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Background. The aim of this study was to identify germline mutation of the RET (rearranged during transfection) gene in patients with medullary thyroid carcinoma (MTC) and their first-degree relatives to find presymptomatic carriers for possible prophylactic thyroidectomy. Methods/Patients. We examined all six hot spot exons (exons 10, 11, 13, and 14–16) of the RET gene by PCR and bidirectional Sanger sequencing in 45 Iranian patients with MTC (either sporadic or familial form) from 7 unrelated kindred and 38 apparently sporadic cases. First-degree relatives of RET positive cases were also genotyped for index mutation. Moreover, presymptomatic carriers were referred to the endocrinologist for further clinical management and prophylactic thyroidectomy if needed. Results. Overall, the genetic status of all of the participants was determined by RET mutation screening, including 61 affected individuals, 22 presymptomatic carriers, and 29 genetically healthy subjects. In 37.5% (17 of 45) of the MTC referral index patients, 8 distinct RET germline mutations were found, including p.C634R (35.3%), p.M918T (17.6%), p.C634Y (11.8%), p.C634F (5.9%), p.C611Y (5.9%), p.C618R (5.9%), p.C630R (5.9%), p.L790F (5.9%), and one uncertain variant p.V648I (5.9%). Also, we found a novel variant p.H648R in one of our apparently sporadic patients. Conclusion. RET mutation detection is a promising/golden screening test and provides an accurate presymptomatic diagnostic test for at-risk carriers (the siblings and offspring of the patients) to consider prophylactic thyroidectomy. Thus, according to the ATA recommendations, the screening of the RET proto-oncogene is indicated for patients with MTC.

Background: The membrane form of a Cluster of Differentiation 14 (CD14) is anchored to the phospholipid bilayer via glycosylphosphatidylinositol anchor. This molecule is expressed on the intrinsic surface of monocytes and neutrophils. This protein is not expressed on the HL60 cell surface. It is believed that the differentiation of HL-60 cells stops in the promyelocytic stage. The differentiation of HL-60 cells with compounds such as vitamin A, D, E results in membrane CD14 expression. Objectives: The objective of this study was an evaluation of CD14 expression by Honey Bee Venom (HBV) in HL60 cells treated by D-alpha-Tocopheryl Succinate (D-α-TS). Methods: HL-60 cells were cultured in RPMI Media 1640 medium and treated with different concentrations of D-α-TS and HBV. Cellular differentiation was tested by Nitro Blue Tetrazolium (NBT) staining, immunocytochemistry, and flow cytometry. The studied data were analyzed using one-way analysis of variance and InStat 3 software. Methods: HL-60 cells were cultured in RPMI medium and treated with different concentrations of D-α-TS and HBV. Cellular differentiation was tested by NBT staining, immunocytochemistry, and flow cytometry. The studied data were analyzed using one-way analysis of variance and InStat 3 software. Results: MTT assay demonstrated that HBV and D-alpha-tocopheryl induce death in HL-60 cell lines in a time and dose-dependent manner. Also, Wright-Giemsa, NBT staining showed morphologically differentiation. Immunocytochemistry and flow cytometry analysis shows that cells treated with 6 µg/mL D-α-TS and 2.5 µg/mL HBV for 5 days significantly increase the expression of CD14 in HL-60 compared to cells treated with D-α-TS. Conclusion: HBV can induce cell death and inhibit cell proliferation. Also, increase differentiation potency of D-α-TS via HBV can increase the differentiation by inhibiting NFκB and COX-2 and increasing the expression of P21 that plays an essential role in increasing CD14 protein expression and subsequently induce differentiation in HL-60 cells to monocytes.

Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by inflammation of the articular tissue. This study aims to evaluate the expression of microRNA (miR)-146a-5p, miR-24-3p, and miR-125a-5p in the plasma of RA patients and compare them with those of healthy controls to obtain a specific expression profile for earlier diagnosis and assistance in treating patients. This study was performed on 50 RA patients and 50 healthy controls. Five microliters of blood were taken from each patient/control. Plasma RNA was extracted using the Trisol solution. cDNAs were synthesized; using moloney murine leukemia virus (MMLV) and deoxynucleoside triphosphate (dNTP). Real-time PCR was performed using SYBR green kit. The mean expression of miR-146a-5p, miR-24-3p, and miR-125a-5p in the RA group were 8.1±1.9, 6.5±1.2, and 6.8±2.2 and in the healthy group were 4.8±1.6, 3.6±2.2, and 3.4±1.7, respectively. Significant differences were also observed in the mean expression of these three miRNAs in four subgroups of RA patients with different disease activity based on disease activity score 28 (DAS28) (p

