NKX2-1 gene is targeted by H19 lncRNA and is found to be overexpressed in benign nodular goiter tissues.

OBJECTIVE: Nodular goiter may increase the risk of thyroid cancer, but the genetic factors contributing to nodular goiter are not well understood. There is an overexpression of H19 lncRNA in goiter tissue and its target remains unknown. In this study, we attempted to identify a new target for H19 in the context of goiter development. METHODS: Using interaction energy calculations, the interaction between NKX2-1 mRNA and H19 lncRNA was examined. Putative microRNAs were found at the H19 lncRNA target site with the highest affinity for NKX2-1. RNAseq data was analyzed to determine the tissue specificity of gene expression. Samples were taken from 18 goiter and 18 normal tissues during thyroidectomy. The expression of NKX2-1 was determined by RT-qPCR using specific primers. RESULTS: The interaction between NKX2-1 and H19 was characterized by six local base-pairing connections, with a maximum energy of -20.56 kcal/moL. Specifically, the sequence that displayed the highest affinity for binding with H19 overlapped with the binding site of has-miR-1827 to NKX2-1. It was found that NKX2-1 is exclusively co-expressed with H19 in normal thyroid tissue. As compared to adjacent normal tissues, nodular goiter tissues have a significant overexpression of NKX2-1 (relative expression = 1.195, p =  0.038). CONCLUSION: NKX2-1 has been identified as the putative target of H19 lncRNA, which is overexpressed in nodular goiter tissues significantly.

FOXE1 Gene is a Probable Tumor Suppressor Gene with Decreased Expression as Papillary Thyroid Cancers Grow, and is Absent in Anaplastic Thyroid Cancers.

Papillary thyroid carcinoma (PTC), the most prevalent cancer of the thyroid, is more common in women than in men. To uncover the expression profile of FOXE1 gene in PTC tumor etiology. Microarray and RNA sequencing data on PTC in humans were analyzed. Eleven PTC tumor tissue samples and their neighboring normal tissue samples were collected. RT-qPCR was performed. Data normality, ROC construction, and logistic regression analysis were conducted. PTC tumors, normal tissues surrounding tumors, patients of different sexes and ages, metastasizing tumors, and tumor variants were assessed for FOXE1 expression. Eleven PTC tissues were obtained from seven women and four men. Among the PTC subtypes, there were two FV-PTCs, four C-PTCs, one microcarcinoma, and four tissues with an unknown subtype. FOXE1 gene expression was significantly increased in PTC tumors with dimensions less than 10 mm (relative expression = 14.437, p = 0.050). A significant increase in FOXE1 gene expression was observed in the normal tissue adjacent to the tumor, which was less than 10 mm in size, compared to the normal tissue adjacent to the tumor, which was larger than 10 mm (relative expression = 41.760, p = 0.0001). Females diagnosed with PTC showed a significant reduction in FOXE1 mRNA levels compared to their male counterparts (relative expression = 0.081, p = 0.042). In contrast to adjacent normal tissue, there was a significant reduction in FOXE1 gene expression in FV-PTC (relative expression = 0.044 and p = 0.0001). PTC tumors under 10mm had higher FOXE1 gene expression than larger tumors; normal tissue adjacent to smaller tumors also had higher FOXE1 expression. Females with PTC, regardless of their subtype, expressed less FOXE1 mRNA than males. FV-PTC tissues exhibited lower expression of FOXE1 mRNA than their adjacent normal tissues.

Overexpression of hsa-miR-30a-5p and non-obstructive azoospermia: A case-control study.

Background: Some previous human and animal studies have supported the idea that KDM3A down-regulation might be the main cause of male infertility, especially in non-obstructive azoospermia (NOA). The regulatory role of micro-RNAs (miRNA) has been investigated in the development of male infertility. Objective: The expression level of hsa-miR-30a-5p in azoospermia was evaluated to reveal its possible association with the etiology of male infertility. Materials and Methods: In this case-control study, 30 men with azoospermia (19 of whom had NOA) were selected as the case individuals, and 11 men with obstructive azoospermia (OA) were selected as control individuals. The best miRNA with the strongest ability to target the KDM3A gene was detected via comprehensive bioinformatics analysis. Reverse transcriptase quantitative polymerase chain reaction was used to assess the expression level of hsa-miR-30a-5p. After analyzing the data, the expression level of hsa-miR-30a-5p wascompared between men with NOA and men with OA. Results: The findings supported the idea that hsa-miR-30a-5p is the miRNA with the best ability to target the KDM3A transcript. The expression analysis of hsa-miR-30a-5p indicated a significant overexpression (p = 0.04) in men with NOA compared to in men with OA. Conclusion: Hsa-miR-30a-5p was overexpressed in men with NOA compared to in control individuals. Hsa-miR-30a-5p could target the KDM3A transcript and may suppress its expression.

Amelioration for an ignored pitfall in reference gene selection by considering the mean expression and standard deviation of target genes.

