Folate receptor alpha autoantibodies in the serum of patients with relapsing-remitting multiple sclerosis (RRMS).

OBJECTIVE: Multiple sclerosis (MS) is a potentially progressive, autoimmune neurologic disorder of the central nervous system (CNS), resulting from an autoimmune attack on central nervous system white matter. Folate deficiencies are linked to DNA instability and breakdown of phospholipid membranes and thus might affect myelin integrity. Folic acid exerts its effects through its receptors (FRs). Folate receptor alpha autoantibodies (FRAA) can block folate transport to the brain. Due to important role of folate in the pathogenesis of MS, in this project we aimed to study FRAA serum levels in patients with relapsing remitting multiple sclerosis (RRMS). METHODS: Fifty-four patients with RRMS and 58 healthy individuals were enrolled in this study. Serum samples were collected from all participants and folate receptor alpha autoantibody (FRAA) serum concentration was measured by Enzyme-linked immunosorbent assay (ELISA). RESULTS: The results showed that FRAA serum levels in patients with RRMS is 67.20 ± 19.79 ng/ml as compared to controls which was 37.32 ± 13.26 ng/ml. Significant increase in folate receptor autoantibody serum concentration was seen in patients with RRMS when compared to control group (P = 0.007). The results showed that a high concentration of folate receptor autoantibody is associated with RRMS. We have also found that 85.18% (46/54) of patients with RRMS were positive for serum FRAA, whereas the prevalence in controls was only 46.55% (27/58). CONCLUSIONS: It is concluded that serum FRAA are more prevalent in RRMS patients than controls. The findings also suggest that FRAA might be involved in the pathophysiology of RRMS.

Effect of in ovo feeding of folic acid on Disabled-1 and gga-miR-182-5p expression in the cerebral cortex of chick embryo.

Folate (vitamin B9) has been shown to reduce the prevalence of neural tube defects (NTDs). Many genes comprising Disabled-1 (DAB1) and miRNAs have been shown to play important role in normal brain development. Reelin-signalling has been shown to play key role in regulating of neuronal migration during brain development. The aim of this study was to evaluate the effects of in ovo administration of folic acid (FA) on DAB1 and gga-miR-182-5p expression in the cerebral cortex of chick embryo. A total number of 30 hatching eggs were used in this study. The number of 10 eggs were injected into the yolk sac with FA (150 µg/egg), 10 eggs by normal saline (sham group) on embryonic day 11 and 10 eggs were left without injection as control. Then the cerebral cortices were collected on E19 and the expression of DAB1 and gga-miR-182-5p was studied by Real-Time PCR. The results showed that DAB1 expression in the cerebral cortex of FA-treated, sham and control were 2.51 ± 0.13, 1.01 ± 0.04 and 1.03 ± 0.04 fold changes, respectively, and this amount for gga-miR-182-5p were 0.54 ± 0.03, 1.09 ± 0.07 and 1.00 ± 0.06-fold change respectively. Statistical analysis showed that there is a significant increase in DAB1 and a decrease in gga-miR-182-5p expression in FA injected cerebral cortex as compared either with either SHAM or control (p < 0.0001). But, no significant change in DAB1 and gga-miR-182-5p expression was observed between sham and the control group (p = 0.99 and p = 0.57 respectively). It is concluded that in ovo feeding of FA increases DAB1 and decreases gga-miR-182-5p expression in the developing chick cerebral cortex.

The importance of SOCS1 - 1478 CA/del polymorphism and expression in breast cancer: a case-control study in the north of Iran.

