SRY Gene Research Articles

Betavulgarin Isolated from Sugar Beet (Beta vulgaris) Suppresses Breast Cancer Stem Cells through Stat3 Signaling.

Breast cancer is a major health problem that affects lives worldwide. Breast cancer stem cells (BCSCs) are small subpopulations of cells with capacities for drug resistance, self-renewal, recurrence, metastasis, and differentiation. Herein, powder extracts of beetroot were subjected to silica gel, gel filtration, thin layer chromatography (TLC), and preparatory high-pressure liquid chromatography (HPLC) for isolation of one compound, based on activity-guided purification using tumorsphere formation assays. The purified compound was identified as betavulgarin, using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI) mass spectrometry. Betavulgarin suppressed the proliferation, migration, colony formation, and mammosphere formation of breast cancer cells and reduced the size of the CD44+/CD24− subpopulation and the expression of the self-renewal-related genes, C-Myc, Nanog, and Oct4. This compound decreased the total level and phosphorylated nuclear level of signal transducer and activator of transcription 3 (Stat3) and reduced the mRNA and protein levels of sex determining region Y (SRY)-box 2 (SOX2), in mammospheres. These data suggest that betavulgarin inhibit the Stat3/Sox2 signaling pathway and induces BCSC death, indicating betavulgarin might be an anticancer agent against breast cancer cells and BCSCs.

Chlorpyrifos-induced toxicity has no gender selectivity in the early fetal brain

Organophosphorus pesticides induce gender-specific developmental neurotoxicity after birth, especially in adolescents and adults. However, whether and when the selectivity occurs in fetus remains unclear. In this study, we analyzed chlorpyrifos (CPF)-induced neurotoxicity in the early fetal brains of male and female mice. The gestational dams were administered 0, 1, 3, and 5 mg/(kg.d) CPF during gestational days (GD)7–11, and brains from the fetuses were isolated and analyzed on GD12. Fetal gender was identified by PCR technique based on male-specific Sry gene and Myog control gene. The body weight and head weight, the activity of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and the content of malondialdehyde (MDA), as well as the oxidative stress-related gene expression were examined. Our results showed that CPF pretreatment induced AChE inhibition in GD12 fetal brain. CPF treatment activated SOD and GPX but not CAT and MDA. For oxidative stress-related gene expression, CPF pretreatment increased mRNA expression of Sod1, Cat, Gpx1, and Gpx2 in the fetal brain on GD12. The statistical analysis did not show gender-selective CPF-induced toxicity. Moreover, our results showed that although the gestational exposure to CPF could elicit abnormalities in the early fetal brain, the toxicity observed was not gender-specific.

Primary amenorrhea with Swyer syndrome: a rare case report

Swyer syndrome with complete gonadal dysgenesis is associated with an absence of testicular differentiation in a phenotypic female with a 46, XY karyotype. The diagnosis is usually made at adolescence when primary amenorrhea is investigated. Here is an interesting case report of 18-year-old unmarried girl, who presented with primary amenorrhea and non-development of breasts. Her body built was musculin with broad shoulders, prominent Adam’s apple and deep voice. Examination of her secondary sexual charaistisctcs revealed no breast development, absent axillary hair and sparse pubic hairs with female type of external genitalia. Laboratoryanalyses revealed serum follicle-stimulating hormone and luteinizing hormone levels compatible with hypergonodotropichypogonadism. Pelvicultrasonography showed an infantile uterus and streak gonads. Chromosome analysis revealed 46, XYkaryotype. Laparoscopic removal of streak gonads was done as there is a risk of gonadoblastoma in such cases. The patient was started on hormonal replacement therapy. Swyersyndrome results mainly due to mutation in certain genes such as SRY gene, which leads to failure of development of testis.
 Birdem Med J 2020; 10(2): 130-132

Resveratrol ameliorates the effect of maternal immune activation associated with schizophrenia in adulthood offspring

