Pax6 Research Articles

A Sensitive and Simplified Classifier of Cervical Lesions Based on a Methylation-Specific PCR Assay: A Chinese Cohort Study.

ObjectiveThe aim of this study is to assess the diagnostic and screening performance of a standardized methylation-specific real-time PCR assay targeting SOX1 and PAX1 genes for cervical cancer in a Chinese cohort.MethodsGenomic DNA was extracted from cervical exfoliated cells and converted by sodium bisulfite and then analyzed by qMSP assay. Ct values were collected for PAX1 and SOX1 as target genes and β-actin as an endogenous reference gene. The samples included 295 cervicitis, 111 LSIL (low-grade squamous intraepithelial lesion), 51 HSIL (high-grade squamous intraepithelial lesion) and 30 cervical cancer.ResultsThe Ct values decreased with the progression of cervical cancer from cervicitis, through LSIL and HSIL to cancer. The difference in Ct values between cytological grades was highly significant (p≤0.01) between grades either for PAX1 or for SOX1 except the difference between cervicitis and LSIL of SOX1. With the Ct cut-off values of PAX1 gene and SOX1 gene 38.6 and 38 and with the PAX1/SOX1 in combination, the positive rate of methylation in invasive cancer tissues was 100%, in contrast to 11.5% (95% CI: 8.67%–14.33%) in cervicitis tissues, 45.1% (95% CI: 40.68%–49.52%) in LSIL tissues, and 68.5% (95% CI: 64.37%–72.63%) in HSIL tissues. The specificity and sensitivity of differentiating tumors from cervicitis were 0.957 (95% CI: 0.939–0.975) and 1.00, respectively. The specificity and sensitivity of differentiation between cervicitis+LSIL and HSIL+cervical cancer were 0.881 (95% CI: 0.852–0.91) and 0.748 (95% CI: 0.709–0.787), respectively.ConclusionPAX1/SOX1 methylation could be translated into clinical practice for cervical neoplasia detection.

Clinical and genetic variability of PAX2-related disorder in the Japanese population.

Pathogenic variants of paired box gene 2 (PAX2) cause autosomal-dominant PAX2-related disorder, which includes renal coloboma syndrome (RCS). Patients with PAX2-related disorder present with renal and ophthalmological pathologies, as well as with other abnormalities, including developmental problems and hearing loss. We sequenced PAX2 in 457 patients with congenital anomalies of the kidney and urinary tract or with renal dysfunction of unknown cause and identified 19 different pathogenic variants in 38 patients from 30 families (6.5%). Thirty-four patients had renal hypodysplasia or chronic kidney disease of unknown cause, and three had focal segmental glomerulosclerosis. Although no obvious genotype-phenotype correlation was observed, six of the seven patients who developed end-stage renal disease in childhood had truncating variants. Twenty-three patients had ocular disabilities, mostly optic disc coloboma. Non-renal and non-ophthalmological manifestations included developmental disorder, electrolyte abnormality, and gonadal abnormalities. Two unrelated patients had congenital cystic adenomatoid malformations in their lungs. Six of ten probands with PAX2 mutation identified by next-generation sequencing did not show typical RCS manifestations. We conclude that PAX2-related disorder has a variable clinical presentation and can be diagnosed by next-generation sequencing even in the absence of typical RCS manifestations.

Lymphoid Enhancer Binding Factor 1 (LEF1) and Paired Box Gene 8 (PAX8): A Limited Immunohistochemistry Panel to Distinguish Solid Pseudopapillary Neoplasms and Pancreatic Neuroendocrine Tumors.