PURPOSE: This project aimed to evaluate the relationship between the suppressor of cytokine signaling-1 (SOCS1) - 1478 CA > del genetic variation and breast cancer susceptibility. Moreover, we investigated the SOCS1 mRNA expression level in cancerous tissues. METHODS: A total of 100 patients with breast cancer and 120 healthy individuals were selected. Genomic DNA was extracted from blood. SOCS1 genotyping and relative gene expression were performed using ARMS-PCR (Amplification-Refractory Mutation System-Polymerase Chain Reaction) and real-time PCR, respectively. RESULTS: In breast cancer patients, the prevalence of genotype frequencies of SOCS1 (- 1478 CA > del) CA/CA, CA/del, and del/del was 52, 31, and 17%, respectively. Among controls, the distribution of CA/CA, CA/del, and del/del was 63, 15, and 22%, respectively. The chi-square test reported that a significant difference was observed in the genotypic distribution of SOCS1 (- 1478 CA > del) polymorphism between cases and controls (χ2 = 8.08, P = 0.01). In addition, the presence of the CA/del genotype was associated with an elevated risk of breast cancer (in the codominant model: OR 2.51; 95% CI 1.27-4.96, P = 0.007 and in the over dominant model: OR 2.54; 95% CI 1.32-4.90, P = 0.005). However, there was no significant difference in allelic distributions between the groups (P > 0.05). There was no significant difference in the breast cancer risk associated with the dominant and recessive genetic models when the reference was CA/CA and CA/CA + CA/del genotype, respectively (P = 0.09 and P = 0.38). Moreover, the expression of SOCS1 decreased in cancerous tissues as compared to the adjacent non-cancerous tissues (P < 0.0001). CONCLUSION: In conclusion, a functional SOCS1 promoter polymorphism (- 1478 CA > del) may affect breast cancer susceptibility.

The Association of Soluble VEGFR-1 Serum Level and Genetic (rs7993418) Polymorphism with In Vitro Fertilization and Embryo Transfer Outcome in the Population of Northern Iran.

Background: Vascular endothelial growth factor receptors (VEGFRS) play an important role in embryo implantation. The aim of the present study was to examine the association of VEGFR1 circulating level and gene polymorphism with in vitro fertilization and embryo transfer (IVF-ET) outcome. Methods: In this case-control study, 120 women who had unsuccessful IVF (IVF-) history and 120 women who had successful IVF outcome (IVF+) as controls were included. Genomic DNA was extracted from blood samples. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The serum levels of soluble VEGFR1 (sVEGFR1) were measured by ELISA. ANOVA test was used for statistical analysis. Results: The frequency of T and C alleles in IVF+ individuals were 87.5%, 12.5% and among IVF- were 75.5%, 24.5%, respectively (p=0.0006). The minor allele (C) was associated with an increased risk of IVF failure based on results from co-dominant (OR=3.86, 95%CI 1.19-12.47), dominant (OR=2.32, 95%CI 1.31-4.10), recessive (OR=3.22, 95%CI 1.00-10.29), and allele models (OR=2.28, 95%CI 1.40-3.69). We also showed that there is a significant decrease in serum sVEGFR1 levels in IVF as compared to IVF+ (p=0.006) groups. Moreover, TT genotype is significantly associated with increased serum sVEGFR1 concentration in IVF group (TT, CT, and CC serum levels were 106.55±11.04, 94.33±10.75, and 83.33±9.13 ng/ml, and in IVF+ group were 156.11±18.08, 120.66±16.51, and 84.66±20.31 ng/ml, respectively). Conclusion: The results of this study indicate that VEGFR1 polymorphism and sVEGFR1 circulating levels are associated with IVF-ET outcome. Moreover, CC genotype is associated with decreased sVEGFR-1 serum concentration and IVF-ET failure.

Association of tissue inhibitor of metalloproteinase-3 (TIMP-3) serum level and its genetic polymorphism with pregnancy outcome of patients undergoing in vitro fertilization and embryo transfer.

OBJECTIVES: Tissue inhibitors of metalloproteinase-3 (TIMP-3) and matrix metalloproteinases (MMPs) play a major role in embryo implantation and placentation. This study aimed to investigate the relationship between TIMP-3 serum level and TIMP-3 genetic polymorphism with pregnancy outcome in patients undergoing in vitro fertilization and embryo transfer (IVF-ET). MATERIAL AND METHODS: This project included 100 infertile women who became pregnant after IVF (IVF+) and 100 infertile women who failed to conceive after IVF (IVF-). Genotyping was performed using Restriction Fragments Length Polymorphism Polymerase Chain Reaction (PCR-RFLP), and the serum level was measured by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: The frequencies of TT, TC, and CC in the IVF+ group were 41%, 37% and 22%, respectively, while in the IVF- group were 18%, 43% and 39%, respectively. The C and T allele frequencies were 40.5% and 59.5% in the IVF+ group and 60.5% and 39.5% in IVF- group, respectively. The C allele conferred a 2.25-fold increased risk of IVF failure (OR 2.25; 95% CI 1.5-3.35; p = 0.0001). Also, there was a significant increase in TIMP-3 serum levels in the IVF- group (193.29 ± 29.50 ng/mL), which was higher than the IVF+ group (166.74 ± 17.60 ng/mL; p = 0.00002), was demonstrated. It was shown that the TT genotype is associated with decreased TIMP-3 serum levels in IVF- group (CC, CT, and TT, values were 143.19 ± 88.49 ng/mL, 117.55 ± 15.73 ng/mL, 61.17 ± 44.36 ng/mL, respectively). CONCLUSIONS: It is concluded that there is a relationship between TIMP-3 gene polymorphism and its serum concentration with IVF-ET outcome. We also suggest that the TT genotype might be involved in IVF-ET outcome.