Maternal exposure to infectious agents such as arboviruses, bacteria, or other protozoans has been associated with an elevated risk of schizophrenia (SZ). Evidence suggests that immunological processes occurring during infection may disturb the neural progenitor, impacting the central nervous system (CNS) functions. Moreover, growing evidence suggests that resveratrol (RSV) has neuroprotective activity through anti-oxidant and anti-inflammatory mechanisms. Therefore, we investigated if the treatment with RSV during pregnancy would prevent the abnormalities associated with a SZ-like phenotype induced by maternal immune activation (MIA). Pregnant dams stimulated with a subcutaneous (s.c.) injection of polyriboinosinic-polyribocytidylic acid (poly I:C; 50 mg/kg), a viral nucleic acid mimetic or vehicle, on gestational day (GD) 12.5, were treated with RSV (40 mg/kg, s.c.) or saline, from GD 9.5 to GD 14.5. On day 45 after birth, the offspring was evaluated using a three-compartment social interaction test, elevated plus maze, and hyperlocomotion test induced by amphetamine. After the behavioral tests, the relative expression of mRNA to synapsin 1 (Syn1), oligodendrocyte transcription factor 1 (Olig1), and SRY (sex-determining region Y)-box 2 (Sox2) was determined in the hippocampus and cortex. Treatment with RSV restored the social behavior and attenuated the hyperlocomotion of the offspring bred by dams submitted to MIA. RSV prevented the effects of MIA on Syn1 and Olig1 expression in the hippocampus and Syn1 in the cortex. The present study showed that maternal treatment with RSV attenuates some of the negative behavioral impacts caused by MIA, with modulation of synaptic and oligodendrogenesis processes.

Sexing of pre-implantation ovine embryos through polymerase chain reaction-based amplification of GAPDH, SRY and AMEL genes.

The ability to identify the sex of embryo and control of sex ratio has a great commercial importance to livestock industry. Prediction of embryonic sex could be useful in the management decisions of sex selection in breeding programs. Several methods have been attempted to determine the sex but the polymerase chain reaction (PCR)-based sexing method is generally favoured, as it is cost effective, simple and reliable. The aim of the present study was to identify sex of sheep embryos produced in vitro through amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sex-determining region Y (SRY) and amelogenin genes present in genomic DNA (gDNA) of embryos through PCR. To avoid false interpretation of the result by no amplification of SRY in female embryos, a duplex PCR was approached to amplify combinedly SRY and GAPDH genes. Sex-specific blood was used in PCR as positive control. In vitro sheep embryos were produced as per standardized protocol of laboratory. Sexing of sex-specific blood and in vitro produced embryos were approached though PCR to amplify the respective genes using gDNA present in the sample without its traditional isolation. The accuracy of sex prediction for embryos was 100% by this procedure.

Identification of novel candidate autoantibodies in Alzheimer's disease.

Accumulated failures in Alzheimer's disease (AD) clinical trials have highlighted an urgent need to identify additional biomarkers involved in AD. Recently, mounting evidence reported that autoantibodies are ubiquitous in human sera. However, it is unknown whether autoantibodies are upregulated in amyloid-tau biomarker-confirmed AD. A total of 40 subjects with mild dementia (Clinical Dementia Rating=1) were stratified into AD (n=16) and non-AD (n=24) groups according to their cerebrospinal fluid levels of tau and Aβ42 . Their sera were collected and analyzed using a microarray containing>1600 potential human autoantigens. Autoantibodies that were present exclusively in the AD group were identified and selected using the penetrance-based fold change method with the following criteria: penetrance fold change(AD) ≥2, frequency(AD) ≥15% and frequency(non-AD) =0%. All controls and samples passed the quality control criteria and were further used for biomarker analysis. Six autoantibodies with elevated responses to the following autoantigens were found exclusively in the AD group: nucleosome assembly protein 1-like 3 (31.3%, 5/16 subjects) and microtubule-associated protein 4, pantothenic acid kinase 3, phosphoinositide-3-kinase regulatory subunit 1, protein tyrosine phosphatase type IVA member 1 and SRY (sex-determining region Y)-box 15 (all 18.8%, 3/16 subjects). Although some identified autoantigens are linked to AD and cognitive dysfunction, the increased autoantibody levels have not been reported in AD. Autoantibodies may provide deeper insights into the pathogenesis of AD and serve as diagnostic biomarkers; their corresponding antigens can be further studied to assess their potential as therapeutic targets.

The heparan sulfate proteoglycan syndecan‐1 regulates colon cancer stem cell function via a focal adhesion kinase—Wnt signaling axis

In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of β1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751.

Gene Copy Number Quantification of SHOX, VAMP7, and SRY for the Detection of Sex Chromosome Aneuploidies in Neonates.