Solid pseudopapillary neoplasms (SPNs) and pancreatic neuroendocrine tumors (PanNETs) are distinctive entities. However, due to overlapping morphologies, distinguishing them remains a diagnostic challenge. Our study investigates the utility of immunohistochemistry for nuclear lymphoid enhancer binding factor 1 (LEF1) and paired box gene 8 (PAX8) in differentiating these 2 entities. LEF1 and PAX8 immunohistochemistry were performed on fine-needle aspiration cell blocks and surgical resection specimens diagnosed as SPN or PanNET at our institution from January 2007 to August 2016. Eight SPN and 25 PanNET cell blocks and 17 SPN and 34 PanNET surgical resection specimens were examined. On cell blocks, positive staining for LEF1 had high frequency, sensitivity, and specificity for SPN (88%, 88%, and 88%) as did positive staining for PAX8 for PanNET (76%, 76%, and 75%). The findings on surgical resection specimens were consistent with those from cell blocks (LEF1+ in SPN: 100%, 100%, and 97%; PAX8+ in PanNET: 59%, 59%, and 100%). A combined LEF1+/PAX8- phenotype showed high sensitivity and specificity for SPN (cell block: 63% and 100%; surgical specimen: 100% and 98%) as did a LEF1-/PAX8+ phenotype for PanNET (cell block: 64% and 100%; surgical specimen: 59% and 100%). SPN and PanNET exhibit opposite immunophenotypic profiles with LEF1+/PAX8- in SPN and LEF1-/PAX8+ in PanNET. The combination of these 2 stains provides an effective means of distinguishing these 2 entities.

An Aggressive Urothelial Carcinoma in a Horseshoe Kidney- A Case Report

Evidence of horseshoe kidney in Indian population is 1 in 600-800 individuals. Horseshoe kidney is predisposed to complications by virtue of its ectopic position, malrotation and associated vascular and ureteral anomalies. Incidence of Renal Cell Carcinoma (RCC) in a horseshoe kidney is same as that in general population. Other pelvic tumours, transitional tumours, Wilms tumour and carcinoids show a greater frequency. High grade urothelial carcinomas are quite rare with a few case reports available. An index case of 75 yeal old male presented with renal mass in an incidentally diagnosed horseshoe kidney on radiologic imaging. The case has been highlighted due to its poorly differentiated tumour morphology and aggressive nature. Further, immunohistochemistry was done to arrive at a correct diagnosis for appropriate treatment. The tumour cells showed positivity for Cytokeratin 7 (CK 7) and CK 5/6. They were negative for p63, PAX-8 (Paired-box gene 8). Also, it is technically difficult to excise large-sized renal mass in an anomalous kidney.

Metanephric adenoma of the right kidney: a challenging diagnosis in a 49-year-old female

Background: Metanephric adenoma of the kidney is a rare, usually solitary, and benign tumor of the kidney, predominately affecting female patients in the fifth or sixth decade of life. It is considered by some specialists to be the hyperdifferentiated benign end of the Wilm’s tumor spectrum. Case Presentation: The case was a 49-year-old woman with abdominal pain, a palpable mass in the right abdomen, and mild hematuria, without any other symptoms or laboratory findings. An ultrasonography and computed tomography revealed a tumor in the upper pole of the right kidney with a diameter of 5.3 cm. There was no infiltration of the neighboring structures and tissues. The differential diagnosis included papillary renal cell carcinoma, adult Wilm’s tumor, and metanephric adenoma. A partial nephrectomy was followed. The histological examination of the tumor revealed relatively small unvarying basophilic epithelial cells with scant cytoplasm, uniform nuclei, and some areas with nuclear grooves, delicate chromatin, and indistinct nucleoli in a loose non-cellular stroma and developed in a tightly packed alveolar, tubular, and rarely papillary pattern. The mitotic rate was extremely low. Tumor cells were positive for paired box gene 8, cluster differentiation 57, and Wilm’s tumor 1 and negative for racemase, cytokeratin 7, and EMA. The cell proliferation rate Ki-67 was extremely low, and there was diffuse, strong cytoplasmic positivity for BRAF V600E staining. Based on morphology and immunohistochemistry, the diagnosis of metanephric adenoma was made. Conclusion: Although metanephric adenomas may be difficult to diagnose clinically, the histological examination and the immunohistochemistry assay, including BRAF V600 staining, can make a safe diagnosis, avoiding the administration of incorrect treatment.

Modified Liu-Jun-Zi decoction alleviates visceral hypersensitivity in functional dyspepsia by regulating EC cell-5HT3r signaling in duodenum.

