Ankyrin Repeat Domain Research Articles

Bioinformatics analysis identifies the protective targets of omentin in mice with focal cerebral ischemia injury

Omentin is known to play a protective role in ischemic stroke. However, its regulatory networks and downstream targets in the pathogenesis of IS are incompletely revealed now. In this study, the model of photochemical brain ischemia was constructed after omentin over-expression. 8 key differentially expressed genes (DEGs) were obtained and analyzed by transcriptome analysis. These DEGs were mainly related to the negative regulation of hormone secretion, cellular phosphate ion homeostasis, and other pathways. Moreover, the mRNA expression of predicted gene 3435 (Gm3435), ankyrin repeat domain 53 (Ankrd53), fibroblast growth factor 23 (Fgf23) and the Fgf23 protein expression were down-regulated after omentin over-expression in HT22 cells injured by oxygen-glucose deprivation (OGD). In conclusion, our findings identified 8 key DEGs regulated by omentin after IS. In vitro models, the Gm3435, Ankrd53, Fgf23 mRNA expression and the Fgf23 protein expression were further verified to consistent with the transcriptomics results.

Perspectives of circular RNAs in diabetic complications from biological markers to potential therapeutic targets (Review).

Chronic complications of diabetes increase mortality and disability of patients. It is crucial to find potential early biomarkers and provide novel therapeutic strategies for diabetic complications. Circular RNAs (circRNAs), covalently closed RNA molecules in eukaryotes, have high stability. Recent studies have confirmed that differentially expressed circRNAs have a vital role in diabetic complications. Certain circRNAs, such as circRNA ankyrin repeat domain 36, circRNA homeodomain‑interacting protein kinase 3 (circHIPK3) and circRNA WD repeat domain 77, are associated with inflammation, endothelial cell apoptosis and smooth muscle cell proliferation, leading to vascular endothelial dysfunction and atherosclerosis. CircRNA LDL receptor related protein 6, circRNA actin related protein 2, circ_0000064, circ‑0101383, circ_0123996, hsa_circ_0003928 and circ_0000285 mediate inflammation, apoptosis and autophagy of podocytes, mesangial cell hypertrophy and proliferation, as well as tubulointerstitial fibrosis, in diabetic nephropathy by regulating the expression of microRNAs and proteins. Circ_0005015, circRNA PWWP domain containing 2A, circRNA zinc finger protein 532, circRNA zinc finger protein 609, circRNA DNA methyltransferase 3β, circRNA collagen typeI α2 chain and circHIPK3 widely affect multiple biological processes of diabetic retinopathy. Furthermore, circ_000203, circ_010567, circHIPK3, hsa_circ_0076631 and circRNA cerebellar degeneration‑related protein 1 antisense are involved in the pathology of diabetic cardiomyopathy. CircHIPK3 is the most well‑studied circRNA in the field of diabetic complications and is most likely to become a biological marker and therapeutic target for diabetic complications. The applications of circRNAs may be a promising treatment strategy for human diseases at the molecular level. The relationship between circRNAs and diabetic complications is summarized in the present study. Of note, circRNA‑targeted therapy and the role of circRNAs as biomarkers may potentially be used in diabetic complications in the future.

A split-luciferase lysate-based approach to identify small-molecule modulators of phosphatase subunit interactions

An emerging strategy for the therapeutic targeting of protein phosphatases involves the use of compounds that interfere with the binding of regulatory subunits or substrates. However, high-throughput screening strategies for such interfering molecules are scarce. Here, we report on the conversion of the NanoBiT split-luciferase system into a robust assay for the quantification of phosphatase subunit and substrate interactions in cell lysates. The assay is suitable to screen small-molecule libraries for interfering compounds. We designed and validated split-luciferase sensors for a broad range of PP1 and PP2A holoenzymes, including sensors that selectively report on weak interaction sites. To facilitate efficient hit triaging in large-scale screening campaigns, deselection procedures were developed to eliminate assay-interfering molecules with high fidelity. As a proof-of-principle, we successfully applied the split-luciferase screening tool to identify small-molecule disruptors of the interaction between the C-terminus of PP1β and the ankyrin-repeat domain of the myosin-phosphatase targeting subunit MYPT1.