Aims: To explore the feasibility of detecting sex chromosome aneuploidies (SCAs) by means of gene copy number quantification of short stature homeobox (SHOX), vesicle-associated membrane protein 7 (VAMP7), and SRY in newborns. Materials and Methods: Gene doses of SHOX, VAMP7, and SRY were determined by quantitative polymerase chain reaction (qPCR) using DNA obtained from dried blood samples from newborns. Relative quantification values were obtained. An aneuploidy profile was established according to cutoff values. Samples with ≥2 gene doses (out of range) were reanalyzed, and those with aneuploidy profiles were confirmed by karyotyping. Sensitivity, specificity, and positive and negative predictive values were obtained. Results: A total of 10,033 samples were collected (4945 females and 5088 males). Of 244 (2.43%) samples with ≥2 gene doses that were retested, 20 cases were confirmed. The overall incidence of SCAs was 1 in 500 live newborns. There were six cases of Turner syndrome (1/824), 3 cases of XXX (1/1648), 7 cases of Klinefelter syndrome (1/726), and 4 cases of of XYY (1/1272). The sensitivity was 0.952 (95.42%); the specificity was 0.975 (97.56%); the positive predictive value was 0.909 (90.91%) and the negative predictive value was 0.987 (98.77%). Conclusions: Gene copy number analyses of the VAMP7, SHOX, and SRY genes by qPCR from blood samples spotted onto filter paper is a highly reliable method for the early detection of male and female SCAs.

Transcriptional analysis of the multiple Sry genes and developmental program at the onset of testis differentiation in the rat

BackgroundThe commonly used laboratory rat, Rattus norvegicus, is unique in having multiple Sry gene copies found on the Y chromosome, with different copies encoding amino acid variations that influence the resulting protein function. It is not clear which Sry genes are expressed at the onset of testis differentiation or how their expression correlates with that of other genes in testis-determination pathways.MethodsHere, two independent E11–E14 developmental RNAseq datasets show that multiple Sry genes are expressed at E12–E13.ResultsThe identified copies expressed during testis initiation include Sry4A, Sry1, and Sry3C, which are conserved in every strain of Rattus norvegicus with genomes sequenced to date.ConclusionsThis work represents a first step in defining the complex environment of rat testis differentiation that can open the door for generating sex reversal model systems using embryo manipulation techniques that have been available in the mouse but not the rat.

48,XXYY syndrome presenting with long-term infertility and newly observed neck deformities: a case report

BackgroundLong-term infertility can be attributed to many factors, with the genetic factor being the most overlooked due to its many nonspecific morphological or endocrine signs. We present a rare case of a patient with progressive testicular failure associated with 48,XXYY syndrome.Case presentationA 39-year-old Arab man presented to our fertility clinic for fertility treatment. He was diagnosed with primary infertility, which had been present for 20 years at the time of presentation. Our patient had nonspecific morphological features of an abnormally wide neck with front slouching neck posture, clinodactyly of the third finger, and had moderate hypoandrogenemic features. A semen analysis revealed azoospermia. Genetic tests for azoospermia, including sex-determining region Y (SRY) detection and chromosome Y microdeletion, revealed no deletion present on the Y chromosome. Karyotyping was used and our patient was diagnosed with 48,XXYY syndrome.ConclusionGenetic testing (karyotyping and so on) played a key role in the diagnosis of our patient with long-term primary infertility secondary to 48,XXYY syndrome, and should play a vital role in all cases of long-term infertility, especially when presentation is accompanied by endocrine, skeletal, or morphological symptoms, signifying an underlying genetic factor.

The capacity of goat epidermal adult stem cells to reconstruct the damaged ocular surface of total LSCD and activate corneal genetic programs.

Epidermal adult stem cells (EpiASCs) have the potential for unlimited proliferation and differentiation, however, the ability of these stem cells to activate corneal genetic programs in response to corneal stroma stimulation needs to be further validated. Herein, a feasible strategy was developed to reconstruct the damaged corneal surface in a goat model with total limbal stem cell deficiency (LSCD) by transplanting EpiASCs, which had been explanted and cultured from the skin of an adult ram goat and were then purified by selecting single cell-derived clones and cultivating them on a denuded human amniotic membrane (HAM). These artificial tissues were then successfully transplanted into ewe goats with total LSCD. Binding of EpiASCs to the base membrane of an EpiASCs-HAM-Sheet (EHS) indicated their proliferating status. After transplantation, the EpiASCs could survive in the host tissue and they reconstructed the damaged ocular surface of total LSCD. The crystal reconstructed corneal epithelium expressed CK3 and Pax-6 similar to normal corneal epithelium and expressed the Sry gene after transplantation. These results demonstrated that EpiASCs could be induced to differentiate into corneal epithelial cell types in a corneal microenvironment and had the ability to activate corneal genetic programs. This work offer a foundation for promoting tissue-engineered cornea into clinical application.

