Anti-cancer effect of metformin on the metastasis and invasion of primary breast cancer cells through mediating NF-kB activity.

Current evidence strongly suggests that aberrant activation of the nuclear factor kappa B (NF-kB) signaling cascade is connected to carcinogenesis. The matrix metalloproteinases (MMP) which are also the key agents for tumor metastasis may be potent candidates for tumor diagnosis in clinics. In this in vitro study, we hypothesized that metformin with an effective dose can inhibit tumor cell proliferation and metastasis by modulating the expressions of MMP-2 and -9 and interfering with NF-kB signaling in primary breast cancer cells (PBCCs). 300 000 cells per ml were obtained from biopsies of breast tumors from five human donors. The cell viability and proliferation were tested. Immunocytochemistry was performed for MMP-2, MMP-9, and NF-kB, and enzyme-linked immunosorbent assay for NF-kB activity, quantitative real-time PCR for RELA/p65, IkBα, MMP-2, and MMP-9. Three different doses of metformin (5, 10, and 25 mM) (Met) reduced the viability and proliferation of PBCCs in a dose-dependent manner, maximum inhibition was observed at 25 mM Met. The expression of RELA/p65 was not affected by 25 mM Met. Nuclear immunoreactivity and activity of NF-kB reduced while cytoplasmic NF-kB (p65) elevated by 25 mM Met compared to non-treatment (P <  0.05). The expression and immunoreactivity of MMP-9 but not MMP-2 were decreased by 25 mM Met treatment, compared with the non-treatment (P <  0.05). Metformin may have an essential antitumor role in the invasion and metastasis pathways of PBCCs by downregulating the MMP-9 expression blocking both the activity and nuclear translocation of NF-kB.

Fas and microRNAs Variations as a Possible Risk for Behçet Disease.

BACKGROUND: Behçet disease (BD) belongs to a disease family that has a transparent borderline between autoinflammatory and autoimmune disorders. Fas and some miRNAs have revealed to display remarkable roles in both autoimmune and autoinflammatory processes, and they can play important roles in defective apoptosis in BD. We investigated the association of the susceptibility of BD with Fas, miRNA variations, and their both single and combined presence in a Turkish population as a case-control study. METHODS: The distributions of FAS-670 A>G rs1800682, mir146a rs2910164, and mir196a rs11614913 polymorphisms are analyzed with the polymerase chain reaction-restriction fragment length polymorphism method in 115 BD patients and 220 controls in 6-month period. RESULTS: Statistical analysis indicates that in the case of Fas-670 A/G rs1800682, AA genotype and A allele have a protective role in BD (p = 0.0004 and p = 0.0009, respectively). The dominant model (AA + AG/GG) also displays a protective effect on BD unlike the recessive model (p = 0.03). In addition, both homozygous genotype (CC) of rs2910164 of mir-146a (p = 0.04) and the dominant model (CC + CG vs. GG) have protective effects on BD unlike the recessive model (p < 0.0001). Both mir-196a2 rs1800682 polymorphism and combined genotype analysis of rs1800682-rs2910164 and rs1800682-rs11614913 gave no statistically significant differences within the groups for genotypes and either of the alleles (p > 0.05). CONCLUSIONS: These findings indicate that both Fas rs1800682 and mir-146a rs2910164 variants might be important factors participating in the protection against BD in the Turkish population.

LEF1 Induces DHRS2 Gene Expression in Human Acute Leukemia Jurkat T-Cells

Objective: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease resulting from the accumulation of genetic changes that affect the development of T-cells. The precise role of lymphoid enhancer-binding factor 1 (LEF1) in T-ALL has been controversial since both overexpression and inactivating LEF1 mutations have been reported to date. Here, we investigate the potential gene targets of LEF1 in the Jurkat human T-cell leukemia cell line. Materials and Methods: We used small interfering RNA (siRNA) technology to knock down LEF1 in Jurkat cells and then compared the gene expression levels in the LEF1 knockdown cells with non-targeting siRNA-transfected and non-transfected cells by employing microarray analysis. Results: We identified DHRS2, a tumor suppressor gene, as the most significantly downregulated gene in LEF1 knockdown cells, and we further confirmed its downregulation by real-time quantitative polymerase chain reaction (qRT-PCR) in mRNA and at protein level by western blotting. Conclusion: Our results revealed that DHRS2 is positively regulated by LEF1 in Jurkat cells, which indicates the capability of LEF1 as a tumor suppressor and, together with previous reports, suggests that LEF1 exhibits a regulatory role in T-ALL via not only its oncogenic targets but also tumor suppressor genes.

p.Ser348Cys mutation in FGFR3 gene leads to "Mild ACH /Severe HCH" phenotype.