Evaluating the Molecular Properties and Function of ANKHD1, and Its Role in Cancer.

Ankyrin repeat and single KH domain-containing protein 1 (ANKHD1) is a large, scaffolding protein composed of two stretches of ankyrin repeat domains that mediate protein-protein interactions and a KH domain that mediates RNA or single-stranded DNA binding. ANKHD1 interacts with proteins in several crucial signalling pathways, including receptor tyrosine kinase, JAK/STAT, mechanosensitive Hippo (YAP/TAZ), and p21. Studies into the role of ANKHD1 in cancer cell lines demonstrate a crucial role in driving uncontrolled cellular proliferation and growth, enhanced tumorigenicity, cell cycle progression through the S phase, and increased epithelial-to-mesenchymal transition. Furthermore, at a clinical level, the increased expression of ANKHD1 has been associated with greater tumour infiltration, increased metastasis, and larger tumours. Elevated ANKHD1 resulted in poorer prognosis, more aggressive growth, and a decrease in patient survival in numerous cancer types. This review aims to gather the current knowledge about ANKHD1 and explore its molecular properties and functions, focusing on the protein's role in cancer at both a cellular and clinical level.

Hprt Serves as an Ideal Reference Gene for qRT-PCR Normalization in Rat DRG Neurons.

To identify suitable reference genes for gene expression studies in rat dorsal root ganglia (DRG) neurons. The raw cycle threshold (Ct) values of 12 selected reference genes were obtained via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in neurons at different developmental stages or under different treatments. Two strategies were employed to screen the most stable reference genes: the genes were ranked according to the coefficient of biological variation and further validated using geNorm and NormFinder programs. The stable and unstable reference genes were subsequently used as internal controls to assess their effects on target gene expression. All reference genes showed varying degrees of fluctuation in Ct values during the growth process of neurons or after different treatments. 18S ribosomal RNA (Rn18s) and β-actin (Actb) exhibited the most significant changes, while ubiquitin C (Ubc), hypoxanthine phosphoribosyl transferase (Hprt), and mitochondrial ribosomal protein L10 (Mrpl10) showed relatively minor changes. The most stable and unstable genes obtained by different evaluation methods varied slightly. Overall, Actb was found to be the most unstable reference gene, while Hprt was the relatively most stable reference gene. The use of unstable reference genes Actb and ankyrin repeat domain 27 (Ankrd27) as internal controls led to high variability within the control group, ultimately affecting the determination of target gene expression. In contrast, the stable reference gene Hprt had small inter-assay variation and high stability. Our observations indicate that Hprt is a proper endogenous reference gene for qRT-PCR analysis in rat DRG neurons and thus provides a critical molecular basis for the genetic characterization in neurological disorders.

ANKRD22 knockdown suppresses papillary thyroid cell carcinoma growth and migration and modulates the Wnt/β-catenin signaling pathway

The incidence of thyroid cancer is escalating globally, particularly among women. Studies have demonstrated the abnormal activation of Ankyrin Repeat Domain 22 (ANKRD22) in various cancers, but it remains uncertain whether it is also highly expressed in papillary thyroid carcinoma (PTC). Our objective was to evaluate the role of ANKRD22 in PTC. The expression of ANKRD22 varies among tissues, as validated by the Cancer Genome Atlas, and further predicted using the Tumor Immune Estimation Resource. Predicted results were examined via polymerase chain reaction and western blotting. Subsequently, the expression of ANKRD22 in cells was suppressed by RNA interference, and changes in cell progression were examined in conjunction with the cell counting kit-8 assay, transwell assay, and colony formation assay. Finally, the effects of ANKRD22 knockdown on the Epithelial-to-Mesenchymal transition and the Wnt/β-catenin signaling pathway were investigated through western blotting. An in vivo mice model was established to validate the effect of ANKRD22. This study discovered that ANKRD22 was highly expressed in PTC, which was validated by polymerase chain reaction and western blotting. Knockdown of ANKRD22, significantly reduced thecell viability, colony formation capability, and cell invasion and migration abilities. Furthermore, we found that knockdown of ANKRD22 impaired both tumor Epithelial-to-Mesenchymal transition and the activation of the Wnt/β-catenin signaling pathway. In conclusion, this study revealed that the knockdown of ANKRD22 inhibits the growth and migration of papillary thyroid cell carcinoma by regulating the Wnt/β-catenin signaling pathway.

