Homozygous Missense Variants in NTNG2, Encoding a Presynaptic Netrin-G2 Adhesion Protein, Lead to a Distinct Neurodevelopmental Disorder.

NTNG2 encodes netrin-G2, a membrane-anchored protein implicated in the molecular organization of neuronal circuitry and synaptic organization and diversification in vertebrates. In this study, through a combination of exome sequencing and autozygosity mapping, we have identified 16 individuals (from seven unrelated families) with ultra-rare homozygous missense variants in NTNG2; these individuals present with shared features of a neurodevelopmental disorder consisting of global developmental delay, severe to profound intellectual disability, muscle weakness and abnormal tone, autistic features, behavioral abnormalities, and variable dysmorphisms. The variants disrupt highly conserved residues across the protein. Functional experiments, including in silico analysis of the protein structure, in vitro assessment of cell surface expression, and in vitro knockdown, revealed potential mechanisms of pathogenicity of the variants, including loss of protein function and decreased neurite outgrowth. Our data indicate that appropriate expression of NTNG2 plays an important role in neurotypical development.

Polycyclic aromatic hydrocarbons exposures and telomere length: A cross-sectional study on preschool children.

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with shorter telomere length (TL), a marker of ageing at cellular level. However, the available evidence on this association among children is still scarce. We therefore aimed to assess, the relationship between urinary 1-hydroxipayrene (1-OHP), a marker of exposure to PAHs, and relative leukocyte TL (LTL) in children at preschool age. Our study was based on 200 children enrolled from 27 randomly-selected kindergartens in the city of Sabzevar, Iran (2017). 1-OHP levels in the participants' urine samples were measured using solid phase extraction (SPE) method and high-performance liquid chromatography (HPLC). Moreover, real-time PCR was used to measure the LTL in the participants' blood samples. Linear mixed effects models, controlled for relevant covariates, were applied to investigate the association of 1-OHP concentration and LTL. The median (interquartile range (IQR)) of relative LTL and urinary 1-OHP were 0.83 (0.7) and 257 (375.5) ng/L, respectively. In the fully adjusted model, an IQR increase in urinary 1-OHP was related to -0.05 (95% confidence interval (CI): 0.09, -0.01, P-value = 0.02) decrease in relative LTL. This association was similar among boys and girls; however, we observed indications for a stronger association for those children whose parents had university education. Our study suggested an inverse relationship between urinary 1-OHP and LTL in children at preschool age. However, further longitudinal research with repeated measures of PAHs and LTL are needed to confirm these findings.

The testicular protective effects of standardised hydroalcoholic extract of Ziziphus jujuba Mill against adriamycin-induced toxicity.

In this study, because of the anti-inflammatory and antioxidant effect of the Ziziphus jujuba (ZJ), we assessed the protective properties of the ZJ extract against testis toxicity caused by Adriamycin in the rat. Twenty rats were grouped into (a) control, (b) Adriamycin, (c) ZJ group and (d) treatment group in which Adriamycin was administrated and the ZJ hydroalcoholic extract was used for three weeks. On the 21st day, two testes were removed to determine the oxidation markers and pathological evaluation. The levels of sex hormones were determined. Epididymis also was crushed, and its spermatozoa were evaluated as concentration, motility and normality. Adriamycin increased oxidative stress markers as well as Luteinising hormone, and follicle-stimulating hormone and decreased testosterone levels compared to control. In the treated group, the levels of the above markers improved. The decreased number and motility of spermatozoa in treatment group increased, and the increased rate of abnormal spermatozoa in this group decreased. Pathological evaluations also show the healing process of damaged testicular tissue in the group receiving the ZJ extract. The ZJ extract relatively improves oxidative stress, sperm characteristics, hormonal alternation and pathological changes. These findings reveal the probable role of ZJ effective compounds in repairing tissue damage.

Dietary pattern and telomere length in preschool children in a middle-income country.