SAT-068 Bewilderment! Sex Chromosome Non-Invasive Prenatal Screening (NIPS) Results Differ from Phenotype and Postnatal Karyotype

BackgroundNIPS uses cell-free DNA in maternal serum to identify fetal aneuploidies including sex chromosome aneuploidies (SCA).1 Despite reports of high sensitivity and specificity, NIPS results, phenotype, and postnatal karyotype may be inconsistent. This situation generates parental anxiety and physician apprehension especially when the apparent genital phenotype differs from the NIPS result. We report four cases involving genotype-phenotype discordance in infants with disorders of sex development (DSD).Case 1:Female infant with complex congenital heart disease had inconclusive NIPS results. Postnatal karyotype was 46,XY karyotype (positive for SRY gene). Ultrasound [US] confirmed female internal genitalia. Postnatal karyotype on uncultured peripheral blood cells with FISH showed mosaicism [46,XY (85%)/45,X (15%)]. Case 2: Female infant conceived via IVF had 46,XY karyotype on NIPS. Prenatal and postnatal US showed female internal genitalia. Postnatal results showed 46,XX karyotype and Urine FISH analysis reported XX in all examined cells. Maternal karyotype is 46,XX; maternal FISH results are pending. Case 3: NIPS reported 45,X. Prenatal US showed male external genitalia. Postnatal exam showed male phenotype with right gonad in inguinal region. Cord blood karyotype and FISH analyses showed mosaicism [45,X (56%)/46,XY (44%)] with 4 different Y chromosome rearrangements. Urine FISH showed mosaicism [45,X (95%)/46,XY (5%)]. Testosterone and AMH concentrations were normal. Case 4: NIPS reported 46,XY karyotype. Prenatal US showed normal female genitalia. Postnatal physical exam revealed low set ears; additional studies revealed horseshoe kidney and atrial septal defect. Cord blood showed mosaicism [45,X (90%)/46,XY (10%)].ConclusionDiscrepancies between NIPS and postnatal karyotype occur more commonly with SCA than for autosomal aneuploidies.2 Such inconsistencies generate anxiety regarding sex of fetus, suggest a DSD diagnosis, and warrant additional prenatal and postnatal investigations. Testing to prevent missed detection of sex chromosome mosaicism is necessary. Early diagnosis of DSDs benefits the infant and the family. In some instances, NIPS expose undiagnosed maternal factors.3 Most importantly, communication involving parents and both prenatal and postnatal health care providers is essential to promote timely holistic health care for the infant and family.

SUN-712 Familial 46, XY Complete Female External Sex Development with a Non-Mosaic Inherited SRY Gene Variation

Context: SRY, an architectural transcription factor containing a SOX-related high-mobility group (HMG) box, initiates testis formation in the mammalian bipotential gonadal ridge. Inherited human SRY mutations can be associated with differences in sexual differentiation (DSD) with variable phenotypes in a family. Objective: To describe the clinical, histopathological and molecular features of a novel inherited SRY allele (pMet64Val; consensus box position 9) observed within an extensive pedigree: two 46, XY sisters with primary amenorrhea (16 and 14 years of age; probands P1 and P2), their normal father and brother, and an affected paternal XY grandaunt. Design, Setting, Participants and Outcome Measurements: Following DNA sequencing to identify the SRY mutation, hormonal studies of the probands and histopathological examination of their gonads were performed. Functional consequences of p.Met64Val (and other mutations at this site) were also investigated. Results: Breast development in P1 and P2was Tanner II and IV, respectively. Müllerian structures and gonads resembling ovaries were found in each sister. Histopathology revealed gonadal dysgenesis, gonadoblastoma and dysgerminoma. AMH/MIS, P450 SCC, and P450 aromatase were expressed in gonadoblastoma tissues. Variant p.Met64Val impaired Sox9 transcriptional activation associated with attenuated occupancy of the testis-specific enhancersEnh13 and TESCO. Conclusion: The partial biological activity of p.Met64Val SRY, maintained at the threshold of SRY function, rationalizes opposing paternal and proband phenotypes (the “the father-daughter paradox”).Sex steroids biosynthesis by gonadoblastoma may delay genetic diagnosis and recognition of gonadal tumors. Quantitative assessment of inherited SRY alleles highlights the tenuous transcriptional threshold of developmental decision-making in the bipotential gonadal ridge.

