Association of MicroRNA Polymorphisms With Hepatocellular Carcinoma in an Iranian Population

BACKGROUND: Single nucleotide polymorphisms (SNPs) can modulate various biological processes by influencing microRNA (miRNA) biogenesis and altering target selection. Common SNPs may alter the processing of miRNA and may be associated with hepatocellular carcinoma (HCC). We investigated the relationship between miR-499A>G, miR-149C>T, miR-196a2T>C, and miR-146aG>C and HCC susceptibility, examining the interaction of the miRNAs with hepatitis B virus (HBV). METHODS: We evaluated the associations of miR-499A>G (rs3746444), miR-149C>T (rs2292832), miR-196a2T>C (rs11614913), and miR-146aG>C (rs2910164) with HCC susceptibility in 100 HCC patients (70 males and 30 females) and 120 healthy controls (70 males and 50 females), using the PCR-restriction fragment length polymorphism method. RESULTS: For miR-499A>G, the frequencies of the AG genotype and G allele were higher in female HCC patients than in female controls (P=0.02 and 0.045, respectively). The frequency of the A allele was higher in HBV-positive HCC patients than in controls (P=0.019). For miR-149C>T, the frequency of the CC genotype was higher in female HCC patients than in female controls (P=0.009). For miR-196a2T>C, the frequencies of the CT and CC genotypes and the C allele were higher in HBV-positive HCC patients than in controls (P C polymorphisms did not differ between HCC patients and controls. CONCLUSIONS: miR-499A>G, miR-149C>T, and miR-196a2T>C were associated with the development of HCC in women and/or that of HBV-related HCC. They can be considered genetic risk factors for the development of HCC among Iranians.

Cloning and expression of Iranian Turkmen thoroughbred horse follicle stimulating hormone in pichia pastoris

Background: follicle stimulating hormone [FSH] plays an essential role in reproductive physiology and follicular development

Objective: a new variant of the equine fsh [efsh] gene was cloned, sequenced, and expressed in Pichia pastoris [P. pastoris] GS115 yeast expression system

Materials and Methods: the full-length cDNAs of the efsh[alpha] and efsh[beta] chains were amplified by reverse transcription polymerase chain reaction [RT-PCR] using the total RNA isolated from an Iranian Turkmen-thoroughbred horse's anterior pituitary gland. The amplified efsh chains were cloned into the pPIC9 vector and transferred into P. pastoris. The secretion of recombined eFSH using P. pastoris expression system was confirmed by Western blotting and immunoprecipitation [IP] methods

Results: the DNA sequence of the efsh[beta] chain accession number JX861871, predicted two putative differential nucleotide arrays, both of which are located in the 3?UTR. Western blotting showed a molecular mass of 13 and 18 kDa for eFSH[alpha] and eFSH[beta] subunits, respectively. The expression of desired protein was confirmed by protein G immunoprecipitation kit

Conclusions: eFSH successfully expressed in P. pastoris. These findings lay a foundation to improve ovulation and embryo recovery rates as well as the efficiency of total embryo-transfer process in mares

Expression and prognostic significance of Bcl-2 and Bax in the progression and clinical outcome of transitional bladder cell carcinoma

To evaluate the mRNA expression ratio of Bcl-2/Bax both in normal and tumoral bladder tissues of patients with transitional cell carcinoma [TCC] of bladder and investigate potential correlation between this expression ratio and clinical outcome. In this experimental study, we used real time-PCR to investigate the expression of Bcl-2 and Bax both in normal and tumoral bladder tissues. The Bcl-2/Bax expression ratio was determined in tumoral bladder tissues of patients with transitional cell carcinoma of the bladder [n=40] and correlation between expression ratios and the emergence of early relapses in a follow-up of 14-30 months was examined. Relapse-free time in 14/31 patients [45.16%] with Bcl-2/Bax>1 was shorter than 9 months [range of 2-9 months] with 5.7 months average median while 17/31 patients [54.84%] with Bcl-2/Bax<1 are currently relapse-free [14-30 months]. Bcl-2 and Bax expression levels were not solely correlated with clinical outcome and progression of carcinogenesis. The mRNA expression ratio of Bcl-2/Bax in tumoral bladder tissues may serve as a significant prognostic indicator in predicting the clinical outcome in low grade non-invasive bladder cancer

Optimization of the electroporation conditions for transfection of human factor IX into the goat fetal fibroblasts

Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk. In this experimental study, a linearized recombinant vector [pBC1] entailing human coagulation factor IX [5.7 kb] cDNA was introduced into goat fetal fibroblast cells [three sub passages] via electroporation in four separate experiments [while other variables were kept constant], beginning with 10 micro g DNA per pulse in the first experiment and increments of 10 micro g/pulse for the next three reactions. Thereafter, polymerase chain reaction [PCR]-positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing. Significance was defined as p< 0.05. The results showed no significant difference among three first concentrations except for group 1 [10 micro g/pulse] and group 3 [30 micro g/pulse], but there was a significant difference between these groups and the fourth group [p<0.05], as this group [40 micro g/pulse] statistically showed the highest rate of transfection. As the fluorescence in situ hybridization [FISH] results indicated the transgene was integrated in a single position in PCR positive cells. Increasing amount of using the vector 40micro g/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role in electroporation efficiency, our results showed an increase in DNA concentration could affect an exponential surge in the electroporation efficiency

Contribution of GJB2 mutations and Four common DFNB loci in autosomal recessive non-syndromic hearing impairment in Markazi and Qom provinces of Iran