Telomere length (TL) has been associated with lifestyle and dietary pattern. However, the available evidence on this association in children is scarce, especially in low- and middle-income countries (LMICs). Therefore, this study aimed to evaluate the association of dietary pattern and leukocyte TL (LTL) in preschool children, Sabzevar, Iran (2017). This cross-sectional study was based on 187 preschool children (aged 5 to 7) recruited from 27 kindergartens. Nutrition information including amounts of consumed dairy products, meat and processed meat products, nuts and seeds, white bread and refined grains, fruits, vegetables, simple sugars, fats and drinks was obtained through a questionnaire. Linear mixed-effects models were fitted with polymerase chain reaction (PCR) plate ID and kindergartens as random effects to estimate the association of each food group consumption with LTL, controlled for relevant covariates. Higher consumption of dairy products and sugar was associated with shorter LTL (ß = -0.180, 95% confidence interval [CI]: -0.276, -0.085, P value <0.001 and ß = -0.139, 95% CI: -0.193, -0.086, P value <0.001, respectively). An increase in consumption of fish, nuts and seeds, coloured fruits, green leafy vegetables, cruciferous vegetables and olive was significantly associated with the increase in relative LTL. The associations for the consumption of legumes, other fruits, yellow and orange vegetables, red meat, egg, white bread and refined grains, solid and liquid fats, processed meats, potato chips, carbonated drinks, tea (black) and soft drinks groups were not statistically significant. Our findings showed that there was an association between the consumption of certain food groups with LTL.

MEIS1 promotes expression of stem cell markers in esophageal squamous cell carcinoma.

BACKGROUND: MEIS1 (Myeloid ecotropic viral integration site 1) as a homeobox (HOX) transcription factor plays regulatory roles in a variety of cellular processes including development, differentiation, survival, apoptosis and hematopoiesis, as well as stem cell regulation. Few studies have established pluripotency and self-renewal regulatory roles for MEIS1 in human esophageal squamous cell carcinoma (ESCC), and our aim in this study was to evaluate the functional correlation between MEIS1 and the stemness markers in ESCC patients and cell line KYSE-30. METHODS: Expression pattern of MEIS1 and SALL4 gene expression was analyzed in different pathological features of ESCC patients. shRNA in retroviral vector was used for constantly silencing of MEIS1 mRNA in ESCC line (KYSE-30). Knockdown of MEIS1 gene and the expression pattern of selected stemness markers including SALL4, OCT4, BMI-1, HIWI, NANOG, PLK1, and KLF4 were evaluated using real-time PCR. RESULTS: Significant correlations were observed between MEIS1 and stemness marker SALL4 in different early pathological features of ESCC including non-invaded tumors, and the tumors with primary stages of progression. Retroviral knockdown of MEIS1 in KYSE-30 cells caused a noteworthy underexpression of both MEIS1 and major involved markers in stemness state of the cells including SALL4, OCT4, BMI-1, HIWI and KLF4. CONCLUSIONS: The results highlight the important potential role of MEIS1 in modulating stemness properties of ESCCs and cells KYSE-30. These findings may confirm the linkage between MEIS1 and self-renewal capacity in ESCC and support probable oncogenic role for MEIS1 in the disease.

Maternal exposure to ambient particulate matter and green spaces and fetal renal function.

Exposure to air pollution has been associated with different harmful effects and exposure to greenspace has been related to improved human health. However, the available evidence on the impact of these exposures on renal function is still scarce. The aim of this study was to determine the relationship between exposure to ambient levels of PM1, PM2.5, PM10 and indicators of exposure to traffic as well as greenspace during pregnancy and fetal renal function based on the umbilical cord blood. This study was based on 150 pregnant women residing in Sabzevar, Iran (2018). Multiple linear regression models were developed to estimate the association of glomerular filtration rate (GFR), creatinine (Cr) and blood urea nitrogen (BUN) with exposure to air pollution, traffic, and greenspace (one at a time) controlled for relevant covariates. There was an inverse significant association between exposure to PM1, PM2.5, PM10 and total street length in a 100 m buffer around the home and eGFR. Increase in distance to major road and residential surrounding greenness (100 m buffer) was associated with increase in eGFR. We observed a significant direct association between exposure to PMs as well as street length in 100 m buffer and serum level of Cr. There was also an inverse association between distance to major road and NDVI in 100 m buffer and Cr. The associations for blood urea nitrogen (BUN) were not statistically significant. Our results suggest that exposure to air pollution during pregnancy could have negative impact and exposure to greenspace could have positive impact on renal function of fetal.

