITED2 can increase the survival of H/R-stimulated H9C2 cells. Additionally, CITED2 restrained H/R-stimulated pyroptosis of H9C2 cells. It also restrained the release of H/R-induced inflammatory factors. Mechanically, CITED2 inhibited HIF-1α expression, thereby suppressing MIRI progression. CITED2 attenuates MIRI in cardiomyocytes via mediating HIF-1α expression.

Cited2 inhibited hypoxia-induced proliferation and migration of PASMCs via the TGF-β1/Cited2/PPARγ pathway.

Proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) contribute to hypoxia-induced pulmonary hypertension (HPH). The transcription factor Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) has been implicated in the control of tumor cells and mesenchymal stem cell (MSC) and cardiomyocyte growth or migration. Whether Cited2 is involved in the proliferation and migration of PASMCs and the underlying mechanisms deserve to be explored. Cited2 expression was detected in rat PASMCs under hypoxia conditions and HPH rat models. The effect of Cited2 on the proliferation and migration of PASMC was detected by overexpression or knockdown of the Cited2 gene. After PAMSCs were treated with recombinant TGF-β1 and the lentivirus vector overexpressing Cited2, expression of peroxisome proliferator-activated receptor gamma (PPARγ) was examined by western blotting. We revealed that hypoxia down-regulated the expression of Cited2 in PASMCs and rat pulmonary arteries. Cited2 overexpression inhibited the proliferation and migration of PASMCs under hypoxia, while Cited2 knockdown induced the proliferation and migration of PASMCs. Cited2 inhibits the negative regulation of the TGF-β1 pathway on PPARγ to inhibit the proliferation and migration of PASMCs. These findings suggest that increased Cited2 expression contributes to the inhibition of PASMCs proliferation and migration by regulating TGF-β1-mediated target gene expression in HPH and provides a new target for molecular therapy of HPH.

Downregulation of circular RNA hsa_circ_0087856 sensitizes bladder cancer cells to cisplatin through targeting miR-1184/CITED2 signaling.

CircRNAs are considered as one of the potential therapeutic targets of multiple cancers. According to accumulating evidence, circRNA regulates cancer progression by acting as a miRNA sponge. In the current work, our data discovered that hsa_circ_0087856 and CITED2 expression was increased, while miR-1184 expression was decreased in BC cell lines and tissues. Hsa_circ_0087856 expression negatively correlated with miR-1184, whereas positively correlated with CITED2. Hsa_circ_0087856 silencing suppressed BC tumor growth, and contributed to the inhibition of cisplatin to tumor growth. In cellular experiments, hsa_circ_0087856 increasing promoted BC cells proliferation, migration and invasion, and inhibited the cells apoptosis. Hsa_circ_0087856 increasing partly reversed the inhibition of cisplatin to BC cell proliferation and the promotion to cell apoptosis. Oppositely, hsa_circ_0087856 silencing could increase the sensitivity of BC cells to cisplatin. Hsa_circ_0087856 promoted CITED2 expression through binding with miR-1184 and inhibiting its expression. CITED2 increasing partly reversed the promotion of hsa_circ_0087856 silencing to cisplatin-induced BC cells apoptosis promotion and proliferation suppression. Overall, our results revealed the role of hsa_circ_0087856 that downregulation its expression could enhance the BC cells sensitivity to cisplatin by facilitating CITED expression via sponging miR-1184. Moreover, our research provided a potential therapeutic target for BC. This article is protected by copyright. All rights reserved.

