OBJECTIVE

To observe MET-associated alteration during the trans-differentiation from MSCs to neuron-like cells, and to explore the possible molecular mechanism.

METHODS

Bone marrow MSCs were isolated from rat femur and purified in continuous cell culture. After induced differentiation to neuron-like cells by the combination of butylated hydroxyanisole (BHA) and dimethyl sulfoxide (DMSO), cells were tested by comparative polymerase chain reaction (PCR) for the relative expression of MET biomarkers and transcription factors, and for cell cycle by flow cytometry. Meanwhile, target genes of Wnt/β-catenin pathway were also analyzed by comparative PCR to determine the possible involvement.

RESULTS

In MSC-induced neuron-like cells, MET-associated transcription factors such as Snail, Slug, ZEB1, ZEB2, and Twist were significantly attenuated in expression level. The Mesenchymal marker Vimentin expression level was increased. Membrane protein E-cad was slightly down-regulated, while N-cad level was marginally elevated. Percentage of proliferating cells (S phase in cell cycle) markedly shrank from 40.42% for MSCs to 6.76% for MSC-derived neuron. Additionally, Wnt/β-catenin target genes β-catenin and c-myc were decreasingly expressed.

CONCLUSIONS

Chemically induced trans-differentiation from MSC to neuron caused similar MET-featured alteration in gene expression and proliferation to known MET, which might be underlied by deactivation of Wnt/β-catenin pathway.

The aim of this study was to determine the gene- and microRNA-expression profile contributing to epithelial to mesenchymal transition in a rat model of experimental colitis. For this, inflammation was induced by injecting 2,4,6-trinitrobenzene sulphonic acid to the colon of male Wistar rats. Samples were taken from both inflamed and uninflamed regions of the same colon, total RNA was isolated, and the mRNA and microRNA expressions were monitored. We have determined that the expression of genes responsible for inducing mesenchymal phenotype, such as Egr1, Fgf2, Fgf7, Jak2, Notch2, Hif1α, Zeb2, Mmp9, Lox, and Vim, was all significantly induced in the inflamed regions of the affected colons while the epithelial marker E-cadherin (Cdh1) was downregulated. In contrast, the expression of microRNAs miR-192, miR-143, miR-375, miR-30a, miR-107, and miR-200b responsible for the regulation of the above mentioned genes was significantly downregulated in inflamed colon. Importantly, we detected moderate induction in the expression of five out of six tested microRNAs in the uninflamed regions. In summary, we identified numerous interacting genes and microRNAs with mutually exclusive expression pattern in inflamed regions of colitis-induced rats. These findings suggest that-among others-an important step in the epithelial to mesenchymal transition in experimental colitis is the dysregulated microRNA expression.

Copy number variants (CNVs) were detected and analyzed in 14 probands with autism and intellectual disability with self-injurious behavior (SIB) resulting in tissue damage. For each proband we obtained a clinical history and detailed behavioral descriptions. Genetic anomalies were observed in all probands, and likely clinical significance could be established in four cases. This included two cases having novel, de novo copy number variants and two cases having variants likely to have functional significance. These cases included segmental trisomy 14, segmental monosomy 21, and variants predicted to disrupt the function of ZEB2 (encoding a transcription factor) and HTR2C (encoding a serotonin receptor). Our results identify variants in regions previously implicated in intellectual disability and suggest candidate genes that could contribute to the etiology of SIB.