MAB21L1 loss of function causes a syndromic neurodevelopmental disorder with distinctive cerebellar, ocular, craniofacial and genital features (COFG syndrome).

BACKGROUND: Putative nucleotidyltransferase MAB21L1 is a member of an evolutionarily well-conserved family of the male abnormal 21 (MAB21)-like proteins. Little is known about the biochemical function of the protein; however, prior studies have shown essential roles for several aspects of embryonic development including the eye, midbrain, neural tube and reproductive organs. OBJECTIVE: A homozygous truncating variant in MAB21L1 has recently been described in a male affected by intellectual disability, scrotal agenesis, ophthalmological anomalies, cerebellar hypoplasia and facial dysmorphism. We employed a combination of exome sequencing and homozygosity mapping to identify the underlying genetic cause in subjects with similar phenotypic features descending from five unrelated consanguineous families. RESULTS: We identified four homozygous MAB21L1 loss of function variants (p.Glu281fs*20, p.Arg287Glufs*14 p.Tyr280* and p.Ser93Serfs*48) and one missense variant (p.Gln233Pro) in 10 affected individuals from 5 consanguineous families with a distinctive autosomal recessive neurodevelopmental syndrome. Cardinal features of this syndrome include a characteristic facial gestalt, corneal dystrophy, hairy nipples, underdeveloped labioscrotal folds and scrotum/scrotal agenesis as well as cerebellar hypoplasia with ataxia and variable microcephaly. CONCLUSION: This report defines an ultrarare but clinically recognisable Cerebello-Oculo-Facio-Genital syndrome associated with recessive MAB21L1 variants. Additionally, our findings further support the critical role of MAB21L1 in cerebellum, lens, genitalia and as craniofacial morphogenesis.

Biallelic loss of function variants in PPP1R21 cause a neurodevelopmental syndrome with impaired endocytic function.

Next-generation sequencing (NGS) has been instrumental in solving the genetic basis of rare inherited diseases, especially neurodevelopmental syndromes. However, functional workup is essential for precise phenotype definition and to understand the underlying disease mechanisms. Using whole exome (WES) and whole genome sequencing (WGS) in four independent families with hypotonia, neurodevelopmental delay, facial dysmorphism, loss of white matter, and thinning of the corpus callosum, we identified four previously unreported homozygous truncating PPP1R21 alleles: c.347delT p.(Ile116Lysfs*25), c.2170_2171insGGTA p.(Ile724Argfs*8), c.1607dupT p.(Leu536Phefs*7), c.2063delA p.(Lys688Serfs*26) and found that PPP1R21 was absent in fibroblasts of an affected individual, supporting the allele's loss of function effect. PPP1R21 function had not been studied except that a large scale affinity proteomics approach suggested an interaction with PIBF1 defective in Joubert syndrome. Our co-immunoprecipitation studies did not confirm this but in contrast defined the localization of PPP1R21 to the early endosome. Consistent with the subcellular expression pattern and the clinical phenotype exhibiting features of storage diseases, we found patient fibroblasts exhibited a delay in clearance of transferrin-488 while uptake was normal. In summary, we delineate a novel neurodevelopmental syndrome caused by biallelic PPP1R21 loss of function variants, and suggest a role of PPP1R21 within the endosomal sorting process or endosome maturation pathway.

Signaling roadmap to epithelial-mesenchymal transition in pterygium, TWIST1 centralized.