Investigation of the Sox-9 and Cited-1 Immune Activity in Placentas of Women with Placenta Accreta

Aim: In this study, we investigated the immune activity of the Sox-9 and Cited-1 in women with placenta accreta. Material and Methods: 20 healthy and 20 placenta accreta were processed for routine histological tissue processing. Placentals samples were dissected and fixed in 10% formaldehyde solution. Samples were embedded in paraffin blocks. Clinical and biochemical parameters were recorded. Placental sections were cut from paraffin blocks and stained with Sox-9 and Cited-1 immunostaining. Results: In our study, control group showed negative Cited-1 expression in decidual cells, root villi and connective tissue areas in general. Placenta accreta group showed increased Cited-1 expression in degenerated decidual cells, fibroblastic cells and endothelium. In control group, Sox-9 expression was negative in the syncytial knots, in the vascular endothelial cells. In placenta accreta group, Sox-9 reaction was positive in the root villi, in the blood vessels, in the connective tissue. Conclusion: It was observed that the Sox-9 reaction was increased and inflammation was induced, depending on the differences in decidual cells, in the syncytial area and in the vascular endothelium in in placentas of women with placenta accreta. It is thought that Sox-9 signaling processes are being determined and Cited-1 may be stimulants that affect cell proliferation and angiogenesis regulation and affect placental development.

Role of cited-1 and caspase-6 expression in HELLP syndrome.

In this study, we investigated the immunohistochemical staining of cited-1 and caspase-6 expression in the placentas of pregnant women with HELLP syndrome. Placentas of 20 normotensive patients and 20 women with HELLP syndrome were processed for routine histological tissue processing. The biochemical and clinical parameters of patients were recorded. Placentas were stained with hematoxylin-eosin and cited-1 and caspase-6 immunostaining. Placentas of normotensive patients showed normal histology. Placentas of women with HELLP syndrome showed degenerated cells, hyalinization and vacuolization. Cited-1 expression was negative in normotensive group; however, it was increased in HELLP group, especially in decidual cells, endothelial cells and other placental cells. Caspase-6 expression was negative in placental structures of normotensive groups. However, it was intense in decidual cells, vacuolar and hyalinized areas, inflammatory cells and connective tissue cells in HELLP group. Cited-1 and caspase-6 are a marker in determining the severity of HELLP syndrome.

Fenofibrate mildly stimulates browning-associated expression in white adipose tissues of young but not old male rats.

The aim of this study was to examine the effects of the hypolipemic drug fenofibrate (FF) and aging on the expression of factors/enzymes involved in brown adipose tissue (BAT) function and browning of white adipose tissue epididymal (eWAT) and subcutaneous (sWAT) depots. Young-adult and old male Wistar rats were fed standard chow (control) or supplemented with 0.1% or 0.5% FF for 30 days. Tissue samples were analysed for gene expression and protein content, and stained with Oil Red O or hematoxylin and eosin. In BAT of young rats, 0.5% FF increased only Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 1 (CITED1) protein content and Fgf21 and Gpr109A mRNA expression. The expression of oxidative metabolism related genes (Pgc1α, Cpt1b, Mcad) decreased after 0.5% FF. In BAT of old rats, FF did not affect UCP1 and CITED1 content and had little effect on gene expression. Oil Red O staining of BAT revealed no changes in lipid droplet area upon treatment in either age group. In eWAT of young rats, 0.1FF elevated UCP1 protein content and Ucp1, Pgc-1α, and Mcad expression, whereas 0.5% FF increased PPARα content and Pgc-1α, Cpt1b, Mcad, and Gpr109A levels. In eWAT of old rats, only 0.1FF increased Pgc1α and Mcad expression. In both age groups median cell area of eWAT adipocytes was reduced after 0.5% FF. In sWAT Ucp1 gene expression was very low and UCP1 protein was undetectable. FF upregulated Ucp1, Cited1, Eva1, and Cpt1b expression in sWAT of young rats, with diminished effects in old rats. In both age groups 0.5% FF increased Fgf21 expression in sWAT. Median cell area of sWAT adipocytes decreased only in young rats treated with 0.5% FF. Our results reveal that fenofibrate differentially affects gene expression in BAT, with diminished effects in old compared to young rats. In WAT of young rats FF modestly stimulates the expression of factors/enzymes involved in lipid oxidative metabolism and browning. Aging reduces both these effects. Gpr109A may present a novel gene target upregulated by FF in BAT and eWAT.