Hypoxia plays an essential role in the development of various cancers. The biological function and underlying mechanism of microRNA-338-3p (miR-338-3p) under hypoxia remain clarified in breast cancer (BC). Herein, we performed bioinformatics, gain- and loss-of-function of miR-338-3p, a luciferase reporter assay, and chromatin immunoprecipitation (ChIP) in vitro and tumor xenograft model. We also explored the potential signaling pathways of miR-338-3p in BC. We detected the expression levels and prognostic significance of miR-338-3p in BC by qRT-PCR and in situ hybridization. MiR-338-3p was lowly expressed in BC tissues and cell lines, and BC patients with underexpression of miR-338-3p tend to have a dismal overall survival. Functional experiments showed that miR-338-3p overexpression inhibited BC cell proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) process, whereas miR-338-3p silencing abolished these biological behaviors. Zinc finger E-box-binding homeobox 2 (ZEB2) was validated as a direct target of miR-338-3p. ZEB2 overexpression promoted, while ZEB2 knockdown abolished the promoted effects of miR-338-3p knockdown on cell biological behaviors through the NF-ĸB and PI3K/Akt signal pathways. HIF1A can transcriptionally downregulate miR-338-3p under hypoxia. In total, miR-338-3p counteracts hypoxia-induced BC cells growth, migration, invasion, and EMT via ZEB2/ NF-ĸB/PI3K signal pathway, implicating miR-338-3p may be a promising target to treat patients with breast cancer.

Keywords: HIF1A; ZEB2; breast cancer; epithelial-mesenchymal transition; miR-338-3p.

Epsin 3 (EPN3) expression is limited to gastric parietal cells and wounded or pathological tissue rather than normal brain tissue, and although it has been identified as an oncogene in estrogen receptor‑positive breast cancer and non‑small cell lung cancer, its function in cancer is poorly understood. The present study aimed to investigate the association of EPN3 expression with the clinicopathological features of patients with glioma, as well as the effects of EPN3 on glioblastoma cells and the potential molecular mechanisms for its effects on glioblastoma cell behavior. EPN3 expression was assessed by immunohistochemistry in tissue samples from 167 patients with glioma, as well as by western blotting in 5 glioblastoma cell lines. The U87 and U251 glioblastoma cell lines were used to investigate the effects of EPN3 on glioblastoma cell invasion and migration through gain and loss of EPN3 expression experiments; expression levels were further investigated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analyses. The results demonstrated that EPN3 expression levels were upregulated in high‑grade glioma tissues compared with low‑grade tissues, and there were varying expression levels of EPN3 in the five glioblastoma cell lines. No significant differences were observed in EPN3 expression in relation to patient age, sex or tumor size. Overexpression of EPN3 promoted glioblastoma cell migration and invasion, which we hypothesized was through affecting epithelial‑mesenchymal transition (EMT). RT‑qPCR and western blotting revealed that EPN3 upregulation increased the expression of Notch1 intracellular domain, β‑catenin, Slug, Twist and zinc‑finger E‑box‑binding homeobox (ZEB)‑1. These results suggested that EPN3 enhances the migration and invasion of glioblastoma cells by activating the transcription factors Slug, Twist and ZEB1, but not Snail 1 or ZEB2, to induce EMT in glioma cells; EPN3 involvement in the Notch and WNT/β‑catenin signaling pathways may contribute to this process.

Snail family zinc finger 2 (SLUG) is related to epithelial-mesenchymal transition (EMT). Quaking (QKI) is an RNA binding protein and has been indicated to have a relationship with EMT by recent studies. The prognostic value of SLUG and QKI in breast cancer patients still needs exploration. We conducted Immunohistochemistry (IHC) to evaluate the protein expression of SLUG and QKI and the prognostic value in 108 breast cancer (BC) patients. The Bc-GenExMiner database was used to compare the mRNA levels of two genes in different subgroups of BC patients. Kaplan-Meier plotter were used for survival data of SLUG and QKI gene. We also mined the cBioPortal database for co-expression analysis of QKI and EMT markers. Our results suggested that patients with higher expression of SLUG and QKI showed shorter overall survival time. The mRNA level of SLUG and QKI were higher in ER negative, PR negative, ≤ 51 y, and TNBC patients. SLUG mRNA showed no survival significance, while higher QKI mRNA expression level was correlated with worse clinical outcome in Kaplan-Meier Plotter database. The cBioPortal database showed that QKI was correlated to SLUG as well as other EMT markers like TWIST2, VIM, and ZEB2. QKI was indicated to be a potential prognostic marker for BC patients, and combined expression of SLUG and QKI showed the best prognostic value. Co-expression analysis indicated that QKI was likely to have a correlation with SLUG and EMT.