Modified Liu-Jun-Zi (MLJZ) is derived from one of the most famous traditional Chinese prescription Liu-Jun-Zi. It exhibits therapeutic effects in functional dyspepsia (FD), but the underlying mechanisms remain not well understood. Enterochromaffin (EC) cells contribute to the pathogeneses of visceral hypersensitivity in functional gastrointestinal disorders. But whether and how EC cells in duodenum participate in the mechanism of FD remain unsettled. To detect the crucial factors related to EC cells, and to evaluate the therapeutic effect of MLJZ and to determine whether MLJZ relieves visceral hypersensitivity in FD by regulating EC cell-5-hydroxytryptamine 3 receptor (5HT3r) signaling. FD rats were established by iodoacetamide gavage combined with tail clamping method. The verification of FD model and the evaluation of the therapeutic effect of MLJZ was taken place by hematoxylin-eosin (HE) staining and visceral sensitivity measurement. The expression of EC cells and 5-hydroxytryptamine (5HT) in duodenum was detected by Immunohistochemistry (IHC) staining and enzyme-linked immunosorbent assay (ELISA). IHC staining and quantitative polymerase chain reaction (qPCR) were applied to measure the expression of tryptophan hydroxylase-1 (TPH1), paired box gene 4 (PAX4), transient receptor potential A1 (TRPA1), transient receptor potential C4 (TRPC4) and 5HT3r. Duodenum sections were stained by double immunofluorescence (IF) to study the synthesis of 5HT in EC cells. The gastric sensitivity increased in FD rats while MLJZ decoction significantly attenuated visceral hypersensitivity. The duodenum of FD rats displayed increased expressions of EC cells, 5HT, TPH1, PAX4 and 5HT3r. And the overexpression was reduced in response to MLJZ decoction treatment. EC cell-5HT3r signaling pathway is abnormally active in FD with visceral hypersensitivity. And MLJZ decoction can alleviates visceral hypersensitivity in FD by regulating EC cell-5HT3r signaling in duodenum.

Corrigendum to “PAX3: A Molecule with Oncogenic or Tumor Suppressor Function Is Involved in Cancer”

[This corrects the article DOI: 10.1155/2018/1095459.].

Genetic screening of the congenital aniridia and genotype-phenotype analysis

Objective To explore the genotype-phenotype correlation among 3 pedigrees affected with congenital aniridia. Methods Clinical data and genomic DNA were collected and genetic variations were screened by whole-exome sequencing, with an emphasis on PAX6-related genes.Suspected variations were verified by Sanger sequencing and quantitative polymerase chain reaction (PCR). Written informed consent was obtained from the parents of each propositus prior to entering study cohort.This study protocol was approved by Ethic Committee of Henan Eye Hospital (No.HNEECKY-2017(6)). Results Genetic analysis identified that a nonsense c. 949 C>T variation and an c. 141+ 1 G>T splicing variation of the PAX6 gene in two of the probands, while the remainder has carried a duplication in 11 p13 (chr11: 31531331-31827959) encompassing the PAX6 and ELP4 genes.Phenotype analysis showed that the probands carrying the nonsense and splicing variations had classical features including complete aniridia, macular hypoplasia, microcornea and nystagmus; the proband carrying the 11p13 duplication had microphthalmos, microcornea, macular dysplasia, iris dysgenesis, and nystagmus. Conclusions The 11p13 duplication involving the PAX6 gene may have caused over-expression of PAX6 gene, resulting in severe eye abnormalities including microphthalmos and microcornea, macular dysplasia and nystagmus.The relatively mild iris dysgenesis has distinguishing it from classical aniridia due to PAX6 haploinsufficiency. Key words: Congenital aniridia; PAX6 gene; Copy number variation; Phenotype

Study of rs12532, rs8670 Polymorphism of Msh Homeobox 1 (MSX1), rs61754301, rs4904155 Polymorphism of Paired Box Gene 9 (PAX9), and rs2240308 Polymorphism of Axis Inhibitor Protein 2 (AXIN2) Genes in Nonsyndromic Hypodontia.

The etiology of hypodontia is complex, in which both genetic and environmental factors can be related. The main objective of our study was to contribute to elucidating the genetic background of nonsyndromic hypodontia (NSH). In this order, we selected 97 NSH subjects (70 females and 27 males) from patients referred to orthodontic treatment, and we matched to each NSH subject a control by age and sex. DNA was obtained from epithelial cells from the oral mucosa. Genotyping of the PAX9 (rs4904155 and rs61754301), MSX1 (rs8670 and rs12532), and AXIN2 (rs2240308) SNPs was performed by using TaqMan SNP Genotyping Assays on a real-time PCR system. Single-nucleotide polymorphisms (SNPs) were studied for the whole NSH group and for frontal and lateral agenesis NSH subjects separately. Our results showed that the variant genotype (p=0.0008, OR = 2.9, 95% CI = 1.58–5.3) and variant T allele (p=0.0002, OR = 2.65, 95% CI = 1.6–4.39) of the MSX1 rs8670 SNP increased the risk of hypodontia in the studied population when the whole NSH group was compared with controls. The variant genotype of the MSX1 rs8670 SNP was the most frequent in frontal agenesis; meanwhile in the lateral agenesis NSH group, the AXIN2 rs2240308 SNP showed a higher frequency of the variant genotype, with a trend towards statistical significance. In conclusion, the results of the present study showed that the variant genotype and variant T allele of the MSX1 rs8670 SNP increased the risk of hypodontia in the studied population. The presence of the variant A allele of AXIN2 rs2240308 is associated with frontal agenesis but not with lateral agenesis.