Astragalus polysaccharide promotes osteogenic differentiation of human bone marrow derived mesenchymal stem cells by facilitating ANKFY1 expression through miR-760 inhibition.

Astragalus polysaccharide (APS) participates in various processes, such as the enhancement of immunity and inhibition of tumours. APS can affect osteoporosis (OP) by regulating the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). This study was designed to elucidate the mechanism of APS in hBMSC proliferation and osteoblast differentiation. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the expression of microRNA (miR)-760 and ankyrin repeat and FYVE domain containing 1 (ANKFY1) in OP tissues and hBMSCs. Cell viability was measured using the Cell Counting Kit-8 assay. The expression of cyclin D1 and osteogenic marker genes (osteocalcin (OCN), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2)) was evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mineral deposits were detected through Alizarin Red S staining. In addition, Western blotting was performed to detect the ANKFY1 protein levels following the regulation of miR-760. The relationship between miR-760 and ANKFY1 was determined using a luciferase reporter assay. The expression of miR-760 was upregulated in OP tissues, whereas ANKFY1 expression was downregulated. APS stimulated the differentiation and proliferation of hBMSCs by: increasing their viability; upregulating the expression levels of cyclin D1, ALP, OCN, and RUNX2; and inducing osteoblast mineralization. Moreover, APS downregulated the expression of miR-760. Overexpression of miR-760 was found to inhibit the promotive effect of APS on hBMSC differentiation and proliferation, while knockdown of miR-760 had the opposite effect. ANKFY1 was found to be the direct target of miR-760. Additionally, ANKFY1 participated in the APS-mediated regulation of miR-760 function in hBMSCs. APS promotes the osteogenic differentiation and proliferation of hBMSCs. Moreover, APS alleviates the effects of OP by downregulating miR-760 and upregulating ANKFY1 expression.

Biallelic ANKS6 null variants cause notable extrarenal phenotypes in a nephronophthisis patient and lead to hepatobiliary abnormalities by YAP1 deficiency.

The ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) gene, encoding an inversin compartment protein of the primary cilium, was recently reported as a pathogenic gene of nephronophthisis (MIM PS256100). Extrarenal manifestations are frequently observed in this disease, however, potential genotype-phenotype correlations and the underlying mechanisms remain poorly understood. Here we described an infant with kidney failure, hepatobiliary abnormalities, and heart disease, in whom whole exome sequencing identified compound heterozygous variants in ANKS6, including a novel nonsense variant p.Trp458* and a recurrent splicing variant c.2394+1G > A. mRNA expression studies showed that the splicing variant caused aberrant mRNA splicing with exon 13 skipping and the biallelic variants were predicted to cause loss of ANKS6 function. We systematically characterized the clinical and genetic spectra of the disease and revealed that biallelic null variants in ANKS6 cause more severe kidney disease and more extrarenal manifestations, thus establishing a clear genotype-phenotype correlation for the disease. Further evaluations showed that ANKS6 deficiency reduced YAP1 expression in the patient's bile duct epithelium and ANKS6 promotes YAP1 transcriptional activity in a dose-dependent manner, indicating that loss of ANKS6 function causes hepatobiliary abnormalities through YAP1 deficiency during biliary morphogenesis and development, which may offer new therapeutic targets.