Objective: An association between the rs10496964 polymorphism and the ZEB2 gene has not yet been reported, and the role of ZEB2 in epilepsy therapy is also unclear. The aims of this research were to evaluate the role of ZEB2 in the therapy of epilepsy and to explore the association between rs10496964 and ZEB2 expression.

Methods: We used the expression quantitative trait loci (eQTL) dataset resource from the Brain eQTL Almanac to evaluate the association between rs10496964 and ZEB2 expression in human brain tissue. Pathway and process enrichment analysis, protein-protein interaction analysis, and PhosphoSitePlus® analysis were then performed to further evaluate the role of ZEB2 in the therapy of epilepsy.

Results: The rs10496964 polymorphism was found to regulate the expression of ZEB2 in human brain tissue. The ZEB2 protein interacts with the targets of approved antiepileptic drugs, and a post-translational acetylation modification of ZEB2 was associated with an epilepsy drug therapy.

Conclusion: Our findings suggest that ZEB2 may be involved in the therapy of epilepsy, and rs10496964 regulates ZEB2 expression in human brain tissue.

Keywords: ZEB2; bioinformatic analysis; biomarker; drug target; epilepsy; expression quantitative trait loci; histone deacetylase; protein–protein interaction; rs10496964; therapy.

Microtubules are polymers of α/β-tubulin, with microtubule organization being regulated by microtubule-associated proteins (MAPs). Herein, we describe a novel role for the epithelial gene repressor, zinc finger E-box-binding homeobox 1 (ZEB1), that switches from a chromatin-associated protein during interphase, to a MAP that associates with α-, β- and γ-tubulin during mitosis. Additionally, ZEB1 was also demonstrated to associate with γ-tubulin at the microtubule organizing center (MTOC). Using confocal microscopy, ZEB1 localization was predominantly nuclear during interphase, with α/β-tubulin being primarily cytoplasmic and the association between these proteins being minimal. However, during the stages of mitosis, ZEB1 co-localization with α-, β-, and γ-tubulin was significantly increased, with the association commonly peaking during metaphase in multiple tumor cell-types. ZEB1 was also observed to accumulate in the cleavage furrow during cytokinesis. The increased interaction between ZEB1 and α-tubulin during mitosis was also confirmed using the proximity ligation assay. In contrast to ZEB1, its paralog ZEB2, was mainly perinuclear and cytoplasmic during interphase, showing some co-localization with α-tubulin during mitosis. Considering the association between ZEB1 with α/β/γ-tubulin during mitosis, studies investigated ZEB1's role in the cell cycle. Silencing ZEB1 resulted in a G₂-M arrest, which could be mediated by the up-regulation of p21^Waf1/Cip1 and p27^Kip1 that are known downstream targets repressed by ZEB1. However, it cannot be excluded the G₂/M arrest observed after ZEB1 silencing is not due to its roles as a MAP. Collectively, ZEB1 plays a role as a MAP during mitosis and could be functionally involved in this process.

Inflammatory breast cancer (IBC) is a rare yet aggressive breast cancer variant, associated with a poor prognosis. The major challenge for IBC is misdiagnosis due to the lack of molecular biomarkers. We profiled dysregulated expression of microRNAs (miRNAs) in primary samples of IBC and non-IBC tumors using human breast cancer miRNA PCR array. We discovered that 28 miRNAs were dysregulated (10 were upregulated, while 18 were underexpressed) in IBC vs. non-IBC tumors. We identified 128 hub genes, which are putative targets of the differentially expressed miRNAs and modulate important cancer biological processes. Furthermore, our qPCR analysis independently verified a significantly upregulated expression of miR-181b-5p, whereas a significant downregulation of miR-200b-3p, miR-200c-3p, and miR-203a-3p was detected in IBC tumors. Receiver operating characteristic (ROC) curves implied that the four miRNAs individually had a diagnostic accuracy in discriminating patients with IBC from non-IBC and that miR-203a-3p had the highest diagnostic value with an AUC of 0.821. Interestingly, a combination of miR-181b-5p, miR-200b-3p, and miR-200c-3p robustly improved the diagnostic accuracy, with an area under the curve (AUC) of 0.897. Intriguingly, qPCR revealed that the expression of zinc finger E box-binding homeobox 2 (ZEB2) mRNA, the putative target of miR-200b-3p, miR-200c-3p, and miR-203a-3p, was upregulated in IBC tumors. Overall, this study identified a set of miRNAs serving as potential biomarkers with diagnostic relevance for IBC.