Effect of in ovo feeding of folic acid on brain derived neurotrophic factor (BDNF) and gga-miR-190a-3p expression in the developing cerebral cortex of chickens.

Folate plays important role in biosynthesis of purine and pyrimidine nucleotides and is therefore crucial for DNA synthesis and neurogenesis in fetal brains. Many genes comprising brain derived neurotrophic factor (BDNF) and miRNAs have been shown to play important role in brain development. Gga-miR-190a-3p targets many genes including BDNF. The aim of this project was to study the effects of in ovo administration of folic acid (FA) on BDNF and gga-miR-190a-3p expression in the cerebral cortex of chick embryo. A total number of 120 hatching eggs with the correct shape and weight were used in this experiment. Forty eggs was injected by FA into the yolk sac at a dose of 150-µg per egg, 40 eggs by PBS (SHAM) on embryonic day 11 and 40 eggs were left without injection as controls. Then the cerebral cortex was collected on E19 and BDNF and gga-miR-190a-3p expression was studied using Real time PCR. The results showed that BDNF expression in the cortex of FA treated, SHAM and controls were 2.06 ± 0.29, 1.12 ± 0.12 and 1.02 ± 0.21 fold changes, respectively and for gga-miR-190a-3p were 0.72 ± 0.08, 0.95 ± 0.09 and 1.007 ± 0.12 fold change, respectively. Statistical analysis showed that there is significant increase in BDNF expression and decreased gga-miR-190a-3p expression in FA injected cerebral cortex as compared either with SHAM or controls. Although, no significant change in BDNF and gga-miR-190a-3p expression were observed between SHAM and controls. It is concluded that in ovo administration of FA increases BDNF and decreases gga-miR-190a-3p expression in the developing chick cerebral cortex.

Polymorphisms of miR-146a and susceptibility to ulcerative colitis risk: a case-control study.

Considering the role of miR-146a in the control of inflammation, we assessed the importance of two miR-146a polymorphisms (rs2910164 and rs57095329) in the development and severity of ulcerative colitis (UC) in Iran. Genomic DNA of 150 cases with UC and 200 healthy individuals were genotyped using the PCR-RFLP technique. Statistical analyses were performed using Med Calc software. The miR-146a rs2910164 C allele was significantly associated with increased risk of UC. Individuals carrying the CC (rs2910164) were more than fourfold higher risk of UC relative to wild type homozygotes. The combined GC + CC genotypes were also associated with increased UC risk. We also found that the rs2910164 CC genotype was associated with a severe form of the disease However, the distribution of variant allele and genotypes of rs57095329 did not differ between the cases and controls. In conclusion, miR-146a rs2910164 polymorphism may play a role in UC. To confirm our findings, additional well-designed studies in diverse ethnic populations are required.

The Impact of STAT3 rs1053005 Variation on Type 1 Diabetes Mellitus Susceptibility: Association Study and in Silico Analysis.