An ankyrin repeat chaperone targets toxic oligomers during amyloidogenesis.

Numerous age-linked diseases are rooted in protein misfolding; this has motivated the development of small molecules and therapeutic antibodies that target the aggregation of disease-linked proteins. Here we explore another approach: molecular chaperones with engineerable protein scaffolds such as the ankyrin repeat domain (ARD). We tested the ability of cpSRP43, a small, robust, ATP- and cofactor-independent plant chaperone built from an ARD, to antagonize disease-linked protein aggregation. cpSRP43 delays the aggregation of multiple proteins including the amyloid beta peptide (Aβ) associated with Alzheimer's disease and α-synuclein associated with Parkinson's disease. Kinetic modeling and biochemical analyses show that cpSRP43 targets early oligomers during Aβ aggregation, preventing their transition to a self-propagating nucleus on the fibril surface. Accordingly, cpSRP43 rescued neuronal cells from the toxicity of extracellular Aβ42 aggregates. The substrate-binding domain of cpSRP43, composed primarily of the ARD, is necessary and sufficient to prevent Aβ42 aggregation and protect cells against Aβ42 toxicity. This work provides an example in which an ARD chaperone non-native to mammalian cells harbors anti-amyloidal activity, which may be exploited for bioengineering.

Circ_PRDM5/miR-25-3p/ANKRD46 axis is associated with cell malignant behaviors in subjects with breast cancer evaluated by ultrasound.

Circular RNAs (circRNAs) are key RNA molecules in cancer biology. CircRNA PR/SET domain 5 (circ_PRDM5, hsa_circ_0005654) was downregulated in breast cancer (BC) tissues. This study is designed to investigate the functional mechanism of circ_PRDM5 in BC. Ultrasound examinations were performed to evaluate BC patients and normal individuals. Circ_PRDM5, miR-25-3p, and Ankyrin repeat domain 46 (ANKRD46) level detection was carried out by reverse transcription-quantitative polymerase chain reaction. 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used for cell viability examination. Cell proliferation was evaluated by ethynyl-2'-deoxyuridine assay and colony formation assay. The protein levels were examined using western blot. Cell migration and invasion abilities were assessed via transwell assay. Target interaction was analyzed via dual-luciferase reporter assay. The role of circ_PRDM5 in vivo was explored via xenograft tumor assay. Circ_PRDM5 expression was downregulated in BC tissues and cells. Overexpression of circ_PRDM5 suppressed proliferation and motility but enhanced apoptosis of BC cells. Circ_PRDM5 served as a sponge of miR-25-3p. Circ_PRDM5 impeded BC cell malignant development via sponging miR-25-3p. Circ_PRDM5 induced ANKRD46 upregulation by targeting miR-25-3p. Inhibition of miR-25-3p retarded BC progression by increasing the ANKRD46 level. Circ_PRDM5 repressed BC tumorigenesis in vivo through mediating the miR-25-3p/ANKRD46 axis. This study evidenced that circ_PRDM5 inhibited cell progression and tumor growth in BC via interacting with mir-25-3p/ANKRD46 network.