Keywords: ZEB2; hub genes; inflammatory breast cancer; miR-1-3p; miR-181b-5p; miR-200b-3p; miR-200c-3p; miR-203a-3p; microRNAs.

Long non-coding RNAs (lncRNAs) are one of the ncRNAs that transcript with length more than 200 nt that are not translated into protein. Studies have shown that lncRNAs have regulatory function in human disease especially cancers. lncRNA dysfunction causes altered cellular behavior including proliferation, invasion, and migration, and also it can inhibit apoptosis. Long non-coding zinc finger E-box binding homeobox 2 antisense RNA1 (lnZEB2-AS1) is one of the lncRNAs that plays the oncogenic role in different cancers. Dysregulation of lncZEB2-AS1 can lead to tumorigenesis and cancer progression. lncZEB2-AS1 may be introduced as a diagnostic marker or therapeutic target for human cancers. In this review, we describe briefly the mechanism of ZEB2-AS1 in cells and its function in cancer progression.

Keywords: TGFβ; biomarker; cancer; lncRNA ZEB2-AS1; non-coding RNA.

Background: Aberrant miRNA expression and DNA methylation are two major epigenetic events in lung adenocarcinoma (LUAD). We conducted a combined analysis of the molecular changes in LUAD.

Methods: We analyzed differentially expressed miRNAs and methylated CpG loci in 489 LUAD tissues versus 49 normal lung tissues of the Cancer Genome Atlas (TCGA). The results were validated in cell lines and xenograft mouse models and additional pairs of 36 LUAD and 36 normal lung tissues.

Results: A total of 125 differentially expressed miRNAs and 145 differentially methylated CpG loci were identified in the LUAD versus normal lung tissues of TCGA data. Expression of the 22 miRNAs was inversely correlated with the 47 differentially methylated sites located in the miRNAs. Molecular and cellular function analysis showed that the abnormally methylated miRNAs were mainly involved in cell-to-cell signaling and interaction in airway cells. The DNA methylation status and altered expressions of miRNAs and their target genes were confirmed in 36 pairs of lung tumor and noncancerous lung tissues. Furthermore, aberrant miRNA expressions or DNA methylations alone could be involved in tumorigenesis of LUAD via different pathways. In addition, elevated miR-132-3p expression, reduced expression of its targeted gene (ZEB2), and decreased cell proliferation was observed in lung cancer cells treated with DNA methyltransferase inhibitor. Moreover, in vitro and in vivo analyses showed that miR-132-3p-3p downregulation via DNA methylation promoted tumorigenicity of lung cancer by directly regulating ZEB2.

Conclusions: The interaction between two epigenetic aberrations could have important functions in LUAD. miR-132-3p might act as a tumor suppressor in the tumorigenicity of LUAD.

Key points: SIGNIFICANT FINDINGS OF THE STUDY: Systemically investigating relationship between aberrant miRNA expression and DNA methylation in lung cancer could improve understanding of lung tumorigenesis and develop diagnostic and therapeutic targets.

What this study adds: Three forms of relationships between the two epigenetic changes are defined. miR-132-3p is further identified as a tumor suppressor in lung cancer.

Keywords: DNA methylation; epigenetics; lung cancer; microRNA.