Pterygium as a complex disease shares common features with other malignant cells in its onset recurrence and especially epithelial-mesenchymal transition (EMT) transition. Although using different approaches including conjunctival autografts, amniotic membrane, radiotherapy, mitomycin C (MMC) has shown promising insights in the inhibition of pterygium recurrence, it needs to be investigated in more details in molecular pathways to present adjuvant target therapy. In this study, we aimed to evaluate the expression of and then illustrate the role of signaling pathways on EMT in pterygium. Using real-time polymerase chain reaction, the twist-related protein 1 (TWIST1) expression was compared in primary pterygium and normal conjunctiva. This study assessed the mRNA expression, as well as the association between the clinicopathological indices and the gene expression level. The expression level of TWIST1 was overexpressed in 36% of our cohort ( n = 76). There was a significant positive correlation between recurrence with grade T, grade V and a significant negative correlation with growth activity. Our vast literature review on different signaling pathways in pterygium showed that EMT has centralization role in recurrence of this disease. Our data confirmed that EMT is important in the recurrence of pterygium samples and different signaling pathways end up activating the EMT markers. It is suggested to evaluate the environmental factors and their correlation with molecular markers to select favorable treatment for this kind of diseases.

Effects of glucosamine against morphine-induced antinociceptive tolerance and dependence in mice.

BACKGROUND: The most important limitations of morphine in pain therapy are its tolerance and dependence. In this study, we evaluated the protective effect of glucosamine against morphine-induced tolerance and dependence in mice. METHODS: Mice received twice daily morphine (20 mg/kg, s.c.) alone, or along with orally administered glucosamine (500, 1000 and 2000 mg/kg), for 9 continuous days. To assess antinociceptive effect of morphine, percentage of maximal possible effect (%MPE) of animals exposed to thermal stimulus was measured in the hot plate test, 30 min after morphine administration. Test was performed on days 1, 3, 5, 7 and 9. The effect of glucosamine on the naloxone (5 mg/kg, i.p.)-precipitated morphine withdrawal, was also evaluated. Changes in brain gene expression levels of induced nitric oxide synthase (iNOS), enzyme responsible for nitric oxide generation, as well as pro-inflammatory mediator, tumor necrosis alpha (TNF-α) were measured in morphine tolerated animals, as well as after withdrawal by real-time polymerase chain reaction (RT-PCR). Protein content of TNF-α was evaluated via ELISA assay. RESULTS: Tolerance to antinociceptive effect of morphine was developed after 7 days of morphine treatment. The concurrent administration of glucosamine (500, 1000 and 2000 mg/kg) with morphine, significantly inhibited tolerance development, on days 7 and 9. In addition, glucosamine ameliorated the naloxone-precipitated opioid withdrawal symptoms (tremor, jumping, teeth chattering, grooming). However, diarrhea was significantly improved only with the dose of 500 mg/kg. Increased mRNA expression of iNOS as well as TNF-α mRNA expression and protein, after both morphine tolerance and withdrawal, were considerably reduced by glucosamine (1000 mg/kg) in the morphine withdrawal animals. CONCLUSION: These data support the utility of glucosamine in attenuating both tolerance to nociceptive effects of morphine as well as withdrawal-induced behavioral profile. Anti-oxidant and anti-inflammatory effects are responsible, at least in part, for the protective effects of this drug.

Thymoquinone alleviates renal interstitial fibrosis and kidney dysfunction in rats with unilateral ureteral obstruction.

Unilateral ureteral obstruction (UUO) causes severe renal tubulointerstitial fibrosis. Because of many pharmacologic properties of thymoquinone (TQ), in this study, the effects of TQ against kidney fibrosis and dysfunction were investigated in rats with UUO. Forty male Wistar rats were divided into five groups: Sham operated, UUO, and the animals with UUO treated with losartan, captopril, or TQ. Collagen IV and transforming growth factor (TGF)-ß1 expressions, interstitial fibrosis, histological changes, and kidney function were assessed. UUO markedly increased renal expression of TGF-ß1 and collagen I and induced interstitial fibrosis (p < .001). Losartan, captopril, or TQ significantly downregulated the expression of these fibrotic markers and interstitial fibrosis (p < .01-p < .001). In UUO group, serum levels of urea and creatinine and protein excretion rate significantly increased, but glomerular filtration rate (GFR) and urine osmolarity showed a significant decrease (p < .001-p < .05). Administration of captopril and TQ caused no significant change in serum urea and protein excretion rate. Unlike losartan and captopril, TQ caused no significant alteration in GFR compared with Day 1. Losartan caused significant increases in serum urea and creatinine but significant decrease in urine osmolarity. TQ could be regarded as a potent therapeutic agent for treatment of UUO-induced kidney fibrosis and dysfunction.