Aging Differentially Affects Axonal Autophagosome Formation and Maturation

ABSTRACT Misregulation of neuronal macroautophagy/autophagy has been implicated in age-related neurodegenerative diseases. We compared autophagosome formation and maturation in primary murine neurons during development and through aging to elucidate how aging affects neuronal autophagy. We observed an age-related decrease in the rate of autophagosome formation leading to a significant decrease in the density of autophagosomes along the axon. Next, we identified a surprising increase in the maturation of autophagic vesicles in neurons from aged mice. While we did not detect notable changes in endolysosomal content in the distal axon during early aging, we did observe a significant loss of acidified vesicles in the distal axon during late aging. Interestingly, we found that autophagic vesicles were transported more efficiently in neurons from adult mice than in neurons from young mice. This efficient transport of autophagic vesicles in both the distal and proximal axon is maintained in neurons during early aging, but is lost during late aging. Our data indicate that early aging does not negatively impact autophagic vesicle transport nor the later stages of autophagy. However, alterations in autophagic vesicle transport efficiency during late aging reveal that aging differentially impacts distinct aspects of neuronal autophagy. Abbreviations: ACAP3: ArfGAP with coiled-coil, ankyrin repeat and PH domains 3; ARF6: ADP-ribosylation factor 6; ATG: autophagy related; AVs: autophagic vesicles; DCTN1/p150Glued: dynactin 1; DRG: dorsal root ganglia; GAP: GTPase activating protein; GEF: guanine nucleotide exchange factor; LAMP2: lysosomal-associated protein 2; LysoT: LysoTracker; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAPK8IP1/JIP1: mitogen-activated protein kinase 8 interacting protein 1; MAPK8IP3/JIP3: mitogen-activated protein kinase 8 interacting protein 3; mCh: mCherry; PE: phosphatidylethanolamine

Loss of ANCO1 Expression Regulates Chromatin Accessibility and Drives Progression of Early-Stage Triple-Negative Breast Cancer.

Mutations in the gene ankyrin repeat domain containing 11 (ANKRD11/ANCO1) play a role in neurodegenerative disorders, and its loss of heterozygosity and low expression are seen in some cancers. Here, we show that low ANCO1 mRNA and protein expression levels are prognostic markers for poor clinical outcomes in breast cancer and that loss of nuclear ANCO1 protein expression predicts lower overall survival of patients with triple-negative breast cancer (TNBC). Knockdown of ANCO1 in early-stage TNBC cells led to aneuploidy, cellular senescence, and enhanced invasion in a 3D matrix. The presence of a subpopulation of ANCO1-depleted cells enabled invasion of the overall cell population in vitro and they converted more rapidly to invasive lesions in a xenograft mouse model. In ANCO1-depleted cells, ChIP-seq analysis showed a global increase in H3K27Ac signals that were enriched for AP-1, TEAD, STAT3, and NFκB motifs. ANCO1-regulated H3K27Ac peaks had a significantly higher overlap with known breast cancer enhancers compared to ANCO1-independent ones. H3K27Ac engagement was associated with transcriptional activation of genes in the PI3K-AKT, epithelial-mesenchymal transition (EMT), and senescence pathways. In conclusion, ANCO1 has hallmarks of a tumor suppressor whose loss of expression activates breast-cancer-specific enhancers and oncogenic pathways that can accelerate the early-stage progression of breast cancer.

SERPINA3-ANKRD11-HDAC3 pathway induced aromatase inhibitor resistance in breast cancer can be reversed by HDAC3 inhibition

Endocrine resistance is a major challenge for breast cancer therapy. To identify the genes pivotal for endocrine-resistance progression, we screened five datasets and found 7 commonly dysregulated genes in endocrine-resistant breast cancer cells. Here we show that downregulation of serine protease inhibitor clade A member 3 (SERPINA3) which is a direct target gene of estrogen receptor α contributes to aromatase inhibitor resistance. Ankyrin repeat domain containing 11 (ANKRD11) works as a downstream effector of SERPINA3 in mediating endocrine-resistance. It induces aromatase inhibitor insensitivity by interacting with histone deacetylase 3 (HDAC3) and upregulating its activity. Our study suggests that aromatase inhibitor therapy downregulates SERPINA3 and leads to the ensuing upregulation of ANKRD11, which in turn promotes aromatase inhibitor resistance via binding to and activating HDAC3. HDAC3 inhibition may reverse the aromatase inhibitor resistance in ER-positive breast cancer with decreased SERPINA3 and increased ANKRD11 expression.

