Simultaneous downregulation of miR-21 and miR-155 through oleuropein for breast cancer prevention and therapy.

Breast cancer (BC) is the leading cause of cancer mortality in women worldwide. It recently was proven that miRNAs play a critical role in BC development. The use of natural agents for control of cancer by modulating miRNAs is promising. Oleuropein is a natural polyphenolic agent with anti-neoplastic properties and is well tolerated by humans. This study was undertaken to determine the therapeutic effects of oleuropein through modulation of master oncomiRs (miR-21 and miR-155) in BC cells. The present study provides the first link between miRNA and oleuropein as a mechanism in BC. MCF-7 cells were tested with and without oleuropein and the cell viability, apoptosis, and migration were examined. The effect of oleuropein on miR-21 and miR-155 expression was assessed through qRT-PCR. It was found that oleuropein induced apoptosis and retarded cell migration and invasion in a dose-dependent manner in the human MCF7 BC cell line. It was observed that oleuropein significantly decreased expression of both miR-21 and miR-155 over time in a dose-dependent manner. These results demonstrate that oleuropein is a potential therapeutic and preventive agent for BC. Oleuropein exhibits an anti-cancer effect by modulation of tumor suppressor gene expression, which is targeted by oncomiRs.

Molecular Study of Deletional and Nondeletional Mutations on the α-Globin Locus in the Azeri Population of Northwestern Iran.

The aim of this study was to determine the molecular spectrum and frequency of deletional and nondeletional α-thalassemia (α-thal) mutations and the genotype-phenotype correlation in common mutations in the Azeri population of Northwestern Iran. A total of 1256 potential carriers with microcytic and hypochromic anemia and normal Hb A2 levels (<3.5%) and without iron deficiency anemia plus three fetuses were identified. Multiplex gap-polymerase chain reaction (gap-PCR) and sequencing for α-thal mutations were carried out. In 606 individuals, the α-globin gene was normal, but in 650 persons (51.6%) and three fetuses, 10 different mutations were detected. The most frequent deletional genotypes were as follows: αα/-α3.7 (61.7%), -α3.7/-α3.7 (11.9%), αα/-α4.2 (4.6%), αα/- -MED (4.3%) and αα/-(α)20.5 (3.8%). The most frequent nondeletional genotypes were αα/αIVS-I (-5 nt)α (HBA2: c.95+2_95+6delTGAGG) and αα/αPoly A2α [polyadenylation signal (polyA2) (AATAAA>AATGAA); HBA2: c.*96G>A] with frequencies of 1.08% and 0.92%, respectively. Meanwhile, 7.71% of individuals with a proven ß-thalassemia (ß-thal) mutation were found to also carry an α-thal mutation. Persons having two functional α-globin genes showed lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (Hb) (MCH) values compared to those with one mutated α-globin gene, provided that they had normal ß-globin genes. Overall, the incidence of α-thal was 2.7% in the Azeri population in Northwestern Iran. Our results showed that the variability of α-thal mutations are high in the Azeri population and that α-thal mutations are highly heterogeneous in both deletional and nondeletional genotype aspects.

Gene induction for the treatment of methylmalonic aciduria.

BACKGROUND: Methylmalonic aciduria is an autosomal recessive inborn error of the propionate metabolic pathway. One form of this disorder is caused by mutations in methylmalonyl-coenzyme A mutase (MCM), resulting in reduced levels of enzyme activity. The pharmacological up-regulation of residual mutase activity is one approach to advance treatment strategies for individuals affected by this disorder. We describe the construction, characterization and use of a cellular genomic reporter assay for MCM expression that will potentially identify therapeutic pharmacological agents for methylmalonic aciduria treatment. METHODS: Homologous recombination was used to insert an enhanced green fluorescent protein (EGFP) cassette inframe before the last codon of exon 13 of the MCM gene (MUT) in a BAC clone. The construct was used to generate stable HeLa cell lines. EGFP expression was measured by flow cytometry and the real-time reverse transcriptase-polymerase chain reaction was used to quantify changes in MUT gene mRNA levels. RESULTS: The genomic reporter assay used to screen a selection of compounds. Cisplatin, zidovudine and adefovir were found to increase the levels of MCM mRNA and EGFP expression, providing support for the possible efficacy of these pharmacological compounds in treating methylmalonic aciduria. CONCLUSIONS: This assay has the potential of being used in high-throughput screening of chemical libraries for the identification of novel compounds that specifically modulate the expression of MCM.

An improved Diagnostic PCR Assay for identification of Cryptic Heterozygosity for CGG Triplet Repeat Alleles in the Fragile X Gene (FMR1).

BACKGROUND: Fragile X syndrome (OMIM #300624) is the most common, recognised, heritable cause of mental retardation. Widespread testing is warranted by the relatively high frequency of the disorder, the benefits of early detection and the identification of related carriers whose offspring are at a 1 in 2 risk of inheriting the expanded pathogenic mutation. However, cost-effective screening of mentally retarded individuals has been impeded by the lack of a single, simple laboratory test. Currently, Fragile X syndrome can be excluded in males and a majority of females using a simple high-throughput PCR test. Due to the limited sensitivity of the PCR test, we find in our diagnostic service that approximately 40% of females appear homozygous and a labour intensive and expensive Southern blot test is required to distinguish these from females carrying one normal allele and an expanded allele. RESULTS: We describe an improved PCR test which displays a high level of precision allowing alleles differing by a single triplet to be resolved. Using the new assay, we detected 46/83 (53%) cryptic heterozygotes previously labelled as homozygotes. The assay also extended the range of repeats amplifiable, up to 170 CGG repeats in males and 130 CGG repeats in females. Combined with the high precision, the assay also improves discrimination of normal (CGG repeats < 45) from grey zone (45 < CGG repeats < 54) alleles and grey zone alleles from small premutations (55 < CGG repeats < 100). CONCLUSION: Use of this PCR test provides significantly improved precision and amplification of longer alleles. The number of follow-up Southern blot tests required is reduced (up to 50%) with consequent improvement in turnaround time and cost.