AIMS: Type 1 diabetes (T1DM) is an autoimmune disorder with multiple genetic and environmental risk factors that are still poorly understood. The signal transducer and activator of transcription (STAT) proteins play a pivotal role in immune-cell genesis and regulation. This study aimed to determine the effect of rs1053005 single nucleotide polymorphism (SNP) in 3'-UTR of STAT3 mRNA on the susceptibility to T1DM in an Iranian population. METHODS: PCR-RFLP was conducted on 250 T1DM patients and 250 control cases to assess STAT3 rs1053005 polymorphism. Moreover, several bioinformatics tools were employed to identify the candidate miRNAs targeting the STAT3 mRNA region under study as well as the effect of rs1053005 on their binding site. RESULTS: Significant variations in the distribution of genotypes and alleles were seen between cases and controls. The comparison results of the frequency of AA, AG, and GG genotypes between T1DM patients and control groups were 49.2% versus 64.8%, 39.2 versus 30%, and 11.6 versus 5.2%, respectively. Individuals who carried GG genotype were at 2.93-fold increased risk of developing T1DM and the G allele was associated with 1.79-fold higher T1DM risk. Bioinformatics analysis demonstrated that due to rs1053005, the interaction of 3 miRNAs were broken, 3 were weakened, 2 were reinforced, and 4 binding sites were created. CONCLUSION: The result of this study indicates an association between STAT3 rs1053005 and T1DM susceptibility which may be due to interference of the SNP with native-binding site of some predicted miRNAs.

miR-203a-3p, ABL1 and TP63 gene expression is altered in the endometrium of women with endometriosis.

OBJECTIVE: Many genes and miRNAs have been shown to be associated with the pathogenesis of endometriosis. TP63 (p63) is implicated in lineage specification, proliferative potential, differentiation, cell death and survival. The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase implicated in cell differentiation, cell division, and cell adhesion. Moreover, hsa-miR-203a-3p was reported to play pivotal roles in tumor progression by targeting multiple genes, including ABL1 and TP63. The aim of this study was to investigate the expression of ABL1, TP63, and miR-203a-3p in endometriosis. METHODS: This study included 30 women with endometriosis (stage III: n = 12 and stage IV: n = 18) and 30 age-matched controls. Total RNA extraction and cDNA synthesis were performed, and a quantitative polymerase chain reaction technique was used to determine the expression of miR-203a-3p, TP63, and ABL1. RESULTS: TP63 and ABL1 were significantly overexpressed in stages III and IV endometriosis as compared to controls (p < .0001). Moreover, overexpression of ABL1 and TP63 was observed in stage IV compared to stage III (p = .0006 and p = .0002, respectively). Furthermore, significant increase miR-203a-3p expression has been seen in stage IV endometriosis compared to controls (p = .006). The expression of miR-203a-3p in stage III was not significant compared to stage IV and control (p = .33 and p = .43, respectively). CONCLUSION: It is concluded that miR-203a-3p, ABL1 and TP63 gene expression is altered in the endometrium of patients with endometriosis. It is also suggested that miR-203a-3p, ABL1, and TP63 might be candidate factors for the pathogenesis of endometriosis and suggesting its therapeutic potential in endometriosis.

Analysis of Insulin-like growth factor-1 serum levels and promoter (rs12579108) polymorphism in the children with autism spectrum disorders.

OBJECTIVES: Autism spectrum disorder (ASD) is a set of neurodevelopmental disorders characterized by a deficit in social behaviors and nonverbal interactions such as reduced eye contact and facial expression. Changes in growth factors expression including Insulin-like growth factors (IGFs) have been shown in ASD. This project aimed to study the association of IGF-1 (rs12579108) promoter polymorphism and its serum concentration with ASD. METHODS: DNA was extracted from blood samples of 200 ASD children and 198 healthy controls and genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and IGF-1 serum concentration was measured by ELISA. RESULTS: The results showed that the prevalence of AA, CA, and CC genotypes were 2%, 61%, 37% in controls and 4%, 31%, and 65% in ASD patients, respectively (P = 0.0005). The prevalence of A and C alleles in the controls were 33% and 67% and in ASD patients were 19% and 81%, respectively (P = 0.001). We have also showed that serum IGF-1 concentration in ASD and control groups was 31.45 ± 9.84 and 54.62 ± 11.63 ng/ml, respectively (P = 0.001). Our results also showed that AA genotype is significantly related to decreased serum IGF-1 concentrations in ASD (CC, CA and AA serum levels were 50.22 ± 12.68, 33.55 ± 9.13 and 22.55 ± 7.26 and in controls were 77.88 ± 17.14, 54.77 ± 15.31 and 31.33 ± 9.91 ng/ml, respectively). CONCLUSION: It is concluded that there is a significant association between IGF-1 (rs12579108) promoter polymorphism and its serum concentration with ASD. We also suggest that AA genotype is linked to lower IGF-1 serum levels and may play as risk factor for ASD.