TRPV4-Rho GTPase complex structures reveal mechanisms of gating and disease

Crosstalk between ion channels and small GTPases is critical during homeostasis and disease, but little is known about the structural underpinnings of these interactions. TRPV4 is a polymodal, calcium-permeable cation channel that has emerged as a potential therapeutic target in multiple conditions. Gain-of-function mutations also cause hereditary neuromuscular disease. Here, we present cryo-EM structures of human TRPV4 in complex with RhoA in the ligand-free, antagonist-bound closed, and agonist-bound open states. These structures reveal the mechanism of ligand-dependent TRPV4 gating. Channel activation is associated with rigid-body rotation of the intracellular ankyrin repeat domain, but state-dependent interaction with membrane-anchored RhoA constrains this movement. Notably, many residues at the TRPV4-RhoA interface are mutated in disease and perturbing this interface by introducing mutations into either TRPV4 or RhoA increases TRPV4 channel activity. Together, these results suggest that RhoA serves as an auxiliary subunit for TRPV4, regulating TRPV4-mediated calcium homeostasis and disruption of TRPV4-RhoA interactions can lead to TRPV4-related neuromuscular disease. These insights will help facilitate TRPV4 therapeutics development.

Structure of human TRPV4 in complex with GTPase RhoA

Transient receptor potential (TRP) channel TRPV4 is a polymodal cellular sensor that responds to moderate heat, cell swelling, shear stress, and small-molecule ligands. It is involved in thermogenesis, regulation of vascular tone, bone homeostasis, renal and pulmonary functions. TRPV4 is implicated in neuromuscular and skeletal disorders, pulmonary edema, and cancers, and represents an important drug target. The cytoskeletal remodeling GTPase RhoA has been shown to suppress TRPV4 activity. Here, we present a structure of the human TRPV4-RhoA complex that shows RhoA interaction with the membrane-facing surface of the TRPV4 ankyrin repeat domains. The contact interface reveals residues that are mutated in neuropathies, providing an insight into the disease pathogenesis. We also identify the binding sites of the TRPV4 agonist 4α-PDD and the inhibitor HC-067047 at the base of the S1-S4 bundle, and show that agonist binding leads to pore opening, while channel inhibition involves a π-to-α transition in the pore-forming helix S6. Our structures elucidate the interaction interface between hTRPV4 and RhoA, as well as residues at this interface that are involved in TRPV4 disease-causing mutations. They shed light on TRPV4 activation and inhibition and provide a template for the design of future therapeutics for treatment of TRPV4-related diseases.

FRET analysis of the temperature-induced structural changes in human TRPV3

Transient receptor potential vanilloid member 3 (TRPV3) is an ion channel that plays a critical role in temperature sensing in skin. There have been active studies on how TRPV3, which is also known as one of the temperature-sensitive transient receptor potential (thermoTRP) channels, responds to temperature. However, the previous studies were mostly based on TRPV3 originating from mice or rats. Here, we focus on human TRPV3 (hTRPV3) and show that which domain of hTRPV3 undergoes conformational changes as temperature increases by Förster resonance energy transfer (FRET) assay. During the heat-induced activation of hTRPV3, the linker domain close to C-terminus, that is, the C-terminal domain shows a largest structural change whereas there is little change in the ankyrin repeat domain (ARD). Interestingly, the activation of hTRPV3 by an agonist shows structural change patterns that are completely different from those observed during activation by heat; we observe structural changes in ARD and S2–S3 linker after ligand stimulation whereas relatively little change is observed when stimulated by heat. Our results provide insight into the thermal activation of hTRPV3 channel.