Breast cancer is the most commonly diagnosed cancer among women and one of the leading causes of cancer mortality worldwide, in which the most severe form happens when it metastasizes to other regions of the body. Metastasis is responsible for most treatment failures in advanced breast cancer. Epithelial-mesenchymal transition (EMT) plays a significant role in promoting metastatic processes in breast cancer. MicroRNAs (miRNAs) are highly conserved endogenous short non-coding RNAs that play a role in regulating a broad range of biological processes, including cancer initiation and development, by functioning as tumor promoters or tumor suppressors. Expression of miR-548m has been found in various types of cancers, but the biological function and molecular mechanisms of miR-548m in cancers have not been fully studied. Here, we demonstrated the role of miR-548m in modulating EMT in the breast cancer cell lines MDA-MB-231 and MCF-7. Expression data for primary breast cancer obtained from NCBI GEO datasets showed that miR-548m expression was downregulated in breast cancer patients compared with healthy group. We hypothesize that miR-548m acts as a tumor suppressor in breast cancer. Overexpression of miR-548m in both cell lines increased E-cadherin expression and decreased the EMT-associated transcription factors SNAI1, SNAI2, ZEB1 and ZEB2, as well as MMP9 expression. Consequently, migration and invasion capabilities of both MDA-MB-231 and MCF-7 cells were significantly inhibited in miR-548m-overexpressing cells. Analysis of 1059 putative target genes of miR-548m revealed common pathways involving both tight junction and the mTOR signaling pathway, which has potential impacts on cell migration and invasion. Furthermore, this study identified aryl hydrocarbon receptor (AHR) as a direct target of miR-548m in breast cancer cells. Taken together, our findings suggest a novel function of miR-548m in reversing the EMT of breast cancer by reducing their migratory and invasive potentials, at least in part via targeting AHR expression.

A disintegrin and metalloproteinase 8 (ADAM8) protein is a multi-domain transmembrane glycoprotein which involves in extracellular matrix remodelling, cell adhesion, invasion and migration. ADAM8 and epithelial-mesenchymal transition (EMT) play an important role in tumour invasion has been well established. However, the interaction between ADAM8 and EMT has remained unclear. The data of colon cancer patients obtained from TCGA (The Cancer Genome Atlas) and GTEx (Genotype-Tissue Expression Project) were analysed by the bioinformatics research method. The expression of ADAM8 in colon cancer cells was up-regulated and down-regulated by transfecting with the expression plasmid and small interfering RNA, respectively. Transwell invasion assay, immunohistochemistry, immunocytochemistry, Western blotting and qRT-PCR were utilized to study the effect of ADAM8 on colon cancer cell's EMT and its related mechanisms. Analysis of TCGA and GTEx data revealed that ADAM8 was linked to poor overall survival in colon cancer patients. Besides, ADAM8 was correlated with multiple EMT biomarkers (E-cadherin, N-cadherin, Vimentin, Snail2 and ZEB2). In vitro, we also proved that the up-regulation of ADAM8 could promote EMT effect and enhance the invasive ability of colon cancer cells. On the contrary, the down-regulation of ADAM8 in colon cancer cells attenuated these effects above. Further studies suggested that ADAM8 modulated EMT on colon cancer cells through TGF-β/Smad2/3 signalling pathway. Our research suggested that ADAM8 could be a potential biomarker for the prognosis of colon cancer and induced EMT to promote the invasion of colon cancer cells via activating TGF-β/Smad2/3 signalling pathway.

Keywords: ADAM8; EMT; Smad; TGF-β; colon cancer.

To elucidate the anti-tumor effects and molecular mechanisms of ING5 on glioma cells, we overexpressed it in U87 cells, and examined the phenotypes and their relevant molecules. It was found that ING5 overexpression suppressed proliferation, energy metabolism, migration, invasion, and induced G2/M arrest, apoptosis, dedifferentiation, senescence, mesenchymal- epithelial transition and chemoresistance to cisplatin, MG132, paclitaxel and SAHA in U87 cells. There appeared a lower expression of N-cadherin, Twist, Slug, Zeb1, Zeb2, Snail, Ac-H3, Ac-H4, Cdc2, Cdk4 and XIAP, but a higher expression of Claudin 1, Histones 3 and 4, p21, p53, Bax, β-catenin, PI3K, Akt, and p-Akt in ING5 transfectants. ING5 overexpression suppressed tumor growth of U87 cells in nude mice by inhibiting proliferation and inducing apoptosis. Down-regulated ING5 expression was closely linked to the tumorigenesis and histogenesis of glioma. These data indicated that ING5 expression might be considered as a good marker for the tumorigenesis and histogenesis of gliomas. It might be employed as a potential target for gene therapy of glioma. PI3K/Akt or β-catenin/TCF-4 activation might be positively linked to chemotherapeutic resistance, mediated by ING5.