The association of stem cell factor and soluble c-Kit (s-cKit) receptor serum concentrations with the severity and risk prediction of autism spectrum disorders.

S tem cell factor (SCF) and its receptor (c-kit) signaling play important role in normal brain physiology including neurogenesis, synapse formation and spatial learning function of the hippocampal region of the brain. Autism spectrum disorder (ASD) is believed to result from abnormal development of neuronal networks and synaptic function. The aim of this study was to evaluate the SCF and its soluble receptor (s-ckit) serum concentrations in ASD. We also studied the serum SCF and s-ckit concentration with the severity of ASD (Levels 1-3; Mild, Moderate and severe, respectively). Ninety five patients with ASD (Mild; n=33, Moderate; n=32 and severe; n=30) and 82 normal controls age matched were included in this study. The serum concentration of SCF and s-ckit were measured by enzyme-linked immunosorbent assay (ELISA). The SCF serum concentration in control subjects was 3.45±1.06 ng/ml and in ASD was 3.41±0.92 ng/ml (P=0.88). The serum levels of s-ckit in control and ASD groups were 56.82±13.22 ng/ml and 67.11±12.00, respectively (P=001). We have also studied serum SCF and s-ckit concentrations with the severity of ASD. The serum concentration of SCF in mild, moderate and severe ASD groups was 3.45±0.93, 3.4±0.87 and 3.43±0.98 ng/ml, respectively (P>0.05) and for s-ckit was 48.77±9.28, 61.66±12.18 and 93.11±14.81ng/ml, respectively (P<0.05). The result of this study suggests that serum s-cKit concentrations may provide a reliable and practical indicator of ASD and positively correlated with disease severity. It is also concluded that s-cKit might be involved in the pathophysiology of ASD.

The effect of mitochondria-targeted antioxidant MitoQ10 on redox signaling pathway components in PCOS mouse model.

PURPOSE: Considerable evidence suggests that mitochondrial dysfunction and oxidative stress contribute to the pathogenesis of Polycystic ovary syndrome (PCOS). We aimed to evaluate the effectiveness of mitochondria-targeted antioxidant, MitoQ10, on the redox signaling pathway's component in PCOS. METHOD: We assessed TXNIP, TRX, and ASK1 expression in granulosa cells (GCs) of the DHEA-induced PCOS mouse model. Female BALB/c mice in five groups of Control, DHEA, and DHEA + MitoQ10 in three doses of 250, 500, and 750 µmol/L MitoQ10 were treated for 21 days. RESULTS: Histological investigation showed a probable improvement in folliculogenesis; besides, ASK1 and TXNIP expression were significantly increased in GCs of the PCOS mouse F4Fmodel as compared to the control groups and decreased steadily in groups treated by MitoQ10. However, TRX expression showed a drop that was restored by MitoQ10 meaningfully (P ≤ 0.05). CONCLUSION: The work presented herein suggests mitochondria-targeted antioxidant, MitoQ10, have modulating effects on folliculogenesis in the ovary and also on the redox signaling pathway in GCs of PCOS mouse model which may have potential to attenuate oxidative stress and its relative damages.

How matrix metalloproteinase (MMP)-9 (rs3918242) polymorphism affects MMP-9 serum concentration and associates with autism spectrum disorders: A case-control study in Iranian population.

The aim of this project was to evaluate the relationship of matrix metalloproteinase-9 (MMP-9) genetic variation and its serum concentration with autism spectrum disorder (ASD). One hundred ASD and 120 controls were enrolled in this study. Genomic DNA was extracted from blood and MMP-9 polymorphism was determined by polymerase chain reaction restriction fragment length polymorphism and serum levels were measured by enzyme-linked immunosorbent assay. The frequencies of CC, CT, and TT genotypes were 72%, 26%, and 2% in controls and 31%, 57%, and 12% in ASD, respectively. The frequencies of C and T alleles in ASD were 59.5% and 40.5%, and controls were 86% and 14%, respectively. There is a significant increase in serum MMP-9 levels in ASD as compared to controls. We have also shown that TT genotype is significantly associated with increase serum MMP-9 levels in patients (TT, CT, and CC serum levels were 91.77 ± 10.53, 70.66 ± 7.21, and 38.66 ± 5.52 and in controls were 55.55 ± 11.39, 42.66 ± 7.85, and 30.55 ± 6.34 ng/ml, respectively). It is concluded that there is a significant association between rs3918242 MMP-9 polymorphism and its serum concentration with autism. We also suggest that TT genotype is associated with increased MMP9 expression and may be a risk factor for ASD.