Prognostic implication of forkhead box protein O1 (FOXO1) and paired box gene 3 (PAX3) in epithelial ovarian cancer.

61 Background: Transcriptional factor, Forkhead box protein O1 (FOXO1) has been reported to play an imported role in human cancer, but the role in epithelial ovarian cancer (EOC) has not yet been clarified. Here, we evaluatedthe expression and clinical significance of FOXO1 in EOC. Methods: Immunohistochemical analyses of FOXO1 and PAX3 in 212 in EOCs, 57 borderline ovarian tumors and 153 benign epithelial ovarian tumors and 79 nonadjacent normal epithelial tissues were performed using tissue microarray analysis. The data were compared with clinicopathological variables including the survival of EOC patients. Also, the effect of FOXO1 on cell growth were assessed in EOC cell lines. Results: The expressions of FOXO1 and PAX3 protein were significantly higher in EOC tissues than in nonadjacent normal epithelial tissues, benign tissues and borderline tumors respectively (all p< 0.001). Overexpression of FOXO1 was significantly associated with poor grade ( p = 0.004). FOXO1 expression showed trend of positive correlation with that of PAX3 in EOC tissues ( Spearman’s rho0.118, p= 0.149). Multivariate survival analysis revealed that the high expression of FOXO1 (hazard ratio = 2.74 [95% CI, 1.22–13.10], p = 0.001) could be an independent prognostic factor for overall survival. Most importantly, high expression of both FOXO1 and PAX3 showed high hazard ratio (hazard ratio = 5.53 [95% CI, 2.47–12.40], p< 0.001) for overall survival. In vitro result revealed that knockdown of FOXO1 was associated decreased cell viability and migration. Conclusions: This study reveals that high expression of FOXO1/PAX3 is an indicator of poor prognosis in EOC. Our results not only suggest the promising potential of FOXO1 and PAX3 as a prognostic and survival marker, but also warrant further studies on a possible link between the biological function of FOXO1 and PAX3 of EOC.

Association between dense PAX1 promoter methylation and HPV16 infection in cervical squamous epithelial neoplasms of Xin Jiang Uyghur and Han women

DNA methylation is an epigenetic alteration that may lead to carcinogenesis by silencing key tumor suppressor genes. Hypermethylation of the paired box gene 1 (PAX1) promoter is important in cervical cancer development. Here, PAX1 methylation levels were compared between Uyghur and Han patients with cervical lesions. Data on PAX1 methylation in different cervical lesions were obtained from the Gene Expression Omnibus (GEO) database, whereas data on survival and PAX1 mRNA expression in invasive cervical cancer (ICC) were retrieved from the Cancer Genome Atlas (TCGA) database. MassARRAY spectrometry was used to detect methylation of 19 CpG sites in the promoter region of PAX1, whereas gene mass spectrograms were drawn by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Human papillomavirus (HPV) 16 infection was detected by polymerase chain reaction. PAX1 methylation in high-grade squamous intraepithelial lesion (HSIL) and ICC was significantly higher than in normal tissues. PAX1 hypermethylation was associated with poor prognosis and reduced transcription. ICC-specific PAX1 promoter methylation involved distinct CpG sites in Uyghur and Han patients HPV16 infection in HSIL and ICC patient was significantly higher than in normal women (p < 0.05). Our study revealed a strong association between PAX1 methylation and the development of cervical cancer. Moreover, hypermethylation of distinct CpG sites may induce HSIL transformation into ICC in both Uyghur and Han patients. Our results suggest the existence of ethnic differences in the genetic susceptibility to cervical cancer. Finally, PAX1 methylation and HPV infection exhibited synergistic effects on cervical carcinogenesis.