Gut microbiota-induced microRNA-206-3p increases anxiety-like behaviors by inhibiting expression of Cited2 and STK39

BackgroundAnxiety disorder is highly prevalent worldwide and represents a chronic and functionally disabling condition, with high levels of psychological stress characterized by cognitive and physiological symptoms. The purpose of this study is to evaluate the clinical significance of gut microbiota regulating microRNA (miR)-206-3p as a biomarker in the anxiety-like behaviors. MethodsInitially, bioinformatics analysis was performed to predict the related factors for gut microbiota affecting anxiety-like behaviors. Next, the anxiety-like behaviors in mice were measured by multiple experiments. Western blot analysis, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were utilized to measure the levels of 5-hydroxytryptamine (5-HT), brain derived neurotrophic factor (BDNF), and neutrophil expressed (NE) in brain tissues and serum and cAMP responsive element binding protein 1 (CREB) phosphorylation in brain tissues of germ-free (GF) mice. Dual-luciferase reporter gene assay was employed to verify the relationship between miR-206-3p and Cbp/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 2 (Cited2)/serine/threonine kinase 39 (STK39). Ectopic expression and depletion experiments of miR-206-3p were conducted to determine the expression of miR-206-3p and mRNA and protein levels of Cited2, and STK39 in HT22 cells and brain tissues. Finally, transmission electron microscope (TEM) was used to observe the effects of miR-206-3p on hippocampal mitochondria and synapses. ResultsGut microbiota could elevate miR-206-3p expression in brain tissues to increase the anxiety-like behaviors. GF mice displayed the increased levels of 5-HT, BDNF, and NE in brain tissues and serum and CREB phosphorylation in brain tissues. Cited2/STK39 was identified as the target genes of miR-206-3p. Upregulated miR-206-3p increased anxiety-like behaviors by promoting degeneration of mitochondria and synapses in hippocampus via downregulation of Cited2 and STK39. ConclusionsIn conclusion, the key findings of the current study demonstrate that gut microbiota aggravated anxiety-like behaviors via the miR-206-3p/Cited2/STK39 axis.

Abstract 9829: Exercise-Induced CITED4 Expression is Necessary for Regional Remodeling of Cardiac Microstructural Tissue Helicity

Background: Exercise promotes cardioprotective benefits for the adult heart; however, the exercise-induced molecular and microstructural remodeling processes have yet to be fully characterized. This study sought to define the spatial interactions between molecular and microstructural remodeling that are induced by exercise and might result in cardiac protection by evaluating regional correlations of expressed cardiac CITED4, a transcriptional co-regulator previously shown to increase in hearts with exercise, and myocardial architecture mapping with diffusion tensor cardiac MRI (DT-CMR). Methods: In the first cohort, male wild-type C57BL/6J mice were housed with or without free access to a running wheel. In the second cohort, CITED4 cardiomyocyte specific knockout (KO) mice were generated along with control littermates (fl/fl mice) and the methods were repeated. After 8-weeks of voluntary wheel running, the mice were euthanized and their hearts were excised, scanned at 14.1 T MRI, and sectioned. RNA Fluorescent in situ hybridization (RNA-FISH) was used to detect CITED4 and all slides were counterstained with DAPI. All experiments were performed under approval of the IACUC of the Massachusetts General Hospital. Results: The exercised group of cohort 1 showed a significant increase in CITED4 expression and microstructural DT-CMR markers in the lateral wall of the left ventricle (p&lt;0.05 for all). In particular, myofiber orientation, evaluated as helicity (helix angle transmurality), was significantly increased within the exercised group (p&lt;0.05), indicating beneficial changes in the myocardial microstructure. Cohort 2 demonstrated that CITED4 was required for these changes to take place, as the CITED4 knock out exercise group did not replicate the same structural changes seen in cohort 1, while the fl/fl control mice reproduced the expected changes in tissue microstructure following exercise. Conclusion: Exercise induces a regional increase in CITED4 expression that coincides with microstructural remodeling. In addition, we have demonstrated that CITED4 is required for these beneficial exercise-induced microstructural changes to occur.