Background: The aim of this study was to identify germline mutation of the RET (rearranged during transfection) gene in patients with Medullary Thyroid Carcinoma (MTC) and their first-degree relatives to find pre-symptomatic carriers for possible prophylactic thyroidectomy.Methods and results: We examined all six hot spot exons (exons 10, 11, 13, and 14-16) of the RET gene by PCR and bidirectional sanger sequencing in 45 Iranian patients with MTC (either sporadic or familial form) from 7 unrelated kindreds and 38 apparently sporadic cases. First-degree relatives of RET positive cases were also genotyped for index mutation. Moreover, pre-symptomatic carriers were referred to the endocrinologist for further clinical management and prophylactic thyroidectomy if needed. Overall, the genetic status of all of the participants was determined by RET mutation screening, including 61 affected individuals, 22 pre-symptomatic carriers and 29 genetically healthy subjects. In 37.5% (17 of 45) of the MTC referral index patients, seven distinct RET germline mutations were found, including p.C634R (35.3%), p.M918T (17.6%), p.C634Y (11.8%), p.C634F (5.9%), p.C611Y (5.9%), p.C618R (5.9%), p.C630R (5.9%), p.L790F (5.9%) and an uncertain variant p.V648I (5.9%). Conclusion: RET mutation detection is a promising/golden screening test and provides an accurate pre-symptomatic diagnostic test for at-risk carriers (the siblings and offspring of the patients) to consider prophylactic thyroidectomy. Thus, mutation screening of the RET hot spot exons could be suggested for any MTC patient to find pre-symptomatic RET carriers in their relatives.

Background: Luteinizing hormone receptor gene shows four nonsynonymous polymorphisms within the exons. Three of these polymorphisms, i.e. rs4539842 (an insertion of 6bp CTGCAG at nucleotide position 54), rs12470652 (c.827A>G/p.Asn 291Ser), and rs2293275(c.935G>A/p.Ser312Asn) have been studied more frequently. Beside other hormones, LH and FSH have an important role in production of competent oocyte and female fertility. Therefore, the objective of the current study was to investigate the prevalence of exons 1(rs4539842) and 10(rs12470652, rs2293275) polymorphisms of the LHCGR gene and its relationship with successful IVF in Iranian infertile women. Methods: SNPs in exons 1 and 10 were analyzed in 100 women of two equally sized groups of IVF failure and IVF success women using genomic DNA. For polymorphisms in exon 10, PCR and direct sequencing were used and for the polymorphism in exon 1, RFLP technique was used. The RFLP technique is confirmed by sequencing. Results: Our results showed significant difference in allelic frequency of SNP rs2293275 among IVF successful and IVF failure groups (p=0.001). For this variation, AA genotype (A allele) was shown to have protective effect against IVF failure (p=0.03 and OR=0.04), while GG genotype (G allele) was a susceptive genotype to IVF failure (p=0.003 and OR=3.88).Allelic frequency of SNP rs4539842 also showed significant difference between the two groups (p=0.0025). For this SNP, subjects with no 6bp insertion (homozygote deletion genotype) were susceptible to failure in IVF (p=0.009 and OR=2.93). Conclusion: It has been revealed that two common SNPs (rs4539842 and rs2293275) in the LHCGR gene are associated with the outcome of IVF in Iranian infertile women. Thus, these two SNPs can be suggested to be used as predictors for IVF outcome in Iranian population.

Cell cycle dysregulation is important in tumorigenesis. Transcriptional silencing of cell cycle regulatory genes, due to DNA methylation, is a common epigenetic event in malignancies. As2O3 has been shown to induce cell cycle arrest and also to be a potential hypomethylating agent. Our study aimed to investigate DNA methylation patterns of cell cycle regulatory genes promoters, the effects of Arsenic trioxide (As2O3) on the methylated genes and cell cycle distribution in colorectal cancer (CRC) cell lines.The methylation-specific PCR (MSP) and/or restriction enzyme-based methods were used to study the promoter methylation patterns of 24 cell cycle regulatory genes in CRC cell lines. Gene expression level and cell cycle distribution were determined by Real-time PCR and flow cytometric analyses, respectively.Our methylation analysis indicated that only promoters of RBL1 (p107), CHFR and p16 genes were aberrantly methylated in three cell lines. As2O3 significantly decreased DNA methylation in promoter regions of these genes and restored their expression. We found that As2O3 significantly reduced the expression of DNA methyltransferase 1 (DNMT1) and increased arsenic methyltransferase (AS3MT). Furthermore, As2O3 altered transcriptional activity of several unmethylated cell cycle regulatory genes including cyclin B1, E1, D1, GADD45A and p21. Cell cycle flow cytometry analysis showed As2O3 induced G2/M arrest in all three cell lines.These data suggest that demethylation and alteration in the expression level of the cell cycle-related genes may be possible mechanisms in As2O3-induced cell cycle arrest in colorectal cancer cells.