ANKRD29, as a new prognostic and immunological biomarker of non–small cell lung cancer, inhibits cell growth and migration by regulating MAPK signaling pathway

BackgroundThe predominant cancer-related deaths worldwide are caused by lung cancer, particularly non-small cell lung cancer (NSCLC), despite the fact that numerous therapeutic initiatives have been devised to improve the outcomes. Ankyrin repeat domain (ANKRD) is one of the widespread protein structural motifs in eukaryotes but the functions of ANKRD proteins in NSCLC progression remains unclear.MethodsWe performed integrative bioinformatical analysis to determine the dysregulated expression of ANKRDs in multiple tumors and the association between ANKRD29 expression and the NSCLC tumor environment. Quantitative real-time PCR (qRT-PCR), western blot, immunohistochemistry (IHC), and tissue microarray (TMA) assays were used to investigate the expression of ANKRD29 in NSCLC cell lines. The role of ANKRD29 in NSCLC cell proliferation and migration in vitro was deteceted by 5-bromodeoxyuridine (BrdU) incorporation, colony formation, flow cytometry, would-healing, trans-well, and western blot experiment. RNA-seq technology was applied to deciper the molecular mechanism regulated by ANKRD29 in NSCLC.ResultsWe constructed a valuable risk-score system for predicting the overall survival outcomes of NSCLC patients based on the expression of five hub ANKRD genes. And we found that the hub gene ANKRD29 was remarkedly decreased in NSCLC tissues and cell lines due to the promoter hypermethylation, and revealed that high ANKRD29 expression obviously correlated with patients’ better clinical outcome. Overexpression of ANKRD29 significantly inhibited cell proliferation and migration, promoted the cancerous cells’ sensitivity to carboplatin and enhanced the killing ability of T cells in NSCLC cells. Interestingly, ANKRD29 can be served as a biomarker to predict the response to immunotherapy in NSCLC. Mechanically, RNA-seq results showed that ANKRD29 could regulate MAPK signaling pathway. Moreover, we screened two potential agonists for ANKRD29.ConclusionsANKRD29 functions as a new tumor suppressor in NSCLC tumorigenesis and could be developed as a biomarker for prognostic prediction, immunotherapy response, and drug susceptibility evaluation of NSCLC in the future.

Circulating tumor DNA in plasma as an early predictor for pathological lymph node involvement and (neo-)adjuvant chemotherapy in patients with colon cancer.

3532 Background: Postoperative chemotherapy is standard for colon cancer patients with pathological lymph node involvement (pN+). If the presence of pN+ could be accurately predicted prior to surgery, the initiation of neoadjuvant chemotherapy could potentially reduce the presence of micrometastasis and enhance survival. The objective of this study was to assess the utility of preoperative methylated circulating tumor DNA (meth-ctDNA) as a predictive marker for pN+ and its clinical value as a marker for the initiation of neoadjuvant chemotherapy. Methods: The clinical data of 203 colon cancer patients (stage I-III) was collected from the Colorectal Cancer Database established at Danish Colorectal Cancer Center South, Vejle Hospital, Denmark. Patients were randomized into a discovery and a validation cohort. Plasma collected preoperatively was analyzed for three different meth-ctDNAs (Neuropeptide Y, Galactose-3-O-Sulfotransferase 3, and KN Motif and Ankyrin Repeat Domains 1) by droplet digital PCR (ddPCR). Results: Based on analysis of the discovery cohort, samples were considered positive if two or more of the three methylation markers had at least three positive droplets in the ddPCR analysis. 40% of patients in the discovery cohort and 46% of patients in the validation cohort were considered positive. When using meth-ctDNA as a diagnostic tool to predict pN+, the sensitivity and specificity in the discovery cohort were 42% and 61% respectively. In the validation cohort the values were 54% and 59%, respectively. AUC was < 60% in both cohorts. Meth-ctDNA was associated to clinical tumor (T)- and node (N)-category in both cohorts (p≤0.01). Four-year disease free survival (DFS) in patients with and without detectable meth-ctDNA was 60% and 90% in the discovery cohort (p = 0.01), and 62% and 78% in the validation cohort (p = 0.04). Meth-ctDNA remained the strongest prognostic factor of DFS in the multivariate analysis compared to clinical T- or N-category, gender and age (p = 0.065, HR 2.46, 95% CI 1.03-5.88), however not statistically significant. Four-year overall survival (OS) in patients with and without detectable meth-ctDNA was 75% and 95% in the discovery cohort (p = 0.006), and 78% and 91% in the validation cohort (p = 0.02). In the multivariate analysis, meth-ctDNA was the strongest prognostic factor of OS compared to clinical T- or N-category, gender and age (p = 0.016, HR 4.54, 95% CI 1.32-15.6). Conclusions: The preoperative measurement of meth-ctDNA was not found to be a clinically significant predictor of pN+ or a reliable indicator for the initiation of neoadjuvant chemotherapy. Nonetheless, the results showed that preoperative ctDNA is a powerful prognostic indicator, suggesting that randomized controlled trials should be conducted to determine the efficacy of neoadjuvant chemotherapy in colon cancer patients with preoperative detectable ctDNA.