VEGF to CITED2 ratio predicts the collateral circulation of acute ischemic stroke.

ObjectiveThe research objective was to evaluate the predicting role of the vascular endothelial growth factor to CBP/P300-interacting transactivator with Glu/Asp-rich C-terminal domain 2 Ratio (VEGF/CITED2) from peripheral blood mononuclear cells (PBMCs) in the collateral circulation of acute ischemic stroke (AIS).MethodsIn an observational study of patients with AIS, the western blot was applied to test the protein expression of VEGF and CITED2. Then, we calculated the VEGF/CITED2 and collected other clinical data. Binary logistic regression analysis between collateral circulation and clinical data was performed. Finally, receiver operating characteristic (ROC) curve analysis was used to explore the predictive value of VEGF/CITED2.ResultsA total of 67 patients with AIS were included in the study. Binary logistic regression analysis indicated the VEGF/CITED2 (OR 165.79, 95%CI 7.25–3,791.54, P = 0.001) was an independent protective factor. The ROC analyses showed an area under the ROC curve of the VEGF/CITED2 was 0.861 (95%CI 0.761–0.961). The optimal cutoff value of 1.013 for VEGF/CITED2 had a sensitivity of 89.1% and a specificity of 85.7%.ConclusionIn patients with AIS, the VEGF/CITED2 was related to the establishment of collateral circulation. The VEGF/CITED2 is a potentially valuable biomarker for predicting collateral circulation.Clinical trial registrationClinicalTrials.gov, identifier: NCT05345366.

Exercise-induced CITED4 expression is necessary for regional remodeling of cardiac microstructural tissue helicity

Both exercise-induced molecular mechanisms and physiological cardiac remodeling have been previously studied on a whole heart level. However, the regional microstructural tissue effects of these molecular mechanisms in the heart have yet to be spatially linked and further elucidated. We show in exercised mice that the expression of CITED4, a transcriptional co-regulator necessary for cardioprotection, is regionally heterogenous in the heart with preferential significant increases in the lateral wall compared with sedentary mice. Concordantly in this same region, the heart’s local microstructural tissue helicity is also selectively increased in exercised mice. Quantification of CITED4 expression and microstructural tissue helicity reveals a significant correlation across both sedentary and exercise mouse cohorts. Furthermore, genetic deletion of CITED4 in the heart prohibits regional exercise-induced microstructural helicity remodeling. Taken together, CITED4 expression is necessary for exercise-induced regional remodeling of the heart’s microstructural helicity revealing how a key molecular regulator of cardiac remodeling manifests into downstream local tissue-level changes.

Regulation of the DNA damage response by E2F4 phosphorylation in its T249/T251 conserved motif and Alzheimer’s disease.

Alzheimer's disease (AD) has a multifactorial etiology that includes DNA damage in neurons. The transcription factor E2F4, which can potentially regulate DNA repair, has two conserved threonines (T249/T251 in the mouse) that can be phosphorylated by p38MAPK (p38). The expression in vivo of the T249A/T251A E2F4 mutant form (E2F4DN) has been shown to be a multifactorial therapeutic agent against AD. In this work, we have analyzed the effects of E2F4 phosphorylation in T249/T251 on the DNA repair response (DDR). To this aim, we used N2a mouse neuroblastoma cells treated with 10 µM camptothecin (CPT), a treatment known to induce Cited2 expression and subsequent cell death. In this paradigm, the repression of Cited2 by E2F4 is abolished upon CPT treatment, thus allowing its E2F1-dependent expression followed by cell death. While E2F4 can be detected in both the nucleus and cytoplasm of N2a cells, phosphoT249-E2F4-specific immunoreactivity is specifically observed in the nucleus 4 h after treatment with CPT. Therefore, the known activation of p38 in response to CPT could lead to T249 phosphorylation of E2F4, thus suppressing its inhibition on E2F1 activity and allowing Cited2 expression. This hypothesis, is being tested in CPT- treated N2a cells co-transduced with adenoviral vectors expressing E2F1 together with either wild-type E2F4 or E2F4DN, in either the presence or absence of p38 inhibitors. In summary, our work provides support for a novel mechanism used by E2F4 to regulate the response to DNA damage in pathological situations, which could participate in the therapeutic capacity of E2F4DN against AD.