Neuroprotective effects of clavulanic acid following permanent bilateral common carotid artery occlusion in rats.

We investigated whether clavulanic acid could improve learning and memory, in rats underwent bilateral occlusion of common carotid artery (2VO). Seventy male Wistar rats were subjected to 2VO, with a 1-week interval between right and left artery occlusions. After 2VO, animals received clavulanic acid (10, 20, 40 mg/kg, intraperitoneally), from day 8 to 20. Spatial memory was assessed in the Morris water maze, 1 week after the induction of 2VO (day 15). The mRNA expression levels of bcl-2, bcl2-associated x protein (bax), caspase-3, inducible nitric oxide synthase (iNOS), and amyloid beta precursor protein (APP) were measured in the neocortex and hippocampus. Clavulanic acid significantly decreased the escape latency and swimming time in the training trial days. As well, it increased time and distance percentage in the target quadrant, while it decreased such factors in the opposite quadrant in the final trial day, compared to 2VO + normal saline animals. Real time-PCR data showed a significant higher mRNA expression of bax, caspase 3, and iNOS in the hippocampus and neocortex of 2VO animal compared to nonoccluded rats. APP increased in the neocortex but not hippocampus. Compared with 2VO animals, clavulanic acid significantly down-regulated the expression of iNOS, caspase 3, and APP, accompanied by diminishing the bax/bcl2 ratio. Our results reveal a potential therapeutic use of clavulanic acid for cognitive dysfunction associated with cerebral hypoperfusion in vascular dementia and Alzheimer disease.

Expression of Pluripotency Markers, SOX2 and OCT4, in Pterygium Development.

Pterygium is a common ocular surface disease characterized by the abnormal epithelial proliferation, matrix remodeling, vascularization and the migration of the lesion. Although the etiology of pterygium is elusive, recent studies have focused on the role of limbal stem cells (LSCs) damage and effects of UVB. This study aimed to determine the expression levels of pluripotent markers of SOX2 and OCT4 in primary pterygium and normal conjunctiva. Using real time polymerase chain reaction (PCR), the SOX2 and OCT4 expressions were compared in primary pterygium and normal conjunctiva. This study assessed the correlation between SOX2 mRNA expression and OCT4 mRNA expression, as well as the association between the clinicopathological indices and both gene expression levels. The relative mRNA expression levels of OCT4 genes in primary pterygium were significantly reduced compared to the normal conjunctiva tissues. The association between OCT4 gene expression and the clinicopathological indices reported significant laterality (P = .004) and marginal growth activity indices (P = .063). The univariate correlation between the SOX2 and OCT4 expressions was statistically significant (P = .001). The present study emphasized the downregulation of pluripotent marker OCT4 genes in the pterygium. It is speculated that these results may predict a new avenue for exploring the role of stem cell deficiency in the development of pterygium.

MPZL2 is a novel gene associated with autosomal recessive nonsyndromic moderate hearing loss.

While recent studies have revealed a substantial portion of the genes underlying human hearing loss, the extensive genetic landscape has not been completely explored. Here, we report a loss-of-function variant (c.72delA) in MPZL2 in three unrelated multiplex families from Turkey and Iran with autosomal recessive nonsyndromic hearing loss. The variant co-segregates with moderate sensorineural hearing loss in all three families. We show a shared haplotype flanking the variant in our families implicating a single founder. While rare in other populations, the allele frequency of the variant is ~ 0.004 in Ashkenazi Jews, suggesting that it may be an important cause of moderate hearing loss in that population. We show that Mpzl2 is expressed in mouse inner ear, and the protein localizes in the auditory inner and outer hair cells, with an asymmetric subcellular localization. We thus present MPZL2 as a novel gene associated with sensorineural hearing loss.

Expanding the clinical phenotype of IARS2-related mitochondrial disease.