Achondroplasia (ACH) and hypochondroplasia (HCH) are genetic bone disorders known to be caused by gain-of-function mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. Both conditions share radiographic and phenotypical features. HCH is a milder form of ACH. Most individuals with ACH have the recurrent mutation (p.Gly380Arg) in the transmembrane (TM) domain of the receptor and individuals with HCH show the common mutation (p.Asn540Lys) in the tyrosine kinase 1 (TK1) region. Other rare mutations have been reported, however no additional hot-spot has been identified. We report an 8-month-old infant, with the heterozygous mutation, c.1043C > G, leading to an amino acid change from serine at 348 to cysteine (p.Ser348Cys). Clinical diagnosis of the patient is intertwined with "mild ACH" or "severe HCH". He did not demonstrate acanthosis nigricans (AN). This mutation has been reported in two different patients and it is located in the Ig-III domain of the FGFR3 region near other mutations associated with ACH. Among the two the 8-year old one also demonstrated AN without evindece of hyperinsulinem. This report emphasizes the benefit of whole gene sequencing for FGFR3 in individuals with suspected "mild ACH/severe HCH". This child will be monitored for future occurrence of AN.

Integrative analysis of mRNA and microRNA expression profiles in laryngeal squamous cell carcinoma.

Larynx cancer is a therapeutically challenging disease. Rapidly evolving experimentally validated data have significantly improved our understanding of the complex role of numerous RNA, DNA, and proteins that play a role in the development and progression of cancer. Based on the insights from approximately two decades of research, it seems clear that microRNAs (miRNAs) have revolutionized our concepts related to the main role of noncoding RNAs in different cancers' progression, development, and metastasis. Mechanistically, miRNAs have been reported to regulate different RNAs and finally protein-coding genes. The expression profiling of miRNAs and messenger RNA (mRNAs) was conducted for a deeper analysis of the miRNAs and mRNAs which play an essential role in larynx cancer. Downregulation or upregulation over twofolds in the miRNAs was considered to be significant, and that of sixfolds or below was considered to be significant for the mRNAs. In accordance with this approach, the expression levels of 43 miRNAs were increased in this study, whereas the expression levels of 129 were decreased. Accordingly, all the genomic expression studies provided evidence of upregulation of 97 genes, whereas 128 genes were found to be downregulated. Among these miRNAs, hsa-miR-20a-3p and hsa-miR-1972 were noted to be important in the etiology of larynx cancer.

Is there a correlation between follicle size and gene expression in cumulus cells and is gene expression an indicator of embryo development?

BACKGROUND: In an article published in 2017, we discussed the results of the first part of our study into the morphokinetic development of embryos in relation to follicle diameter and homogeneity of follicular development. Our findings showed that embryos coming from small follicles in heterogeneous cycles had significantly higher rates of arrest or failure to reach blastocyst than embryos coming from large follicles in homogenous cycles. The aim of this further study was to investigate the relationship between follicular size and gene expression of cumulus cells (CCs) and evaluate whether gene expression could be an indicator of embryo development. METHODS: This study was based on 2495 COCs from 184 patients. CC expressions of five genes (TNFAIP6, PTGS2, HAS2, PTX3 and GDF9) were studied by generalized linear mixed models (GLMMs) regarding follicular size. CC expressions were then separately analysed regarding patient-specific variables (age, BMI, AMH and follicular size) in relation to embryos reaching blastocyst (eRB) or top or good quality blastocysts (TQ + GQ) using GLMMs with logit link. RESULTS: Follicular size significantly correlated with the potential of an oocyte to develop into a blastocyst: oocytes developing from large follicles were more than twice as likely to develop into an eRB than oocytes from small follicles (p < 0.001). Gene expression of HAS2 and GDF9 correlated with blastocyst quality when separately evaluated with follicular size and the patient specific variables of age, BMI and AMH. However, no such correlation was found in other gene expressions studied. CONCLUSIONS: Our findings suggest that differences in the expression of genes studied could be related to follicular size rather than to embryo quality. Although gene expression of HAS2 and GDF9 correlated with blastocyst quality, the only variable correlating with eRB and TQ and GQ blastocysts for each of these five models was follicular size. TRIAL REGISTRATION: This prospective cohort study was registered at clinicaltrials.gov (NCT02230449).

MGMT gene variants, temozolomide myelotoxicity and glioma risk. A concise literature survey including an illustrative case.