1632 Rectal Endometriosis: An Atypical Presentation

INTRODUCTION: Endometriosis is a condition in which endometrial tissue is implanted outside of the uterus. Patients with gastrointestinal involvement experience pelvic pain, rectal bleeding and abdominal pain which may be associated with menstruation. Often patients are misdiagnosed or have significant delays in diagnosis, especially in the setting of negative imaging. While endometrial lesions that penetrate the bowel mucosa are rare, endoscopy can play a vital role in the correct clinical setting. We present a rare case of rectal endometriosis that was diagnosed primarily through colonoscopy. CASE DESCRIPTION/METHODS: A 46-year-old woman with a history of uterine fibroids was referred to our center for evaluation of severe intermittent rectal pressure for the previous four months. Patient reported that her pain was associated with bowel movements and improved after evacuation. She denied blood in her stool. She had undergone a transvaginal ultrasound and magnetic resonance imaging (MRI) of her pelvis that demonstrated a decrease in size of her uterine fibroids from prior imaging. The bowel was noted to be unremarkable on MRI. She was referred to our clinic for further management of possible irritable bowel syndrome. The patient had a benign abdominal exam and rectal exam revealed internal hemorrhoids. Colonoscopy was subsequently scheduled and showed unremarkable ileum and colon, however approaching the anal verge, a nodularity was found in the rectum with a somewhat hard consistency. Several biopsies were collected. Ink was injected proximal to the nodularity for reference. Pathology review of the biopsies were consistent with endometriosis with no evidence of dysplasia or malignancy. Immunohistochemical stains were positive for estrogen receptor (ER), paired-box gene 8 (PAX 8) and pankeratin, markers for endometriosis. The patient was referred for rectal endoscopic ultrasonography (EUS) to determine the depth of invasion. She follows with gynecology where further treatment options are being discussed. DISCUSSION: Gastrointestinal endometriosis serves as the most common site for extra-gynecologic localization and affects 3-38% of women presenting with the endometriosis. Endometriosis is commonly misdiagnosed and often mistaken for other pathologies, resulting in delays in diagnosis, unnecessary testing and mismanagement. For women of childbearing age who present with persistent pelvic pain and associated symptoms of endometriosis, colonoscopy may serve as a primary method for diagnosis.

Cancer of Unknown Primary: A Review on Clinical Guidelines in the Development and Targeted Management of Patients with the Unknown Primary Site.

Cancer of unknown primary (CUP) is a malignant widespread metastatic disease without an identifiable primary site after extensive clinical investigation. Recently, a decline is observed in the diagnosis of CUP, mainly due to improvement in detection of the primary tumors, thus decreasing the unknown primaries. Worldwide, CUP is the sixth to eighth most common malignancy, accounting for 2.3% to 5% of a new cancer diagnosis. CUP is third to fourth most common cause of death due to cancer-related mortality. The prognosis of CUP is depressing with the median survival of three to six months in the previous studies, but according to recent studies, median survival is less than one year. High risk for developing CUP is seen in heavy smokers (26 or more cigarettes/day) and individuals with the lowest quartiles of waist circumference. A weak association is observed with the use of alcohol consumption and low level of education. Human papillomavirus DNA plays a role in those with squamous cell carcinoma of unknown primaries in head and neck regions. In the diagnosis of CUP, comprehensive medical history, complete physical examination (including genitourinary, rectal exam, and breast examination in women) and necessary laboratory tests are crucial.Whole-body positron emission tomography-computed tomography (PET/CT) is the investigation of choice to assess the entire body for CUP. Multiparametric 3T-MRI (MP-MRI) is used to examine the local soft tissue status, helps in the staging of the tumor, and to determine the extent of involvement of tissue for medical as well as prognostic purposes. Immunohistochemistry outlines the specific markers, including caudal-related homeobox protein (CDX2), homeobox protein Nkx-3.1 (NKX3-1), paired box gene 8 (PAX8), special AT-rich sequence-binding protein 2 (SATB2), thyroid transcription factor 1 (TTF-1), and splicing factor 1 (SF1) with the focus on the effectiveness of lineage-restricted transcription factors. Patients response to treatment can be evaluated by the gene expression profiling (GEP) test that also predicts tissue of origin (TOO). Tumor identified through gene profiling is sensitive to platinum/taxane therapy, others that are not TOO tumors are resistant to platinum/taxane. The new therapeutic method based on molecular profiling is associated with higher treatment response. In comprehensive genomic profiling, it is observed that there is at least one clinically appropriate genomic alteration in CUP that can influence the targeted therapy. The targeted therapeutic approach will not only improve the disease outcome but will also be cost-effective and save time from finding the primary site.