A Rare Case of Sry-Negative 46,XX Testicular Disorder of Sex Development with Complete Masculinization: A Case Report and Literature Review

46,XX testicular disorder of sex development is a rare cause of male infertility. There is a disagreement between the manifested sexual phenotype (male) and the genotype (female). It occurs in males with 46,XX karyotype who may have normal or ambiguous male genitalia, testosterone deficiency and infertility. With adequate hormonal treatment, the chance of having a good quality of life is high. In the diagnosis, the genetic study is fundamental for the identification of the gene that causes the disorder. The role of SRY gene is important because it allows classifying these males into two groups: those with SRY gene, which activates male sexual differentiation, translocated from Y chromosome to X (SRY-positive), and those without this translocation (SRY-negative). 80-90% of cases are SRY-positive. In the 1020% SRY-negative it is difficult to identify the gene responsible for the alteration. This would indicate the involvement of other genes linked to the X chromosome. This is a review of the first case of SRY-negative 46,XX testicular disorder of sex development with complete masculinization reported in Basque Country in which the cause of the disorder is being investigated, even without specifying.

Unraveling of Diagnosis Odyssey in A Girl with Primary Amenorrhea: A case report

ABSTRACT Background:Primary amenorrhea may result from congenital abnormalities in the development of the gonads, genital tract, or external genitalia or from a disturbance within the hypothalamic-pituitary-ovarian axis. Gonadal dysgenesis is a disorder of sex development in which the diagnosis is based on the histology of gonads and is the main cause of primary amenorrhea. Optimal protocol of management for phenotypic female with 46, XY gonadal dysgenesis involves prophylactic gonadectomy at diagnosis.Case Presentation: The patient was referred to our hospital at the age of 15 years old for primary amenorrhea. She was obese with no secondary sex sign. Gynecologic examination revealed a normal vagina and clitoris. Rectal Toucher examination revealed no internal genitalia structure. The laboratory data: FSH levels was above normal range, LH and testosterone levels were within normal range. Pelvic Ultrasonography uterus and vaginal structure and testis were not visualized. Cytogenetic and ARgene analysis found a 46, XY karyotype and no pathogenic variants. On laparoscopy, Mullerian structure and Wolffian remnant structure were identified and biopsies were performed. Based on histopathological examination and immunohistochemical markers of the right and left gonad showed the impression of Malignant Mixed Germ Cell-Sex Cord Stromal Tumor. SRY gene examination was positive.Examination of other DSD gene analysis has not been done. Second laparoscopy for gonadectomy and removal of Mullerian and Wolfiian remnant structure were performed. Conclusion:Chromosomal analysis should become the first line testing in primary amenorrhea followed by advanced molecular test. Multidisciplinary managements recommended for DSD cases.

Cell Survival Is Regulated via SOX9/BCL2L1 Axis in HCT-116 Colorectal Cancer Cell Line.

Colorectal cancer (CRC) is one of the most frequent types of malignancies and one of the major causes of cancer-related death worldwide. Sex-determining region Y (SRY)-box 9 protein (SOX9) is a member of the SOX family of transcription factors which are involved in the regulation of differentiation and development. Recently, several reports suggest an important role of SOX9 in tumorigenesis since its overexpression correlates with tumor progression and poor outcome in several types of cancer; however, its role in CRC is not clear until now. Therefore, in this work, we searched for novel SOX9-regulated genes involved in cell survival of CRC. We silenced SOX9 in the poorly differentiated HCT-116 cell line, using a specific siRNA, to identify differential expressed genes by DNA microarrays and analyzed the role or candidate genes in apoptosis and autophagy. Transcriptome analysis showed that diverse cellular pathways, associated with CRC carcinogenesis such as Wnt/β-catenin, MAPK, TGF-β, and mTOR, were modulated after SOX9 silencing. Interestingly, we found that SOX9 silencing promotes downregulation of BCL2L1 and overexpression of CASP3, proteins related to apoptosis, which was further confirmed in SW-480, a moderated-differentiated cell line, but not in HT-29, well-differentiated cell line. Moreover, inhibition of BCL2L1 by ABT-737 (BH3 mimetic) in SOX9-silenced HCT-116 cells resulted in an increased apoptosis percentage. However, downregulation of BCL2L1 was not enough to induce autophagy. This is the first report, suggesting that cell survival in poorly and moderated-differentiated CRC cells lines is regulated by SOX9/BCL2L1 axis, but not in well-differentiated cell lines.