Routine tissue-specific reference genes are often used in expression studies, but target genes are not taken into account. Using the relative RT-qPCR approach, we evaluated the expression of three target genes. At the same time, meta-analyses were conducted in various ethnic groups, genders, and thyroid cancer subtypes. When eight common reference genes were examined, it was discovered that some of them not only lacked consistent expression but also had considerable expression variance. It is worth noting that while choosing a reference gene, the mean gene expression and its standard deviation should be carefully addressed. An equation was developed based on this, and it was used to perform statistical analysis on over 25,000 genes. According to the subtype of thyroid cancer and, of course, the target genes in this investigation, appropriate reference genes were proposed. The intuitive choice of GAPDH as a common reference gene caused a major shift in the quantitative expression data of target genes, inverting the relative expression values. As a result, choosing the appropriate reference gene(s) for quantification of transcription data, and especially for relative studies of the expression of target gene(s), is critical and should be carefully considered during the study design.

The role of melatonin on caspase-3-like activity and expression of the genes involved in programmed cell death (PCD) induced by in vitro salt stress in alfalfa (Medicago sativa L.) roots.

BACKGROUND: Alfalfa (Medicago sativa L.) is the most cultivated forage plant as a model in legumes. Salinity stress due to Na+ toxicity causes severe, oxidative stress as a main reason for program cell death (PCD) in plants. Melatonin application can increase plant productivity in response to diverse stressors via modulating plant antioxidant mechanisms and PCD inhibition in plants. RESULTS: Alfalfa roots were subjected to different concentrations of in vitro salinity supplemented with melatonin (0.1, 10 and 15 µM) for ten days. Application of melatonin under salinity stress reduced ROS, H2O2 and [Formula: see text] content and showed a dramatic impact on TTC reduction and augmented cell viability. Interestingly, melatonin inhibited caspase 3-like protease activity and could decrease DNA fragmentation induced by salinity while increased expression of anti-apoptotic genes BI-1, UCP1-UCP2 involved in PCD pathway. In contrast, in 300 mM salinity, γVPE gene as a proapoptotic of PCD down-regulated significantly. CONCLUSIONS: For the first time, present data showed that, melatonin plays a major function in preventing PCD in alfalfa root meristem cells. We attempted to offer a mechanism for the function of melatonin as an anti-apoptotic agent by demonstrating significant actions of melatonin on mitochondria proteins, such as UCPs, in a manner similar to animal cells.

TXNDC2 joint molecular marker is associated with testis pathology and is an accurate predictor of sperm retrieval.

The association of PRM1/2 with male azoospermia is well-documented, but the relationship between TXNDC2 deficiency and the azoospermia phenotype, sperm retrieval, and pathology has not been elucidated. Here we identified the association of TXNDC2 and protamines in evaluating testis pathology and sperm retrieval. An extensive microarray meta-analysis of men with idiopathic azoospermia was performed, and after undergoing several steps of data quality controls, the data passing QC were pooled and batch effect corrected. As redox imbalance has been shown to have a variable relationship with fertility, our relative expression studies began with candidate protamination and thioredoxin genes. We constructed a logistic regression model of TXNDC2 with PRM1 and PRM2 genes, and collective ROC analysis indicated a sensitivity of 96.8% and specificity of 95.5% with a ROC value of 0.995 (SE = 0.0070, 95% CI 0.982-1.000). These results demonstrate that TXNDC2, PRM1, and PRM2 combined have a robust power to predict sperm retrieval and correlate with severe azoospermia pathology.

Associations of BCL2 CA-Repeat Polymorphism and Breast Cancer Susceptibility in Isfahan Province of Iran.

BCL2 apoptosis regulator (BCL2) is a cause of tumorigenesis whose CA-repeat promoter polymorphisms has inconsistent association with various types of cancers. The association of BCL2 polymorphism with breast cancer was investigated in the Isfahan province of Iran. PCRamplification of the CA-repeat was followed by polyacrylamide gel electrophoresis and direct sequencing for 120 breast cancer women and an equal number of corresponding healthy control individuals. Seven different alleles, ranging from 11 to 17 CA-repeats were observed. Short alleles with 11 to 14 repeats were protective (OR 0.363, P = 0.001), but large alleles with 15 to 17 repeats were threatening against breast cancer development (OR 2.780, P = 0.001). Accordingly, genotypes with large alleles showed a higher risk of breast cancer development (OR 3.400, P = 0.004). ERS1\ERBB2 positive breast cancer patients, but not PGRpositive ones, showed protection against breast cancer (OR 0.405, OR 0.346 respectively). In conclusion, women with at least one large allele of BCL2 were 3.4 times at higher risk of breast cancer development in the Isfahan province of Iran.

Hsa-miR-27a-3p overexpression in men with nonobstructive azoospermia: A case-control study.