MicroRNA-204-3p modulates epithelial-mesenchymal transition by targeting paired box gene 2 in human melanoma A-375 cells.

BackgroundPaired box 2 gene promoted the progression of subsequent epithelial-mesenchymal transition (EMT) in malignant melanoma, which is the most dangerous type of skin cancer. We aimed to investigate the functional role of miR-204-3p in metastatic melanoma.MethodsTo study the effects of overexpressed miR-204-3p on the melanoma cells, miR-204-3p mimic vector were transfected into A-375 cells for detecting the cell viability using CCK-8 kit. Wound healing and Transwell assays were performed to assess the migration and invasion capacities of melanoma cells. TargetScan and miRTarBase were used to predict the potential target of miR-204-3p, which was subsequently verified by dual-luciferase assay. The expressions of several EMT-associated genes were determined by quantitative real-time polymerase chain (qRT-PCR) and Western blot.ResultsThe expression of miR-204-3p in several melanoma cell lines was lower than that in HaCaT cells. After transfecting with the miR-204-3p mimic vector, the cell viability of A-375 cells decreased over time and significantly reduced at 48 h (P<0.01). Overexpression of miR-204-3p noticeably suppressed the migration and invasion abilities of A-375 cells (P<0.01). PAX2 and guanine nucleotide binding protein (G protein) q polypeptide (GNAQ) were the potential targets of miR-204-3p, which could effectively silence PAX2 rather than GNAQ. MiR-204-3p overexpression could upregulate the epithelial cadherin (E-cadherin) and downregulate the vimentin, matrix metalloproteinase 2 (MMP2) and MMP-9 at transcriptional and posttranscriptional levels.ConclusionsOur results revealed that miR-204-3p had inhibitory effects on the migratory and invasive capacities and EMT progression in malignant melanoma through targeting PAX2.

Aberrant Methylation Status of Tumour Suppressor Genes in Ovarian Cancer Tissue and Paired Plasma Samples.

Ovarian cancer is a highly heterogeneous disease and its formation is affected by many epidemiological factors. It has typical lack of early signs and symptoms, and almost 70% of ovarian cancers are diagnosed in advanced stages. Robust, early and non-invasive ovarian cancer diagnosis will certainly be beneficial. Herein we analysed the regulatory sequence methylation profiles of the RASSF1, PTEN, CDH1 and PAX1 tumour suppressor genes by pyrosequencing in healthy, benign and malignant ovarian tissues, and corresponding plasma samples. We recorded statistically significant higher methylation levels (p < 0.05) in the CDH1 and PAX1 genes in malignant tissues than in controls (39.06 ± 18.78 versus 24.22 ± 6.93; 13.55 ± 10.65 versus 5.73 ± 2.19). Higher values in the CDH1 gene were also found in plasma samples (22.25 ± 14.13 versus 46.42 ± 20.91). A similar methylation pattern with positive correlation between plasma and benign lesions was noted in the CDH1 gene (r = 0.886, p = 0.019) and malignant lesions in the PAX1 gene (r = 0.771, p < 0.001). The random forest algorithm combining methylation indices of all four genes and age determined 0.932 AUC (area under the receiver operating characteristic (ROC) curve) prediction power in the model classifying malignant lesions and controls. Our study results indicate the effects of methylation changes in ovarian cancer development and suggest that the CDH1 gene is a potential candidate for non-invasive diagnosis of ovarian cancer.

Genetic analyses in a cohort of Portuguese pediatric patients with congenital hypothyroidism.