The Prognostic and Predictive Value of SOX2+ Cell Densities in Patients Treated for Colorectal Cancer.

SOX2 (sex-determining region-Y homeobox-2) is a transcription factor essential for the maintenance of pluripotency and is also associated with stem-cell-like properties in preclinical cancer models. Our previous study on a cohort of stage III colon cancer patients demonstrated high SOX2+ cell densities were associated with poor prognosis. However, most patients were treated with adjuvant chemotherapy so the prognostic value of SOX2 could not be assessed independently from its value as a predictive marker for non-response to chemotherapy. This study aimed to assess whether SOX2 was a true prognostic marker or a marker for chemotherapy response in a historical cohort of patients, a high proportion of whom were chemotherapy-naïve. SOX2 immunostaining was performed on tissue micro-arrays containing tumor cores from 797 patients with stage II and III colorectal cancer. SOX2+ cell densities were then quantified with StrataQuest digital image analysis software. Overall survival was assessed using Kaplan–Meier estimates and Cox regression. It was found that high SOX2+ cell densities were not associated with poor overall survival. Furthermore, all patients had a significant improvement in survival after 5-fluorouracil (5-FU) treatment, irrespective of their SOX2+ cell density. Therefore, SOX2+ cell densities were not associated with prognosis or chemotherapy benefit in this study. This is in contrast to our previous study, in which most patients received oxaliplatin as part of their treatment, in addition to 5-FU. This suggests SOX2 may predict response to oxaliplatin treatment, but not 5-FU.

Modification and significance of histone acetylation associated with interleukin-4 gene in pediatric Kawasaki disease

Objective To investigate the histone acetylation of interleukin-4(IL-4) gene and its roles in immunological pathogenesis of Kawasaki disease (KD). Methods Thirty-six children with KD and 28 age-matched healthy children in Shenzhen Children′s Hospital from October 2016 to December 2018 were recruited in this study.Peripheral venous blood samples were collected from healthy controls (28 cases) and patients with KD during acute phase and 4 to 5 days after effective intravenous immunoglobulin (IVIG) treatment.Co-immunoprecipitation followed by real-time PCR was used to assess histone H4 acetylation levels of IL-4 promoter and Va enhancer, and binding abilities of p300 and CREB-binding protein (CBP) with promoter and Va enhancer of IL-4 gene in peripheral blood CD4+ T cells.Flow cytometry was performed to analyze the proportion of CD4+ IL-4+ T cells (Th2) and protein le-vels of phosphorylated signal transducer and activator of transcription 6 (pSTAT6), GATA binding protein 3 (GATA3), nuclear factor 1 of activated T cells(NFAT1), transforming growth factor-β receptor Ⅱ (TGF-βRⅡ), and phosphorylated L-type amino acid transporter 1(pLAT1). Quantitative real-time PCR was used to evaluate the transcription levels of IL-4, IL-5, IL-13, IL-4 receptor α (IL-4Rα), transforming growth factor-β receptor Ⅰ (TGF-βRⅠ) and sex-determining region Y(SRY)-box 4 (SOX4) in CD4+ T cells.Plasma concentrations of IL-4 and transforming growth factor-β(TGF-β) were measured by enzyme-linked immunosorbent assay. Results (1)Compared with control group, the proportion of Th2 cells, expression levels of Th2-associated cytokines (IL-4, IL-5 and IL-13) and histone H4 acetylation levels associating with IL-4 promoter and Va enhancer, increased remarkably during acute KD(all P<0.05), and restored after IVIG therapy(all P<0.05). Meanwhile, all the former items in KD patients with coronary artery lesions (CAL) were higher than those in patients with non-coronary artery lesions (NCAL) (all P<0.05). (2) Compared with control group, binding abilities of p300 and CBP with IL-4 promoter and Va enhancer in CD4+ T cells were up-regulated significantly during acute KD (all P<0.05), and decreased in varying degrees after IVIG treatment (all P<0.05). Positive correlations between binding abilities of p300 with IL-4 (promoter and Va enhancer) and the expression of IL-4 promoter and Va enhancer were detected in patients with acute KD (r=0.72, 0.43, all P<0.05). Furthermore, binding abilities of p300 and CBP with IL-4 promoter and Va enhancer in CAL group were higher than those in NCAL group (all P<0.05). (3) Compared with control group, patients with acute KD had remarkably increased plasma concentration of IL-4, and expression levels of IL-4Rα/STAT6/GATA-3 and pLAT1/NFAT1 in CD4+ T cells (all P<0.05), and significantly down-regulated plasma concentration of TGF-β and expression level of TGF-βRⅡ/TGF-βRⅠ/SOX4 (all P<0.05). All the items mentioned above restored in varying degrees after IVIG treatment (all P<0.05). Simultaneously, the 6 items aforementioned in CAL group were found to be higher than those in NCAL group (all P<0.05), while the latter four items were lower than those in NCAL group (all P<0.05). Conclusion Histone hyperacetylation of IL-4 gene may be related to immune dysfunction in patients with KD. Key words: Kawasaki disease; Type Ⅱ helper T cells; Cytokine; Histone acetylation; Immune