Overexpression of Hepatocyte growth factor and its soluble receptor (s-cMet) in the serum of patients with different grades of meningioma.

OBJECTIVES: Meningiomas are the most common primary intracranial tumor. Hepatocyte growth factor (HGF) and its receptor, cMet, were shown to be involved in meningioma. This study was aimed to determine the concentration of HGF and soluble cMet (s-cMet) in the serum of patients with different grades of meningioma. METHODS: Ninety serum samples from different grades of meningioma patients (42 cases of grade I, 28 grade II, 20 grade III) and 51 controls were included in this study. The serum total protein concentration (TPC) was measured by a Bio-Rad protein assay and serum concentration of HGF and s-cMet by enzyme linked immunosorbent assay (ELISA). RESULTS: No significant change in the serum TPC of patients was seen as compared to controls. We also showed that serum HGF and s-cMet concentration in meningioma patients was higher than in controls. The results showed that starting from grades I to III meningioma, a significant increase in HGF and s-cMet serum concentration was observed (HGF; 380 ± 57.69, 430.27 ± 48.72, 596.36 ± 104.49 pg/ml, respectively, as compared to controls which was 327.72 ± 49.68 pg/ml and for s-cMet was 274.45 ± 45.05, 314.81 ± 38.71, 433.54 ± 51.81 ng/ml, respectively, as compared to controls which was 213.72 ± 29.13 ng/ml). The results showed that a high concentration of HGF and s-cMet is associated with advanced grades of meningioma. CONCLUSION: It is concluded that HGF and s-cMet serum levels increased in meningioma patients and their concentration was significantly higher in more advanced grades of the disease. It is also suggested that HGF/s-cMet might be involved in the progression of meningioma.

The association of SHANK3 gene polymorphism and autism.

BACKGROUND: Autism spectrum disorder (ASD) and Autism are both general terms for a group of complex disorders of brain development. These disorders are characterized by difficulties in social interaction, verbal and nonverbal communication and repetitive behaviors. Many genes have been shown to be involved in Autism. SHANK3 (SH3 and multiple ankyrin repeat domain 3) is a member of the highly conserved Shank/ProSAP family of synaptic scaffolding proteins. SHANK3 is suggested as a strong candidate gene for the pathogenesis of Autism and its loss results in disruption of synaptic function. The rs9616915 SNP, which directly affects SHANK3 gene function of splicing regulation and protein structure damage, is a non-synonymous SNP (T>C) that found in exon 6, leads to substitution of Isoleucine to Threonine. The present study was aimed to evaluate whether rs9616915 polymorphism of SHANK3 are related with the susceptibility to Autism. METHODS: Samples were obtained from 90 patients diagnosed with Autism and 100 controls subjects and genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP). The results of this study showed that there is a significant association in genotype distribution between cases and controls (P=0.0001). RESULTS: Our findings revealed that individuals with TC genotypes were associated with increased risk of Autism disorder (OR=4.35, 95% CI: 2.15-8.80, P=0.0001) but no significant differences were found in allele distributions (P=0.1). CONCLUSIONS: Our results indicated that the SHANK3 rs9616915 polymorphism is associated with increased risk of Autism. Larger studies with more patients and controls are needed to confirm the results.

The reaggregation of normal granulosa-cumulus cells and mouse oocytes with polycystic ovarian syndrome in vitro: An experimental study.