Studies have found that Lnc LINC00461 is an important regulator of cancer. However, the function of Lnc LINC00461 in NSCLC is not known. Therefore, this experimental design was based on Lnc LINC00461 to explore the pathogenesis of Non-small cell lung cancer (NSCLC). RT-qPCR was used to detect the expression of lnc LINC00461 and miR-30a-5p in NSCLC. The CCK-8 method and Transwell assay were used to detect the effects of lnc LINC00461 and miR-30a-5p on proliferation, migration in NSCLC. Target gene prediction and screening, luciferase reporter assays were used to validate downstream target genes of lnc LINC00461 and miR-30a-5p. The protein expression of ZEB2 was detected by Western blot. The tumor changes in mice were detected by in vivo experiments. Lnc LINC00461 was significantly elevated in NSCLC. Lnc LINC00461 knockdown significantly inhibited proliferation and migration in NSCLC. miR-30a-5p was a direct target of lnc LINC00461 and miR-30a-5p was significantly reduced in NSCLC. shLINC00461 and miR-30a-5p inhibitor partially eliminated the effect of shLINC00461 on cell proliferation. And lnc LINC00461 was negatively correlated with miR-30a-5p expression. ZEB2 was a direct target of miR-30a-5p, and miR-30a-5p mimic and sh lnc LINC00461 significantly reduced ZEB2 expression levels. Finally, In vivo, lnc LINC00461 promoted tumor growth by modulating the miR-30a-5p / ZEB2 axis. In conclusion, LncLINC00461 promoted the progression of NSCLC by the miR-30a-5p / ZEB2 axis, and lnc LINC00461 may be a potential therapeutic target for NSCLC.

Genotypic and phenotypic data of 1,562 animals were analyzed to find genomic regions that potentially influence the birth weight (BW), weaning weight at seven months of age (WW) and yearling weight (YW) of Colombian Brahman cattle, with genotyping conducted using Illumina Bead chip array with 74,669 SNPs. A Single Step Genomic BLUP (ssGBLP), approach was used to estimate the proportion of variance explained by each marker. Multiple regions scattered across the genome were found to influence weights at different ages, also dependent on the trait component (direct or maternal). The most interesting regions were connected to previously identified QTLs and genes, such as ADAMTSL3, CAPN2, CAPN2, FABP6, ZEB2 influencing growth and weight traits. The identified regions will contribute to the development and refinement of genomic selection programs for Zebu Brahman cattle in Colombia.

MIR22HG has a reported involvement in the tumorigenesis of a variety of cancers, including hepatocellular carcinoma (HCC). However, the exact molecular mechanism of MIR22HG in HCC has not been clarified.

In the present study, we integrated data from in-house RT-qPCR, RNA-sequencing, microarray, and literature studies to conduct a comprehensive evaluation of the clinico-pathological and prognostic significance of MIR22HG in an extremely large group of HCC samples. We also explored the potential mechanism of MIR22HG in HCC by analyzing the alteration profiles of MIR22HG in HCC to predict transcription factors (TFs) that may interact with MIR22HG and to annotate the biological functions of genes co-expressed with MIR22HG. MIR22HG expression was also compared in HCC nude mice xenografts before and after a treatment with nitidine chloride.

We found that MIR22HG was downregulated in HCC and that this downregulation correlated with the malignant phenotype of HCC. Comprehensive analysis of the prognostic impact of MIR22HG in HCC revealed a beneficial effect of MIR22HG on the survival outcome of HCC patients. Seven cases of MIR22HG deep deletion occurred in 360 of the cancer genome atlas (TCGA) provisional HCC samples. A total of 22 MIR22HG-TF-mRNA triplets in HCC were predicted by the lncRNAmap. Co-expressed genes of MIR22HG, identified by weighted correlation network analysis (WGCNA), mainly participated in the pathways involving osteoclast differentiation, chemokine signaling pathways, and hematopoietic cell lineage. In vivo experiments demonstrated that nitidine chloride could stimulate MIR22HG expression in HCC xenografts.