Single Nucleotide Polymorphism of Sex Determining Region-Y Gene Coding Sequences in Belgian Blue Bull and Wagyu Bull Crossbred Cattle

Crossbreeding using exotic bulls have been developed widely in Indonesia. Y-chromosome markers have high value for the evaluation of paternal genetic and phylogeny history. The aim of this study was to investigate the coding sequence polymorphism of SRY gene in crossbred cattle using Belgian Blue Bull and Wagyu Bull. Polymerase chain reaction method was used to identify the target sequence 690 bp of SRY gene coding sequences. Then, direct DNA sequencing with forward primer was performed to identify the single nucleotide polymorphism (SNP) differences between individual sample. The results of the alignment coding sequence of SRY gene from four GenBank data, Brahman cross cattle, Belgian Blue Bull Cross and Wagyu Bull cross revealed that there are 21 SNPs variation. Six SNPs out of 21 SNPs (29%) was synonymous and 15 SNPs out of 21 SNPs (71%) was change the amino acid (non-synonymous). Eight out of 21 SNPs (38%) were transition mutation and 13 SNPs out of 21 SNPs (62%) were transversion mutation. Crossbred cattle using Brahman cross cow with Belgian Blue Bull and Wagyu Bull show the SNP at position 1707T→G (transversion mutation). This SNP change the amino acid from Phe(TTT) to Cys(TGT). Thus, the result can be used as molecular marker for identifying the paternal lineage of crossbred cattle which recently develop in Indonesia.

Long non-coding RNA PVT1 contributes to cell growth and metastasis in non-small-cell lung cancer by regulating miR-361-3p/SOX9 axis and activating Wnt/β-catenin signaling pathway

Lung cancer is the most frequent cause of cancer-related mortality in men, and 85 % of lung cancer is diagnosed as non-small-cell lung cancer (NSCLC). Plasmacytoma variant translocation1 (PVT1) serves as an oncogenic factor in NSCLC. However, the pathogenesis of PVT1 in NSCLC is still vague. The expression levels of PVT1, sex determining region Y (SRY)-related high mobility group (HMG)-box9 (SOX9), and microRNA (miR)-361-3p in NSCLC tissues and cells were detected by quantitative real-time polymerase chain reaction (RT-qPCR). The protein levels of SOX9, β-catenin, and c-Myc were detected by western blot assay. Cell proliferation, apoptosis, migration and invasion were measured by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, transwell assays, severally. The interaction between miR-361-3p and PVT1 or SOX9was predicted by starBase, and then verified by the dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. PVT1 and SOX9 was highly expressed in NSCLC tumor tissues and cells. PVT1 facilitated proliferation, migration, invasion and hindered apoptosis of NSCLC cells. MiR-361-3p was a target of PVT1 in NSCLC cells. SOX9 acted as a target of miR-361-3p. PVT1 worked as a miR-361-3p sponge to regulate SOX9 expression. PVT1 modulate the Wnt/β-catenin Signaling Pathway by miR-361-3p/SOX9 axis. Our studies disclosed that PVT1 boosted proliferation, migration, invasion and curbed apoptosis by miR-361-3p/SOX9 axis, providing the possible therapeutic strategy for NSCLC.