BACKGROUND: The role of KDM3A and its downstream genes in male fertility has been approved in animal models. Additionally, the expression shrinkage of KDM3A is significantly correlated with human azoospermia phenotype. Aberrant expression of micro-RNAs could mislead spermatogenesis and mostly lead to diverse phenotypes of male infertility. OBJECTIVE: The aim of this study was to evaluate the expression level of hsa-miR-27a-3p in azoospermic men to reveal its possible association with infertility. MATERIALS AND METHODS: This case-control study was conducted on 30 azoospermic men, of whom, 19 had non obstructive azoospermia (NOA) and 11 obstructive azoospermia (OA) according to the pathological examinations. Comprehensive bioinformatics investigations were performed securely and hsa-miR-27a-3p was selected afterward. Reverse Transcriptase-quantitative polymerase chain reaction (RT-qPCR) method was used and statistical analysis was performed to compare the expression level of hsa-miR-27a-3p in both OA and NOA individuals. RESULTS: In silico analysis suggested hsa-miR-27a-3p, with its potential binding ability to target KDM3A transcripts. The expression analysis of candidate hsa-miR-27a-3p indicated its significant overexpression in NOA men. CONCLUSION: The hsa-miR-27a-3p was overexpressed in NOA men compared to OA-control individuals. As a consequence, the overexpressed micro-RNA could downregulate directly KDM3A and indirectly TNP1 and PRM1. Therefore, spermatogenesis could be misled and male infertility could be developed.

The pseudogene problem and RT-qPCR data normalization; SYMPK: a suitable reference gene for papillary thyroid carcinoma.

In RT-qPCR, accuracy requires multiple levels of standardization, but results could be obfuscated by human errors and technical limitations. Data normalization against suitable reference genes is critical, yet their observed expression can be confounded by pseudogenes. Eight reference genes were selected based on literature review and analysis of papillary thyroid carcinoma (PTC) microarray data. RNA extraction and cDNA synthesis were followed by RT-qPCR amplification in triplicate with exon-junction or intron-spanning primers. Several statistical analyses were applied using Microsoft Excel, NormFinder, and BestKeeper. In normal tissues, the least correlation of variation (CqCV%) and the lowest maximum fold change (MFC) were respectively recorded for PYCR1 and SYMPK. In PTC tissues, SYMPK had the lowest CqCV% (5.16%) and MFC (1.17). According to NormFinder, the best reference combination was SYMPK and ACTB (stability value = 0.209). BestKeeper suggested SYMPK as the best reference in both normal (r = 0.969) and PTC tissues (r = 0.958). SYMPK is suggested as the best reference gene for overcoming the pseudogene problem in RT-qPCR data normalization, with a stability value of 0.319.

TP53 rs1625895 is Related to Breast Cancer Incidence and Early Death in Iranian Population.

Breast cancer as the second most common cancer worldwide tend to be experienced by Iranian women 10 years earlier with a peak incidence at the premenopausal stage. Genetic mutations of TP53 tumor suppressor gene has been shown to be related to early onset of breast cancer. It has been shown already that rs1625895 polymorphic site is related to glioma as well as lung cancer. In this study, we have investigated the role of rs1625895 polymorphism in breast cancer incidence in Iranian women. DNA extraction of 86 breast cancer patients and 96 control individuals have been used for allele-specific primer-PCR and genotyping of allele A and allele G of the TP53 rs1625895. Genotypes frequencies have been shown that GG homozygosis as the most frequent genotype is a significant association with increased risk of breast cancer development in Iranian women (odds ratio = 6, p = 0.002). On the other hand and in comparison to allele G, allele A could cause early death of breast cancer patients by threefolds significantly (p = 0.011). As a conclusion, we show that allele A is the minor allele in both breast cancer patients and also control individuals and major allele G, is related to the increased risk of breast cancer development in Iranian women.

Identification of Two Novel Mutations in KDM3A Regulatory Gene in Iranian Infertile Males

Background: KDM3A is a key epigenetic regulator expressed in the testis and is required for packaging and condensation of sperm chromatin. To this point, the association of the KDM3A gene with infertility has not been studied in human. The aim of this study was to screen any new mutation in KDM3A gene to explore more details of human male infertility. Methods: In this work, 150 infertile men (oligozoospermia and azoospermia) and 150 normal healthy fathers were studied. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing were used to screen any mutation in exons 12, 22, and 24 of KDM3A. Results: The infertile men showed various SSCP patterns for the exons 12 and 24, but not for exon 22. A transversion point mutation in exon 12 and a single nucleotide deletion in exon 24 were detected using sequencing analysis. The transversion mutation was located in the preceding exon of lysine-specific demethylase1 and Jumonji (Jmj)-C domain and the later one (deletion) in the cupin-like motif of KDM3A protein. Neither Y chromosome microdeletions nor partial azoospermia factor deletion was found in these patients. Conclusion: The mutations found in infertile men with otherwise unexplained severe spermatogenic failure could be considered as the origin of their abnormalities.