In summary, MIR22HG may play a tumor-suppressive role in HCC by coordinating with predicted TFs and co-expressed genes, such as NLRP3, CSF1R, SIGLEC10, and ZEB2, or by being controlled by nitidine chloride.

Introduction: A paucity of literature exists on genotype- phenotype correlates of 'unknown-etiology' infantile-onset developmental-epileptic encephalopathies (DEE) from India. The primary objective was to explore the yield of genetic testing in identifying potential disease causing variants in electro-clinical phenotypes of DEE METHODS: An observational hospital-based study was undertaken on children with unexplained refractory seizure-onset ≤12 months age and developmental delay, whose families consented and underwent genetic testing during a three year time period (2016-2018) by next-generation sequencing (NGS) or multiplex ligand protein amplification. Yield was considered based on demonstration of pathogenic/likely pathogenic variants only and variants of unknown significance (VUS) were documented.

Results: Pathogenic/likely pathogenic variants were identified in 26 (31.7 %) out of 82 children with DEE. These included those variants responsible for primarily DEE- 21(76.7 %); neuro-metabolic disorders- 3(18.6 %) and chromosomal deletions- 2(4.7 %). Of these patients, early-infantile epilepsy onset ≤ 6 months age was noted in 22(84.6 %). The DEE studied included Ohtahara syndrome associated with STXBP1 and SCN8A variants with yield of 50 % (2/4 tested); early myoclonic encephalopathy (no yield in 2); West syndrome with CDKL5, yield of 13.3 % (2/15 tested); epilepsy of infancy with migrating partial seizures due to CACNA1A and KCNT1 variants, yield of 67 % (2/3 tested); DEE-unclassified with KCNQ2, AP3B2, ZEB2, metabolic variants (SUOX, ALDH7A1, GLDC) and chromosome deletions (chr 1p36, chr2q24.3); yield of 32 % (8/25 tested). Patients with Dravet syndrome/Dravet-like phenotypes (N = 33) had variants in SCN1A (N = 10), SCN1B, CHD2; yield of 36.4 % (12/33 tested; 57.1 % from NGS). Eighteen patients with potential variants (SCN1A, SCN2A, SCN8A, KCNQ2, ALDH7A1 which also included VUS) could be offered targeted therapy.

Conclusions: Our study confirms a good yield of genetic testing in neonatal and infantile-onset DEE provided robust phenotyping of infants is attempted with prognostic and therapeutic implications, particularly relevant to centres with resource constraints.

Keywords: Developmental & epileptic encephalopathy; Electro-clinical syndromes; Genetics; Next generation sequencing; Variants.

Targeting self-renewal and tumorigenicity has been proposed as a potential strategy against cancer stem cells (CSCs). Epigenetic proteins are key modulators of gene expression and cancer development contributing to regulation and maintenance of self-renewal and tumorigenicity. Here, we have screened a small-molecule epigenetic inhibitor library using 3D in vitro models in order to determine potential epigenetic targets associated with self-renewal and tumorigenicity in Canine Mammary Cancer (CMC) cells. We identified inhibition of BET proteins as a promising strategy to inhibit CMC colonies and tumorspheres formation. Low doses of (+)-JQ1 were able to downregulate important genes associated to self-renewal pathways such as WNT, NOTCH, Hedgehog, PI3K/AKT/mTOR, EGF receptor and FGF receptor in CMC tumorspheres. In addition, we observed downregulation of ZEB2, a transcription factor important for the maintenance of self-renewal in canine mammary cancer cells. Furthermore, low doses of (+)-JQ1 were not cytotoxic in CMC cells cultured in 2D in vitro models but induced G2/M cell cycle arrest accompanied by upregulation of G2/M checkpoint-associated genes including BTG2 and CCNG2. Our work indicates the BET inhibition as a new strategy for canine mammary cancers by modulating the self-renewal phenotype in tumorigenic cells such as CSCs.