Temozolomide may cause thrombocytopenia or neutropenia in 3-4% of glioblastoma patients, respectively. However, pancytopenia is rarely reported. MGMT (O6-methylguanine-DNA-methyltransferase) enzyme repairs temozolomide-induced DNA mutations and associates both with antitumour efficacy and myelosuppression. Many studies on the effects of MGMT gene-methylation on temozolomide's effects exist, but much fewer publications concerning MGMT variants were documented. A full sequencing of the MGMT gene was performed in a female glioblastoma patient, who developed pancytopenia following temozolomide treatment. Results indicated the presence of all the rs2308321 (I143 V), rs2308327 (K178R) and rs12917 (L84F) MGMT-variants, which were previously associated with temozolomide myelotoxicity. rs12917 (L84F) variant was reported as associating with lesser risk of gallbladder tumours, yet with higher risk of non-Hodgkin lymphomas related with exposure to chlorinated solvents or hair dyes. DNA repair proteins may exert diverging effects on DNA injuries caused by different chemicals and therefore exerting complex effects on myelotoxicity, antitumour activity and carcinogenesis.

Polymorphisms in the Matrix Metalloproteinase-9 Promoters and Susceptibility to Glial Tumors in Turkey.

AIM: Evidence suggests an association between MMP-9 functional gene polymorphisms and several tumors. The aim of this study was to investigate the possible role of single-nucleotide polymorphisms (SNP) at MMP-9 R279Q A/G, P574R G/C and R668Q G/A and R668Q (rs17577) genotypes with glial tumors in Turkey. MATERIAL AND METHODS: The present series consisted of tissue samples obtained from 100 cancer-free controls and 100 patients who had undergone glial tumor resection from 2007 to 2011 at the Cerrahpasa Medical Faculty of Istanbul University. Blood samples were collected to extract the genomic deoxyribonucleic acid (DNA) of each subject by polymerase chain reaction (PCR) and DNA sequencing. The genotypes of MMP-9 P574R, R279Q and R668Q SNPs were determined by using the PCR-RFLP assay. Genotypic distributions between patient and control groups were compared for correlations with glial tumor occurrence. RESULTS: SNPs in MMP-9 were not found to be significantly associated with glial tumor risk among participants except R279Q (G-G) which showed high risk only in multivariate analysis (OR adjusted, 3.15 95% CI, 1.10-9.01). The comparisons between the grade of tumor and the genotypic polymorphisms also showed no significant associations in the case group (all p values > 0.05). CONCLUSION: The current study showed a significant association between the R279Q G/G polymorphism and formation of glial tumor in advanced age. Changed protein features may cause triggering of some subcellular mechanisms that may have a role in activating oncogenic processes over the years. These data add to the growing epidemiological and experimental evidence that MMP-9 may play a role in glial tumors.

Seven years of experience of preimplantation HLA typing: a clinical overview of 327 cycles.

Preimplantation human leukocyte antigen (HLA) typing allows the birth of healthy children who are potential donors of stem cells for their affected siblings. This technique can be used for acquired diseases such as leukaemia or can be used for single-gene disorders such as thalassaemia. This retrospective study presents clinical data obtained from 171 couples who had undergone 327 preimplantation HLA typing cycles: 262 cycles for HLA typing in combination with mutation analysis and 65 cycles for the sole purpose of HLA typing. Of the diagnosed embryos 17.6% were found to be HLA matched. Embryo transfer was performed in 212 cycles, 34.9% clinical pregnancy rate per transfer was achieved and 59 healthy and HLA-compatible children were born. Twenty-one sick children have been cured through haemopoietic stem cell transplantation. The effect of maternal age and ovarian reserve on reproductive outcome was assessed retrospectively. The data demonstrated that, once a mutation-free and HLA-compatible embryo was found, clinical pregnancy rate did not differ statistically significantly despite the presence of some cycle-related limitations such as advanced maternal age and/or diminished ovarian reserve. Preimplantation HLA typing is an effective therapeutic tool for curing an affected sibling even for poor-prognosis patients. Preimplantation human leukocyte antigent (HLA) typing allows the birth of healthy children who are potential donors of stem cells for their affected siblings. This technique can be used for acquired diseases such as leukaemia or can be used for single-gene disorders such as thalassaemia. This study presents clinical data obtained from 171 couples who underwent 327 preimplantation HLA-typing cycles. Of these, 262 cycles were performed for HLA typing in combination with mutation analysis and 65 cycles were performed for the sole purpose of HLA typing. A total of 17.6% of the diagnosed embryos were found to be HLA matched. Embryo transfer was performed in 212 cycles. The clinical pregnancy rate per transfer was 34.9% and 59 healthy and HLA compatible children were born. Twenty-one sick children have been cured through haemopoietic stem cell transplantation. The effect of maternal age and ovarian reserve on reproductive outcome was assessed retrospectively. The data demonstrated that, once a mutation-free and HLA-compatible embryo was found, clinical pregnancy rates did not differ statistically significantly by the presence of some cycle-related limitations such as advanced maternal age and/or diminished ovarian reserve. Preimplantation HLA typing is an effective therapeutic tool for curing an affected sibling even for poor-prognosis patients.

Sperm fluorescence in situ hybridization analysis reveals normal sperm cells for 14;14 homologous male Robertsonian translocation carrier.