BACKGROUND: The dialogue between oocytes and their surrounding cells plays a major role in the progress of oocyte meiosis and their developmental potential. OBJECTIVE: This study aimed to evaluate the effect of co-culture of normal granulosa-cumulus cells (GCCs) with oocytes from polycystic ovarian syndrome (PCOS) mice. MATERIALS AND METHODS: Normal GCCs were collected from 10 virgin adult Naval Medical Research Institute female mice (30-35 gr, 7-8 wk old), and were cultured in an alpha-minimum essential medium supplemented with 5% fetal calf serum for 24-48 hr (1 × 10 6 cells/well). Then, germinal-vesicle oocytes from PCOS mice were cultured in the presence of cultured normal GCCs (experimental group) and without GCCs (control group). The maturation rate and quality of the PCOS oocytes were examined by evaluating TFAM and Cx43 gene expression (real-time PCR) and the connection among PCOS oocytes and normal GCCs after 24 hr of culture. RESULTS: The co-culture of normal GCCs and PCOS oocytes in the experimental group led to the formation of a complex called a PCOS oocyte-normal GCCs complex. The maturation rate of these complexes was significantly increased compared to that of the control group (p ≤ 0.001). A significant difference was also found in the expression of Cx43 (p ≤ 0.001) and TFAM (p < 0.05) genes in the experimental group compared with the control group. The connection between PCOS oocytes and normal GCCs was observed in the scanning electron microscope images. CONCLUSION: Co-culture with normal GCCs improves the capacity of PCOS oocytes to enter meiosis, which may result in the promotion of assisted reproduction techniques.

Influence of a 5-bp Indel Polymorphism at Promoter of the GAS5 lncRNA and Risk of Breast Cancer.

Long non-coding RNAs (lncRNAs) are RNA molecules (>200 nucleotides in length) with no protein-coding capacity. Recent studies have demonstrated that lncRNAs involve in the regulation of their target genes at transcriptional, post-transcriptional and epigenetic levels. The aim of this case-control study was to explore whether growth arrest-specific 5 (GAS5) lncRNA 5-bp Ins/Del (rs145204276) polymorphism is involved in the breast cancer susceptibility. A total of 170 cases and 220 age matched controls were recruited in this study. GAS5 lncRNA polymorphism was genotyped using tetra primers amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) method. Statistical analysis was performed using SPSS. The distribution of the genotype ins/ins, ins/del and del/del were %75.29, 21.76% and 2.94% and 52.27%, 39.55% and 8.81% in the cases and controls, respectively. The ins/del or del/del genotype had a significantly decreased risk of breast cancer as compared with the ins/ins genotype under a codominant model (OR=0.38, 95%CI 0.24-0.60, p=0.0001; OR= 0.25, 95%CI 0.09-0.69, p=0.008, respectively). Moreover, the deletion allele of this polymorphic site is associated with a protective effect (OR=0.41, 95%CI 0.28-0.60, p=0.0001). Our study provided the first evidence that the deletion allele of GAS5 rs145204276 may have a protective role in mediating individual susceptibility to breast cancer. However, further comprehensive studies are warranted in a larger sample.
.

Matrix metalloproteinase (MMP)-1 and MMP-3 gene variations affect MMP-1 and -3 serum concentration and associates with breast cancer.

Matrix metallopeptidases (MMPs) 1 and 3 have been shown to contribute to the initiation, and progression of different cancers, including breast cancer (BC). In this study, we aimed to examine the relations between polymorphisms of MMP1 (rs1799750) and MMP3 (rs632478) and their circulating levels with BC. The polymorphisms were genotyped by PCR-based Restriction Fragment Length Polymorphism (RFLP) and Allele-Specific PCR (AS-PCR) among 100 patients and 100 controls. MMP1 and MMP3 serum levels were measured by enzyme-linked immunosorbent assay (ELISA). Genotype distributions of MMP1 and MMP3 genes showed significant difference between patients and controls. The distribution of 2G/2G, 1G/2G and 1G/1G genotypes for MMP1 was 74%, 2% and 24% in the patients and 38%, 2% and 60% in the controls, respectively (P = 0.0001). For MMP3 the distribution of C/C, A/C and A/A genotypes was 28%, 54% and 18% in patients and 48%, 40% and 12% in controls, respectively (P = 0.01). For MMP1, the 2G/2G genotype was linked with a higher risk of BC when compared with that of the 1G/1G genotype (OR = 4.86; 95% CI = 2.63-8.99; P = 0.0001). For MMP3, in co-dominant model, there was a higher risk of BC in A/A and A/C genotype carriers (A/A: OR = 2.57; 95% CI = 1.08-6.11; P = 0.03) (A/C: OR = 2.31 95% CI = 1.24-4.30; P = 0.008). We also showed that MMP1 and MMP3 serum level was significantly increased in BC patients compared to controls. MMP1 and MMP3 genetic variations and their circulating levels are both significantly related to BC.