Background Permanent primary congenital hypothyroidism (CH) can be caused by thyroid dysgenesis or dyshormonogenesis. A molecular genetic study is recommended in dyshormonogenesis, in syndromic hypothyroidism and when there is a family history of CH. The aim of this study was to identify a monogenic etiology for CH in selected individuals from a cohort of primary permanent CH. Methods From an initial cohort of 79 patients with permanent CH (3-19 years), 11 patients were selected for molecular analyses. Nine patients with dyshormonogenesis (normal in-situ gland or goiter) were screened for causative variants, by next-generation sequencing (NGS), in 28 genes known to be responsible for CH. One patient with a family history of CH was screened for the paired-box gene 8 (PAX8) gene and another patient with a syndromic CH was screened for the NKX2-1 gene. Results We found a monogenic basis of disease in eight patients, involving the thyroid peroxidase (TPO) gene (four patients), the thyroglobulin (TG) gene (two patients), and the PAX8 and NKX2-1 genes (one patient each). Two patients were heterozygotes, one harboring a variant in the TG gene and the other in the SLC5A5 gene. In one patient, we found no potential causative variants in any of the 28 genes screened. We described five novel variants: three in the TG gene, one in the NKX2-1 and one in the SLC5A5 gene, all of them classified as pathogenic. Conclusions In eight of the 11 screened patients, a monogenic disease was found. These results highlight the advantage of using an NGS panel and provide further data regarding the molecular basis of CH.

The Synergistic Effect of Glucagon-Like Peptide-1 and Chamomile Oil on Differentiation of Mesenchymal Stem Cells into Insulin-Producing Cells.

Objective Glucagon-like peptide-1 (GLP-1) has attracted tremendous attention for treatment of diabetes. Likewise, it seems that active ingredients of chamomile oil might have anti-diabetic effects. This work was conducted to investigate the effects of the combination of GLP-1 and chamomile oil on differentiation of mesenchymal stem cells (MSCs) into functional insulin-producing cells (IPCs). Materials and Methods In this experimental study, adipose MSCs derived from the adult male New Zealand white rabbits were assigned into four groups: control (without any treatment); GLP-1 (in which cells were treated with 10 nM GLP-1 every other day for 5 days); chamomile oil (in which cells were treated with 100 ug/ml Matricaria chamomilla L. flower oil every other day for 5 days); and GLP-1+ chamomile oil (in which cells were treated with 10 nM GLP-1 and 100 µg/ml M. chamomilla flower oil every other day for 5 days). Characterization of isolated MSCs was performed using flow cytometry, Alizarin red S staining and Oil red O staining. The expressions of genes specific for IPCs were measured using reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Measurement of insulin and the cleaved connecting peptide (C-peptide) in response to different concentrations of glucose, were performed using ELISA kits. Results Our results demonstrated that isolated cells highly expressed MSC markers and were able to differentiate into osteocytes and adipocytes. Additionally, using GLP-1 in combination with chamomile oil exhibited higher levels of IPCs gene markers including NK homeobox gene 2.2 (NKX-2.2), paired box gene 4 (PAX4), insulin (INS) and pancreatic duodenal homeobox-1 (PDX1) as well as insulin and C-peptide secretion in response to different glucose concentrations compared to GLP-1 or chamomile oil alone (P<0.05). ConclusionCollectively, these findings establish a substantial foundation for using peptides in combination with natural products to obtain higher efficiency in regenerative medicine and peptide therapy.

Forkhead box protein O1 (FOXO1) and paired box gene 3 (PAX3) overexpression is associated with poor prognosis in patients with cervical cancer.

Forkhead box protein O1 (FOXO1) and paired box gene 3 (PAX3) have been reported to play an imported role in human cancers, but their role in cervical cancer has not yet been clarified. In this study, we evaluated the functional role of FOXO1 in cervical cancer cells and investigated the expression and clinical significance of FOXO1 and PAX3 in cervical lesions. In vitro assessment of cell function by cell viability, migration, and invasion assays were performed on FOXO1-knockdown cervical cancer cells. Immunohistochemical (IHC) staining analyses of FOXO1 and PAX3 were performed with a tissue microarray (TMA). The clinical significance was evaluated by comparing the data with various clinicopathologic characteristics, including survival of patients with cervical cancer. In vitro results revealed that knockdown of FOXO1 is associated with decreased cell viability (p < 0.001), migration (p < 0.001), and invasion (p < 0.05), supporting the oncogenic role of FOXO1 in cervical cancer. FOXO1 and PAX3 expression was significantly higher in CIN (both p < 0.001) and cancer tissue (both p < 0.001) than in normal tissue. Multivariate analysis indicated that FOXO1 expression (hazard ratio 4.01 [95% CI 1.22-13.10], p = 0.021) and an advanced FIGO stage (hazard ratio 3.89 [95% CI 1.35-11.19], p = 0.012) were independent prognostic factors for overall survival. This study reveals increased FOXO1 and PAX3 expression in cervical cancers and indicates an oncogenic role of FOXO1 in cervical cancer cells that correlates with poor patient survival.