OBJECTIVE: To report the presence of normal sperm cells for chromosome 14 in a homologous 14;14 Robertsonian translocation carrier. DESIGN: Case report. SETTING: In vitro fertilization clinic and genetics laboratory in a private hospital. PATIENT(S): Infertile couple referred for IVF. INTERVENTION(S): Conventional cytogenetic and fluorescence in situ hybridization (FISH) techniques were used in karyotype and sperm FISH analysis. Three IVF treatments were performed, two of which included preimplantation genetic diagnosis (PGD). MAIN OUTCOME MEASURE(S): Cytogenetic analysis revealed pure 45,XY,t(14;14)(q10;q10) karyotype. Sperm FISH analysis for chromosome 14 revealed 13% normal sperm cells in the sperm sample. RESULT(S): Sperm FISH analysis revealed 13% normal sperm cells for chromosome 14 in the homologous 14;14 Robertsonian translocation carrier. The couple underwent two IVF cycles together with PGD. In the first trial there was no suitable embryo for transfer. In the second trial one normal blastocyst was transferred on day 6. However, pregnancy was not established in this second PGD cycle. CONCLUSION(S): To our knowledge, this is the first sperm FISH study revealing the presence of normal sperm in the ejaculate of a pure homologous translocation carrier. The PGD study performed for this couple is also unique in the literature.

Preimplantation genetic diagnosis (PGD) for extremes--successful birth after PGD for a consanguineous couple carrying an identical balanced reciprocal translocation.

OBJECTIVE: To report a healthy birth after preimplantation genetic diagnosis (PGD) performed for a consanguineous couple carrying an identical familial reciprocal translocation in both partners. DESIGN: Case report. SETTING: In vitro fertilization (IVF) clinic and genetic laboratory in a private hospital. PATIENT(S): Consanguineous couple carrying the same balanced reciprocal translocation: 46,XX,t(1;16)(q12;q11.2) and 46,XY,t(1;16)(q12;q11.2). INTERVENTION(S): 25 oocyte-cumulus complexes were retrieved 36 hours after human chorionic gonadotropin injection; metaphase II oocytes were fertilized by intracytoplasmic sperm injection; single blastomere biopsy was performed on 15 embryos on day 3; one embryo was found to be normal or balanced according to fluorescent in situ hybridization studies, embryo transfer was performed on day 4. MAIN OUTCOME MEASURE(S): Healthy birth of homozygous double translocation carrier twins with 46,XY,t(1;16)(q12;q11.2)mat,t(1;16)(q12;q11.2)pat karyotype. RESULT(S): Healthy monozygotic male twins were born at 36 weeks of gestation. Karyotype studies of the babies revealed that they are double translocation homozygotes: 46,XY,t(1;16)(q12;q11.2)mat,t(1;16)(q12;q11.2)pat. They are healthy and more than 4 years old later show no physical or mental abnormalities. CONCLUSION(S): To our knowledge, this is the first PGD study performed for a couple who carry the same reciprocal translocation. The twins born after this study are rare examples in the literature of healthy balanced reciprocal translocation homozygotes.

Association between reduced levels of MEFV messenger RNA in peripheral blood leukocytes and acute inflammation.

OBJECTIVE: Familial Mediterranean fever (FMF) is associated with more than 70 missense mutations in the MEFV gene. The purpose of this study was to investigate the relative expression of messenger RNA (mRNA) for the MEFV gene in peripheral blood leukocytes (PBLs) obtained from patients with FMF during attacks of acute abdominal inflammation as well as during asymptomatic periods. METHODS: We studied 16 patients with FMF during an attack of acute peritonitis and 17 otherwise healthy individuals who were undergoing surgery because of acute appendicitis. Blood samples were collected from both groups of patients during both acute inflammatory and asymptomatic periods. Relative levels of MEFV mRNA in PBLs were detected with real-time reverse transcriptase-polymerase chain reaction using LightCycler, with 2 sets of primers for the MEFV gene (exons 7-10 and exons 2-3) and with primers for CIAS1 and PSTPIP1 genes. Expression levels were compared with beta(2)-microglobulin as an internal control. RESULTS: MEFV expression was reduced in FMF patients during asymptomatic periods as compared with the non-FMF controls (P < 0.001). We observed a further decrease in MEFV expression in FMF patients during periods of inflammation (P = 0.01). Reduced levels of MEFV mRNA were also noted during the preoperative period as compared with asymptomatic periods in control patients with acute appendicitis (P = 0.01). CIAS1 expression in PBLs from patients with FMF was also found to be lower than that in the control patients. However, CIAS1 expression did not change with acute inflammation. CONCLUSION: This study confirmed that reduced expression of the MEFV gene is associated with inflammation and that it may be one of the pathogenic mechanisms of the attacks of inflammation in FMF patients, along with disease-associated variations in pyrin.