A33512C and Intronic Poly(AT) Insertion/Deletion (PAT-/+) Polymorphisms of the XPC Gene and Their Association With the Risk of Breast Cancer.

BACKGROUND: Several lines of evidence suggest that A33512C and intronic poly(AT) insertion/deletion (PAT-/+) polymorphisms of the XPC gene is associated with various types of malignancy. This case-control study aimed to determine the possible association between A33512C and PAT-/+ polymorphisms of the XPC gene and breast cancer (BC). PATIENTS AND METHODS: A total of 200 women diagnosed with BC as cases and 200 ethnically matched healthy controls were genotyped for A33512C and PAT-/+ polymorphisms of the XPC gene by PCR-restriction fragment length polymorphism and PCR methods, respectively. The possible association between XPC A33512C and PAT-/+ polymorphisms with the risk of BC were also analyzed. RESULTS: PAT-/+ polymorphism of the XPC gene was significantly associated with increased risk of BC (P < .05), whereas there was no association between XPC A33512C polymorphism and BC (P > .05). The frequency of the XPC PAT+ allele in BC patients was significantly higher than those in healthy controls (odds ratio, 0.561; 95% confidence interval, 0.403-0.779; P < .05). The combined genotypes AC/PAT+/+ and CC/PAT+/+ were significantly associated with increased risk of BC. CONCLUSION: The prevalence of XPC PAT+ allele was significantly higher in patients with high-tumor-stage disease compared to healthy controls. Overall, the significantly higher frequency of the PAT+ allele in the BC group compared to the control group may suggest an etiologic link between the presence of the PAT+ allele and the risk of BC.

Targeting signaling pathways of VEGFR1 and VEGFR2 as a potential target in the treatment of breast cancer.

Tumor angiogenesis allows tumor cells to grow and migrate toward the bloodstream and initiate metastasis. The interactions of vascular endothelial growth factors (VEGF) A and B, as the important regulating factors for blood vessel growth, with VEGFR1 and VEGFR2 trigger angiogenesis process. Thus, preventing these interactions led to the effective blockade of VEGF/VEGFRs signaling pathways. In this study, the inhibitory effect of a 23-mer linear peptide (VGB4), which binds to both VEGFR1 and VEGFR2, on VEGF-stimulated Human Umbilical Vein Endothelial Cells (HUVECs) and highly metastatic human breast cancer cell MDA-MB-231 proliferation was examined using MTT assay. To assess the anti-migratory potential of VGB4, HUVECs and also MDA-MB-231 cells wound healing assay was carried out at 48 and 72 h. In addition, downstream signaling pathways of VEGF associated with cell migration and invasion were investigated by quantification of mRNA and protein expression using real-time quantitative PCR and western blot in 4T1 tumor tissues and MDA-MB-231 cells. The results revealed that VGB4 significantly impeded proliferation of HUVECs and MDA-MB-231 cells, in a dose- and time-dependent manner, and migration of HUVECs and MDA-MB-231 cells for a prolonged time. We also observed statistically significant reduction of the transcripts and protein levels of focal adhesion kinase (FAK), Paxillin, matrix metalloproteinase-2 (MMP-2), RAS-related C3 botulinum substrate 1 (Rac1), P21-activated kinase-2 (PAK-2) and Cofilin-1 in VGB4-treated 4T1 tumor tissues compared to controls. The protein levels of phospho-VEGFR1, phospho-VEGFR2, Vimentin, ß-catenin and Snail were markedly decreased in both VGB4-treated MDA-MB-231 cells and VGB4-treated 4T1 tumor tissues compared to controls as evidenced by western blotting. These results, in addition to our previous studies, confirm that dual blockage of VEGFR1 and VEGFR2, due to the inactivation of diverse signaling mediators, effectively suppresses tumor growth and metastasis.