Abstract 5204: ICAM-1 and IL-17E upregulate FOXO1 to suppress the expression of oncogenic PKC-ι in human melanoma

Abstract Throughout the span of the last 15 years, the number of cases of melanoma has been on a steady increase. Approximately, 92,000 new melanoma cases will be diagnosed in the United States in 2018. Our previous studies demonstrated that protein kinase C-iota (PKC-ι) is crusial for melanoma progression, which promotes the activation of nuclear factor (NF)-κB through a PI3K/AKT manner, thereby supporting survival and progression. In addition, PKC-ι induced the metastasis of melanoma cells by stimulating ephithilial-mesenchmal transition. Results also showed that PKC- specific inhibitors diminished the expression of both PKC-ι and phospo-PKC-ι, suggesting that PKC-ι plays a role in regulating its own expression in melanoma cells in-vitro {Ratnayake, W.S., et al. Cell Adhes. Migr. (2018) https://doi.org/10.1080/19336918.2018.1471323}. In this study, we report the transcriptional regulation mechanisms of PRKCI (PKC-ι gene) expression in melanoma. PROMO and Genomatix Matinspector methods were used to identify c-Jun, interferon-stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI and they were analyzed for their roles in PKC-ι expression. siRNAs were used to silence selected transcription factors, and the results revealed that, silencing of FOXO1 and c-Jun significantly altered the expression of PRKCI. In addtion, we knocked down NF-κB in order to demonstrate changes on activities of c-Jun and FOXO1. Both phosphorylated and total PKC-ι levels increased upon FOXO1 silencing and decreased upon c-Jun silencing, suggesting that c-Jun turns is an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. Multiplex ELISA assays were used to analyze multiple pathways and the expression of cytokines with respect to crosstalk between signaling pathways affected by specific inhibiton PKC-ι. Western blot data for all siRNA treatments of PKC-ι, NF-κB, FOXO1 and c-Jun were supported by real-time qPCR. Taken together, results suggested that the regulation of PKC-ι expression was strongly associated with FOXO1 and c-Jun through the trascriptional deactivation/activation. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)-6 and IL-8, with a significant increase in the levels of IL-17E and intercellular adhesion molecule 1 (ICAM-1) upon the knockdown of expression of PKC-ι in both cell lines. This suggested that PKC-ι expression was affected by said cytokines in an autocrine modus. Overall, the findings suggest that PKC-ι inhibition suppresses its own expression, diminishing oncogenic signaling such as NF-κB, PI3K/AKT and JNK/c-Jun while upregulating anti-tumor signaling such as ICAM-1/FOXO1, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma metastasis. Citation Format: Wishrawana Sarathi Ratnayake, Christopher Apostolatos, Sloan Breedy, André Apostolatos, Mildred Acevedo-Duncan. ICAM-1 and IL-17E upregulate FOXO1 to suppress the expression of oncogenic PKC-ι in human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5204.

Somite development in the avian tail.

Somites are epithelial segments of the paraxial mesoderm. Shortly after their formation, the epithelial somites undergo extensive cellular rearrangements and form specific somite compartments, including the sclerotome and the myotome, which give rise to the axial skeleton and to striated musculature, respectively. The dynamics of somite development varies along the body axis, but most research has focused on somite development at thoracolumbar levels. The development of tail somites has not yet been thoroughly characterized, even though vertebrate tail development has been intensely studied recently with respect to the termination of segmentation and the limitation of body length in evolution. Here, we provide a detailed description of the somites in the avian tail from the beginning of tail formation at HH-stage 20 to the onset of degeneration of tail segments at HH-stage 27. We characterize the formation of somite compartment formation in the tail region with respect to morphology and the expression patterns of the sclerotomal marker gene paired-box gene 1 (Pax1) and the myotomal marker genes MyoD and myogenic factor 5 (Myf5). Our study gives insight into the development of the very last segments formed in the avian embryo, and provides a basis for further research on the development of tail somite derivatives such as tail vertebrae, pygostyle and tail musculature.