BACKGROUND: IARS2 encodes a mitochondrial isoleucyl-tRNA synthetase, a highly conserved nuclear-encoded enzyme required for the charging of tRNAs with their cognate amino acid for translation. Recently, pathogenic IARS2 variants have been identified in a number of patients presenting broad clinical phenotypes with autosomal recessive inheritance. These phenotypes range from Leigh and West syndrome to a new syndrome abbreviated CAGSSS that is characterised by cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysplasia, as well as cataract with no additional anomalies. METHODS: Genomic DNA from Iranian probands from two families with consanguineous parental background and overlapping CAGSSS features were subjected to exome sequencing and bioinformatics analysis. RESULTS: Exome sequencing and data analysis revealed a novel homozygous missense variant (c.2625C > T, p.Pro909Ser, NM_018060.3) within a 14.3 Mb run of homozygosity in proband 1 and a novel homozygous missense variant (c.2282A > G, p.His761Arg) residing in an ~ 8 Mb region of homozygosity in a proband of the second family. Patient-derived fibroblasts from proband 1 showed normal respiratory chain enzyme activity, as well as unchanged oxidative phosphorylation protein subunits and IARS2 levels. Homology modelling of the known and novel amino acid residue substitutions in IARS2 provided insight into the possible consequence of these variants on function and structure of the protein. CONCLUSIONS: This study further expands the phenotypic spectrum of IARS2 pathogenic variants to include two patients (patients 2 and 3) with cataract and skeletal dysplasia and no other features of CAGSSS to the possible presentation of the defects in IARS2. Additionally, this study suggests that adult patients with CAGSSS may manifest central adrenal insufficiency and type II esophageal achalasia and proposes that a variable sensorineural hearing loss onset, proportionate short stature, polyneuropathy, and mild dysmorphic features are possible, as seen in patient 1. Our findings support that even though biallelic IARS2 pathogenic variants can result in a distinctive, clinically recognisable phenotype in humans, it can also show a wide range of clinical presentation from severe pediatric neurological disorders of Leigh and West syndrome to both non-syndromic cataract and cataract accompanied by skeletal dysplasia.

Correction to: Metoclopramide role in preventing ileus after cesarean, a clinical trial.

The original publication of this paper contains an error.

Mortality and morbidity due to exposure to ambient particulate matter.

The aim of this study was to investigate spatial variation and health risk of the exposure to PM2.5 (particulate matter with a diameter of 2.5 µm or less) and PM10 (particulate matter with a diameter of 10 µm or less) in Sabzevar, Iran. PM2.5 and PM10 were measured during three campaigns from April to November 2017, in 26 sampling points. Spatial analysis was performed using kriging and autocorrelations (Moran's index) model in Arc GIS software. Relationship between exposure to the PM2.5 and PM10 and their health impacts were investigated by AirQ 2.2.3 software. The mean concentrations (and standard deviation) of PM 2.5 and PM10 over the entire study period were 32.54 (37.28) and 42.61 (47.76) µg/m3, respectively, which were higher than the guideline of World Health Organization. According to the spatial analysis, the maximum concentrations of PM2.5 and PM10 were around the main highway (beltway) which placed all over the south of Sabzevar. According to the Moran's index, the emission patterns for PM2.5 (Z-score = 2.53; P-value = 0.011) and PM10 (Z-score = 2.82; P-value = 0.004) were clustered. The attributable percentage (AP) of total mortality related to PM2.5 and PM10 were 3.544% (95% confidence interval (CI): 2.623-4.447%) and 2.055% (95% CI: 1.379-2.721%) per increasing each 10 µg/m3 of these pollutants, respectively. According to observed results, it is suggested that the beltway and other pollution sources, such as industries, should be placed at a greater distance from the city, to reduce PM amounts in residential areas.

Nigella sativa extract is a potent therapeutic agent for renal inflammation, apoptosis, and oxidative stress in a rat model of unilateral ureteral obstruction.