In silico analysis of autism spectrum disorder through the integration of DNA methylation and gene expression data for biomarker search

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by apparent communication deficits, repetitious unusual rituals, and social dysfunction. The epigenetic mechanism of the disorder has been researched meticulously yet not fully understood. Furthermore, there is yet no diagnostic marker that is associated with the condition. This study aimed at enhancing the understanding of the epigenetic mechanism of ASD and exploring biomarker candidates for the disorder using an in-silico approach.METHODS: ASD associated blood specific DNA methylation datasets were analyzed and overlapped with gene expression data to determine differentially regulated and methylated genes. Enrichment analysis was performed to identify signaling pathways, gene ontology terms, and disease associations. Additionally, metabolites and metabolic pathways associated with differentially methylated genes were demonstrated.RESULTS: The results revealed 42 differentially regulated and methylated genes, the majority of which were previously not associated with ASD. Particularly downregulated NFATC1 (nuclear factor of activated T cells 1) and upregulated MANBA (mannosidase beta) and ARAP1 (ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 1) may be researched further to assess their biomarker features. The MAPK signaling pathway and gene ontology terms for protein/serine kinase activity and protein kinase binding came into prominence as a result of enrichment analysis. Metabolic alterations in ASD including tyrosine, tryptophan and asparagine were identified. Loss of amino acid homeostasis as well as dysregulation in related metabolic pathways were also observed.CONCLUSIONS: This in-silico study brings a comprehensive outlook on epigenetic alterations complementing governance by DNA methylation through integrating data across omics. DNA methylation, gene expression as well as metabolomics signatures of ASD that may be regarded as biomarker candidates were reported. The results of this study can guide future experimental studies, which will eventually lead to diagnostic tools and therapeutics designed for individuals suffering from the conditions of this complex disorder.

LncASAP1-IT1 promotes hepatocellular carcinoma progression through the regulation of the miR-1294/TGFBR1 pathway in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the most common tumor with severe morbidity and high mortality. The lncRNA ASAP1-IT1 [the intronic transcript 1 (IT-1) of ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1)] have been shown to promote tumor formation in a variety of cancers. This study sought to investigate the effects of dysregulated ASAP1-IT1 on the biological processes of HCC. The expression levels of ASAP1-IT1 in 30 paired HCC and adjacent non-tumor tissues were measured by real-time-quantitative polymerase chain reaction (RT-qPCR). Several functional tests were performed to investigate the molecular mechanism of ASAP1-IT1 in HCC progression. Our study showed that ASAP1-IT1 was highly expressed in the HCC tissues and cell lines. The knockdown of ASAP1-IT1 inhibited cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) progression and enhanced the sorafenib sensitivity of the HCC cells. Further investigations revealed that ASAP1-IT1 served as a sponge of microRNA-1294 (miR-1294) to promote transforming growth factor beta receptor 1 (TGFBR1) expression. In addition, the tumor-promoting effect of ASAP1-IT1 was blocked by inhibiting miR-1294/TGFBR1. Tumorigenic assays in nude mice demonstrated that the inhibition of ASAP1-IT1 inhibited the growth of HCC in vivo. These results suggest that lncASAP1-IT1 promotes HCC development by targeting TGFBR1 through miR-1294, which provides a potential target for HCC diagnosis and treatment.