FSCN1 gene encodes an actin-bundling protein, FSCN1, which is involved in formation of actin-based structures that contribute to cell migration. High levels of FSCN1 expression is observed in cells with extended membranes and protrusions. Moreover, up-regulation of FSCN1 has been reported in several epithelial carcinomas. Therefore, FSCN1 is thought to play a role in cell movement and invasion. However, the mechanism behind FSCN1 up-regulation is not known. We investigated the expression of FSCN1 using immunohistochemistry. Methylation-specific PCR was adopted to analyze the methylation status of FSCN1 promoter as a potential regulatory mechanism in FSCN1 expression. The samples included papillary thyroid carcinoma, follicular thyroid carcinoma and goiter samples (controls). Methylation of FSCN1 promoter was observed in 50% of follicular, 48.6% of papillary and 60% of controls. The promoter was unmethylated in 16.7% of follicular samples, 5.7% of papillary samples and 26.7% of controls. In the remaining 33.3% of follicular and 45.7% of papillary samples as well as 13.3% of controls, both methylated and unmethylated alleles were amplified, a condition referred to as semi-methylation. The results showed that FSCN1 promoter was significantly hypomethylated in papillary cases while the methylation status was not significantly altered in follicular cases. On the other hand, FSCN1 was expressed in only nine papillary samples. Regarding protein expression and methylation status, we suggest that hypomethylation of FSCN1 promoter in papillary thyroid carcinoma does not lead to overexpression of FSCN1 and that there might be other regulatory mechanisms involved in FSCN1 up-regulation.

Numerous links exist between inflammation and tumor development. Toll-like receptor 4 (TLR4) expression by tumor cells can be a contributing factor that promotes tumor cell proliferation, survival, migration, and metastasis. In this study, we explored the impact of TLR4 inhibition using TAK-242, a specific inhibitor of TLR4, on the invasion properties of ovarian (A2780CP, 2008C13, SKOV3, and A2780S) and breast (MCF7, SKBR3, MDA-MB-231, and BT-474) cancer cell lines. Six out of eight cell lines expressed TLR4 and its downstream mediators (MyD88, NF-ĸB1, and RELB), indicating that these cell lines could be proper candidates for the TLR4 inhibition. TAK-242 induced a cytotoxic effect on all tested cell lines; however, a different cell sensitivity pattern was noticeable. Interestingly, in the TLR4-expressing cell lines, there was a significant correlation between the TLR4/MyD88 expressions and the cancer cell response to TAK-242: the higher the expression, the higher the IC50. To the best of our knowledge, no study has addressed the effects of TAK-242 on invasive abilities of cancer cells and our study suggests for the first time that TAK-242 could considerably decrease invasion properties of ovarian and breast cancer cell lines. We found that not only did TAK-242 reduce the enzymatic activity of MMP2 and MMP9, but also down-regulated gene expressions of epithelial-mesenchymal transition (EMT)-related genes. In sum, it seems that targeting TLR4 using TAK-242 possesses novel promising potential in cancer treatment strategies and may prevent invasion in patients suffering from ovarian and breast cancers, especially in those with over-expression of TLR4.

Background: Follicle-Stimulating Hormone Receptor (FSHR) gene shows five Single Nucleotide Polymorphisms (SNPs) in the promoter region at positions -29, -37, - 114, -123 and -138 that have been reported to be associated with higher levels of FSH and various ovarian responses to FSH in IVF (In-vitro fertilization) treatment at different populations. Hence, they are important regulators of hormone activity at the target level in IVF process. This study was performed to investigate the association between FSHR gene polymorphisms and IVF failure in Iranian women.Methods: SNPs in the promoter region of FSHR gene were analyzed by PCR and direct sequencing technique in 90 women in three equally sized groups of IVF failure, IVF success and normal fertile women, using genomic DNA extracted from white blood cells.Results: No significant differences were found in allelic variants frequency and genotype distribution between each category of subjects when analyzing the FSHR SNPs in the promoter region (p-value >0.05). However, analysis of the data revealed that the subjects with A/A genotype at the –29 position received higher amount of exogenous FSH for ovulation induction compared to G/G genotypes.Conclusions: These results indicate that the FSHR SNP at position –29 may influence sensitivity of the FSHR to FSH for ovulation induction in IVF treatment. It may be concluded that the A/A genotype at position –29 is associated with poor ovarian response to FSH so that subjects with A/A genotype at the –29 position may require higher doses of exogenous FSH for ovulation induction during IVF process.

DNAzymes are nucleic acid enzymes that can recognise specific RNA substrate via Watson-Crick base pairing and cleave it with multiple turnovers. We have designed and examined the effects of DNAzymes targeting the PML/RARalpha fusion gene in acute promyelocytic leukaemia (APL). The DNAzymes (DZ1 and DZ3) were designed to cleave the PML/RARalpha transcript at the GC nucleotides at the fusion point and three nucleotides upstream of that respectively. Disabled DNAzymes were synthesised and used as controls. Cell-free cleavage reactions were performed on total RNA from NB4 cell line and PML/RARalpha and RARalpha-amplified RNA fragments (aRNA). Postcleavage examination showed that DZ1 and DZ3 cleave PML/RARalpha efficiently and specifically. NB4 APL cells transfected with DZ1 or DZ3 showed a significant suppression of PML/RARalpha protein expression. These DNAzymes also inhibited the proliferation of NB4 cells, reduced the viability rate, and induced apoptosis in these cells. The disabled DNAzymes showed no effect on NB4 cells. The two DNAzymes did not produce any significant effect on K562 cells, which were used as control cells. DNAzymes are more resistant to serum than ribozymes. These data show that targeting the PML/RARalpha fusion gene with DNAzymes can induce apoptosis in APL cells and may have a role in the treatment of APL. They also show DNAzymes are promising tools for targeting specific genes in leukaemia.