Variants and Cellular Functional Verification of CITED2 Gene Promoter Region in Patients with Atrial Septal Defect

ObjectivesCongenital heart disease (CHD) is a common disease of human birth defects, and atrial septal defect (ASD) is the most common form of ASD. Genetic variants in the coding region of the CITED2 gene is known to be significantly correlated with cardiac malformations, but variants of the CITED2 promoter region and its relationship with the formation of ASD are still unclear. We hypothesize that the variants of the CITED2 promoter may be related to pathogenesis of ASD. The purpose of this study was to screen variants in the promoter region of the CITED2 gene and to verify the effect of the variants on gene expression at the cellular level.MethodsThe blood samples of 332 ASD patients and 293 unrelated healthy children used as controls were studied. The total DNA of all subjects was extracted, and the CITED2 promoter variants were screened by PCR combined with Sanger sequencing. The luciferase activity of the CITED2 promoter was measured by a dual luciferase reporter at the cellular level. Electrophoretic mobility change assay (EMSA) and bioinformatics analysis were used to test the effect of CITED2 promoter changes on transcription factor binding sites.ResultsFour variants in the promoter region of the CITED2 gene were found only in the 332 ASD patients with zero occurrence in the 293 control subjects. These included one novel heterozygous variant (g.4933C&gt;A) and three SNPs [1 case of g.4078 A&gt;C(rs1165649373), 4 cases of g.4935C&gt;T(rs111470468), 2 cases of g.5027C&gt;T (rs112831934)]. The cellular functional analysis showed that these four variants significantly changed the transcriptional activity of the CITED2 gene promoter in HEK‐293 cells (P&lt;0.05). In addition, EMSA results and database analysis showed that these variants created or destroyed a series of possible transcription factor binding sites, resulting in changes of the expression of CITED2 protein.ConclusionThe variants of CITED2 promoter sequence in ASD patients affect transcriptional activity and are likely involved in the occurrence and development of ASD. These findings may provide new perspectives on the molecular pathogenesis and potential therapeutic insights of ASD patients.

Melatonin induces fat browning by transdifferentiation of white adipocytes and de novo differentiation of mesenchymal stem cells.

The role of melatonin in obesity control is extensively accepted, but its mechanism of action is still unclear. Previously we demonstrated that chronic oral melatonin acts as a brown-fat inducer, driving subcutaneous white adipose tissue (sWAT) into a brown-fat-like function (beige) in obese diabetic rats. However, immunofluorescence characterization of beige depots in sWAT and whether melatonin is a beige-fat inducer by de novo differentiation and/or transdifferentiation of white adipocytes are still undefined. Lean (ZL) and diabetic fatty (ZDF) Zücker rats were subdivided into two groups, control (C) and oral melatonin-supplemented (M, 10 mg kg-1 day-1) for 6 weeks. Mesenchymal stem cells (MSCs) were isolated from both rat inguinal fat and human lipoaspirates followed by adipogenesis assays with or without melatonin (50 nM for 12 h in a 24 h period, 12 h+/12 h-) mimicking the light/dark cycle. Immunofluorescence and western-blot assays showed the partial transdifferentiation of white adipocytes in both ZL and ZDF rats, with increasing thermogenic and beige markers, UCP1 and CITED1 and decreasing white adipocyte marker ASC-1 expression. In addition, melatonin increased UCP1, CITED1, and PGC1-α expression in differentiated adipocytes in both rats and humans. These results demonstrate that melatonin increases brown fat in obese diabetic rats by both adipocyte transdifferentiation and de novo differentiation. Furthermore, it promotes beige MSC adipogenesis in humans. This may contribute to the control of body weight attributed to melatonin and its metabolic benefits in human diabesity.