Unilateral ureteral obstruction (UUO) is a well-established experimental model to evaluate renal interstitial fibrosis. Current study is aimed to investigate the effects of Nigella sativa (NS) extract and renin-angiotensin system (RAS) blockade against kidney damage following UUO in rats. In this study, the rats received intraperitoneal injection of losartan (15 mg/kg), captopril (30 mg/kg), and two doses of NS extract (200 and 400 mg/kg) for 18 consecutive days. At the fourth day of the experiment, laparotomy was performed, and the left ureter was ligated. Sham-operated animals received saline as vehicle, and laparotomy without ureteral ligation was done. UUO was associated with significant increase in the expression of renal angiotensin II and monocyte chemoattractant protein-1, concentration of malondialdehyde and tumor necrosis factor-α, and the number of apoptotic cells when compared with sham group. Renal total thiol content and the activity of antioxidant enzymes were significantly reduced as compared with the sham group. However, treatment of obstructed rats with losartan, captopril, and NS extract significantly improved these renal impairments when compared with UUO group. Thus, NS extract, a potent antioxidant and anti-inflammatory herb, is a therapeutic agent to treat the UUO-induced kidney damage comparable with the well-known RAS inhibitors captopril and losartan.

TWIST1 upregulates the MAGEA4 oncogene.

Overexpression of MAGEA4 oncogene has been demonstrated in different malignancies; however, little is known about its exact mechanism for overexpression. TWIST1, as a bHLH transcription factor, activates a cell migration-invasion program involved in both embryonic and tumor development. Since MAGEA4 overexpression was statistically correlated to TWIST1, we aimed to elucidate the probable regulatory role of TWIST1 on MAGEA4 expression in KYSE30 cells. METHODS: Expression pattern of MAGEA4 and TWIST1 was analyzed in 55 ESCC patients using relative comparative real-time PCR. In silico analysis of the MAGEA4 gene was performed. Methylation status of MAGEA4 promoter was determined by quantitative methylation specific PCR (qMSP). Using a retroviral system, KYSE30 cells were transduced to ectopically express TWIST1, followed by qRT-PCR, Western blot analysis, chromatin immunoprecipitation (ChIP), and luciferase assays to elucidate the regulatory role of TWIST1 on MAGEA4 gene expression. RESULTS: Concomitant overexpression of MAGEA4 and TWIST1 was detected in ESCC in significant correlation with each other in different clinicopathological indices of poor prognosis (P < 0.05). The TWIST1-expressing cells showed significantly higher MAGEA4 expression compared to control cells. ChIP and luciferase assays results confirmed indirect binding of TWIST1 to the E-boxes of MAGEA4 promoter sequence and revealed a novel regulatory role of TWIST1 in MAGEA4 upregulation. CONCLUSION: Since MAGEA4 is a highly expressed oncogene in a variety of malignancies in significant correlation with tumor cell invasiveness and aggressiveness, our finding may help understand one regulatory mechanism of increased expression in tumor cells. © 2016 Wiley Periodicals, Inc.

Predicting the molecular role of MEIS1 in esophageal squamous cell carcinoma.

The three amino acid loop extension (TALE) class myeloid ecotropic viral integration site 1 (MEIS1) homeobox gene is known to play a crucial role in normal and tumor development. In contrast with its well-described cancer stemness properties in hematopoietic cancers, little is known about its role in solid tumors like esophageal squamous cell carcinoma (ESCC). Here, we analyzed MEIS1 expression and its clinical relevance in ESCC patients and also investigated its correlation with the SOX2 self-renewal master transcription factor in the ESCC samples and in the KYSE-30 ESCC cell line. MEIS1 mRNA and protein expression were significantly decreased in ESCC disease (P < 0.05). The inverse correlation between MEIS1 mRNA expression and tumor cell metastasis to the lymph nodes (P = 0.004) was significant. Also, MEIS1 protein levels inversely correlated to lymph node involvement (P = 0.048) and high tumor stage (stages III/IV, P = 0.030). The low levels of DNA methylation in the MEIS1 promoter showed that this suppression does not depend on methylation. We showed that downregulation of EZH2 restored MEIS1 expression significantly. Also, we investigated that MEIS1 downregulation is concomitant with increased SOX2 expression. To the best of our knowledge, this is the first report on the MEIS1 gene in ESCC. The inverse correlation of MEIS1 with metastasis, tumor staging, and the role of EZH2 in methylation, together with its correlation with stemness factor SOX2 expression, led us to predict cancer stemness properties for MEIS1 in ESCC.