Neurodevelopmental disorders (NDDs) are clinically and genetically extremely heterogeneous with shared phenotypes often associated with genes from the same networks. Mutations in TCF4, MEF2C, UBE3A, ZEB2 or ATRX cause phenotypically overlapping, syndromic forms of NDDs with severe intellectual disability, epilepsy and microcephaly. To characterize potential functional links between these genes/proteins, we screened for genetic interactions in Drosophila melanogaster. We induced ubiquitous or tissue specific knockdown or overexpression of each single orthologous gene (Da, Mef2, Ube3a, Zfh1, XNP) and in pairwise combinations. Subsequently, we assessed parameters such as lethality, wing and eye morphology, neuromuscular junction morphology, bang sensitivity and climbing behaviour in comparison between single and pairwise dosage manipulations. We found most stringent evidence for genetic interaction between Ube3a and Mef2 as simultaneous dosage manipulation in different tissues including glia, wing and eye resulted in multiple phenotype modifications. We subsequently found evidence for physical interaction between UBE3A and MEF2C also in human cells. Systematic pairwise assessment of the Drosophila orthologues of five genes implicated in clinically overlapping, severe NDDs and subsequent confirmation in a human cell line revealed interactions between UBE3A/Ube3a and MEF2C/Mef2, thus contributing to the characterization of the underlying molecular commonalities.

Gastric-type mucinous carcinoma (GAS) is a recently established variant of endocervical mucinous adenocarcinoma that is characterized as being unrelated to HPV and having aggressive behavior and chemoresistance. GAS has a distinct morphology resembling nonneoplastic gastric glands or pancreaticobiliary adenocarcinoma, and their possible genetic similarity has been posed. In this study, next-generation sequencing was performed in 21 GAS cases using a customized panel including 94 cancer-associated genes. A total of 54 nonsynonymous somatic mutations were detected with an average mutation rate of 2.6 per lesion (range: 0-9). The most frequently mutated gene was TP53 (11/21, 52.4%), followed by STK11, HLA-B, PTPRS (4/21, 19.0%), FGFR4 (3/21, 14.3%), GNAS, BRCA2, ELF3, ERBB3, KMT2D, SLX4 (2/21, 9.5%), CDH1, EPCAM, KRAS, MLH1, RNF43, SNAI1, TWIST1, ZEB1, ZEB2, and so on (1/21, 4.8%). The mutated genes were mostly involved in signal transduction, DNA damage repair, and epithelial-mesenchymal transition (EMT). Correlation of TP53 mutation and p53 protein expression demonstrated that 31.3% with abnormal p53 expression harbored wild-type TP53. Compared to genetic features of gastric and pancreaticobiliary adenocarcinoma, TP53 mutations were frequent in both GAS and gastrointestinal adenocarcinoma. While KMT2D, ERBB3, and RNF43 mutations were shared between GAS and gastric adenocarcinoma, highly mutated genes in pancreatic ductal adenocarcinoma such as KRAS, SMAD4, and CDKN2A were rarely mutated in GAS. Of frequently mutated genes in cholangiocarcinoma, BAP1 and HLA-B were identified in GAS. Frequent EMT-related gene mutations suggested a possible role of EMT-related pathways in tumor dissemination and chemoresistance of GAS. In addition, GAS shared some genetic features with gastrointestinal adenocarcinoma. These findings provide a clue in understanding the biological basis of GAS.

GATA3 is a transcriptional factor involved in the development of multiple organs. Post translational modifications of GATA3 are critical to its function. Here, we report that GATA3 interacts with and is acetylated by the acetyltransferase CBP. Class I deacetylases HDAC1, HDAC2 and HDAC3 deacetylate GATA3. The major acetylated site of GATA3 in lung adenocarcinoma cells was determined at lysine 119 (AcK119). Functionally, GATA3-acetylation mimics K119Q mutant was found to inhibit lung adenocarcinoma cell migration and invasion with concomitant downregulation of EMT-controlling transcriptional factors Slug, Zeb1 and Zeb2. Taken together, we demonstrated that GATA3 acetylation at lysine 119 by CBP hinders the migration and invasion of lung adenocarcinoma cells.