CircSNHG5 Sponges Mir-495-3p and Modulates CITED2 to Protect Cartilage Endplate From Degradation.

BackgroundIntervertebral disc degeneration (IDD) is a highly prevalent degenerating disease that produces tremendous amount of low back and neck pain. The cartilage endplate (CEP) is vitally important to intervertebral discs in both physiological and pathological conditions. In addition, circular RNAs (circRNAs) have been shown to be involved in the regulation of various diseases, including IDD. However, the particular role of circRNAs in cervical vertebral CEP degeneration remains unclear. Here, we examined the unique role of circRNAs in CEP of patients with cervical fracture and degenerative cervical myelopathy (DCM).MethodsHuman competitive endogenous RNA (ceRNA) microarray was performed by previous research. Western blot (WB), immunofluorescence (IF), quantitative RT-PCR (qRT-PCR), luciferase assay, and fluorescence in situ hybridization (FISH) were employed to analyze the function of circSNHG5 and its downstream effectors, miR-495-3p, and CITED2.ResultsWe demonstrated that circSNHG5 expression was substantially low in degenerative CEP tissues. Knockdown of circSNHG5 in chondrocytes resulted in a loss of cell proliferation and followed by degradation of extracellular matrix (ECM). In addition, circSNHG5 was shown to sponge miR-495-3p and modulate the expression of the downstream gene CITED2. This mechanism of action was further validated via overexpression and knockdown of CITED2.ConclusionOur findings identified a novel circSNHG5-miR-495-3p axis responsible for IDD progression. Future investigations into IDD therapy may benefit from targeting this axis.

Ginsenoside Rb1 Facilitates Browning by Repressing Wnt/β-Catenin Signaling in 3T3-L1 Adipocytes.

BackgroundThe discovery of browning in white adipose tissue has provided new ideas for treating obesity. Many studies have reported that ginsenoside Rb1 (G-Rb1) has activity against diabetes, inflammation, and obesity, but further investigation is needed on the effect and mechanism of G-Rb1 on browning.Material/MethodsWe treated 3T3-L1 adipocytes with 0–200 μM G-Rb1, and 0.5 μM Compound 3f and 30 μM SKL2001 were used to activate Wnt/β-catenin signaling. Adipocyte activity was evaluated by Cell Counting Kit-8. Oil Red O staining was used to detect the lipid droplets. Quantitative real-time polymerase chain reaction was used to measure the expression of Cd-137, Cited-1, Txb-1, Prdm-16, and Ucp-1 mRNA. Western blotting was used to measure the expression of Ucp-1, pGSK-3β (Ser 9), GSK-3β, and β-catenin proteins. The expression of Ucp-1 was also detected with immunofluorescence.ResultsAdipocyte activity was not affected by 0–100 μM G-Rb1. However, G-Rb1 dose-dependently reduced the accumulation of lipid droplets; increased the expression of Cd-137, Cited-1, Txb-1, Prdm-16, and Ucp-1 mRNA; and increased the expression of Ucp-1, pGSK-3β (Ser 9), GSK- 3β, and β-catenin proteins. The accumulation of lipid droplets and the expression of Ucp-1 protein decreased as β-catenin increased.ConclusionsG-Rb1 at various concentrations (0–100 μM) promoted the browning of adipocytes in a dose-dependent manner. Further, we confirmed that activation of Wnt/β-catenin signaling could inhibit browning. Therefore, the browning promoted by G-Rb1 may be associated with the inhibition of Wnt/β-catenin signaling.