This study aimed to investigate the contribution of four common DFNB ["DFN" for deafness and "B" for autosomal resessive locus] loci and GJB2 gene mutations [exon 2] in hearing impairment in individuals living in Markazi and Qom provinces of Iran. Forty consanguineous Iranian families with at least three affected individuals in family or pedigree who suffer from an autosomal recessive non-syndromic congenital hearing impairment were the subjects of this study. Blood samples were taken from both hearing and non-hearing individuals, DNA was extracted and amplified by using specific primers for the coding region of GJB2 gene [exon 2]. The PCR product of GJB2 gene was then sequenced. Also short tandem repeat [STR] markers amplified by using specific primers for loci DFNB2, DFNB3, DFNB4 and DFNB21. At least 2 microsatellite markers [STR] for each DFNB locus exceeding to 4-6 markers for the linked families were used. The amplified markers were analyzed by conventional Polyacrylamide Gel Electrophoresis followed by silver staining. Six families were homozygous or compound heterozygous for GJB2 mutations and were excluded from further studies. Linkage analysis was carried out for the remaining 34 families by genotyping the flanked STR markers of DFNB2, DFNB3, DFNB4 and DFNB21 loci. Six families showed linkage; including one family to DFNB2, two families to DFNB3 and three families to DFNB4 locus while no family showed linkage to DFNB21 locus. Undoubtedly, the best understanding of the genetic basis of hearing loss in Iranian population will be achieved by performing similar experiments in other provinces and also by analyzing more loci

autoimmune diseases manifested by production of autoantibodies: the autoantigens identified by random peptide library

Phage-displayed random peptide libraries [RPL] provide a powerful technique for identification, structural and functional analysis of ligands for many different target molecules, including, antibodies, receptors or other proteins. This strategy has been verified to be an effective tool for research in immunology and successfully has been used to determine the target sequence for monoclonal and polyclonal antibodies. The peptide library approach provides great promise for characterization of ligands with no prior information concerning antibody specificity. This would allow the recognition of candidate antigens involved in initiation or perpetuation of autoimmune diseases. This technology also offers the potential for new therapeutic opportunities, production of diagnostic reagents, or even development of effective new vaccines. This review focuses on studies regarding the identification of autoantigens recognized by antibodies in autoimmune diseases using phage-display peptide libraries

[Investigation of interferon-gamma receptor-1 polymorphism in Iranian patients affected with pulmonary tuberculosis]

Tuberculosis is one of the most common infectious diseases in the world. In recent years, genetically approach has been developed. One of the interesting gene for investigator is IFN- gamma R1. In this study we determind susceptibility to tuberculosis with polymorphism of IFN- gamma R1 gene. Fifthly patients with smear positive tuberculosis have been chosen randomly. They were matched with 54 healthy controls with no history of TB. Polymorphism at 395 codon of IFN- gamma R1 gene was detected with Newport method. Mean age of patients and control were 55 13.5 years respectively. Demographic characteristic had no +/- 20 and 53 +/- difference within two groups. One patient in case group had heterozygote mutation at IFN- gamma R1 gene. In control group there were no mutations. Genetically susceptibility to TB was not seen in 395 colon of IFN- gamma R1 in Iranian TB sample and polymorphism of this loci has occur in 2% of TB patients and 0.96% of total study population

Use of D11S2179 and D11S1343 markers as informative markers for prenatal diagnosis in Iranian ataxia-telangiectasia patients

Ataxia-telangiectasia is an autosomal recessive disorder affecting 1/40000 to 1/100000 of reported populations. There is 25% possibility for having an affected child when parents are carriers for ATM gene mutation. There is no cure available for this disease and prenatal testing is strongly recommended to prevent this disease. Although preferred method is the direct mutation analysis of ATM gene, but large size of the ATM gene with 63 exons and the large number of possible mutations in patients considerably limit efficiency of mutation analysis as a choice in diagnosis. Indirect method is a better tool when parents are not carriers of founder mutation and pass different mutations to their children. Indirect molecular diagnosis using ATM related molecular markers facilitates prenatal diagnosis of AT children. In this study, four molecular markers: D11S2179, D11S1787, D11S535, D11S1343 are genotypes in 12 unrelated families [19 patients] from different regions of Iran. Those markers are amplified using extracted sequence primers from Gene Bank with their described PCR conditions. The amplified products were separated using denaturing PAGE gels, and the data were analyzed to detect their pattern of inheritance in each family. In all families, segregation of alleles were according to mandelian inheritance and affected chromosomes were distinguishable from unaffected ones. All carriers and affected patients were diagnosed accurately. Thus, this method is effectively usable in prenatal diagnosis of ataxia telangiectasia

Role of mitochondria in ataxia-telangiectasia: investigation of mitochondrial deletions and haplogroups

Ataxia-Telangiectasia [AT] is a rare human neurodegenerative autosomal recessive multisystem disease that is characterized by a wide range of features including, progressive cerebellar ataxia with onset during infancy, occulocutaneous telangiectasia, susceptibility to neoplasia, occulomotor disturbances, chromosomal instability and growth and developmental abnormalities. Mitochondrial DNA [mtDNA] has the only non-coding regions at the displacement loop [D-loop] region that contains two hypervariable segments [HVS-I and HVS-II] with high polymorphism. We investigated mt-DNA deletions and haplogroups in AT patients. In this study, 24 Iranian patients suffering from AT and 100 normal controls were examined. mt-DNA was extracted from whole blood and examined by 6 primers for existence of mitochondrial deletions. We also amplified and sequenced the mtDNA HVS-I by standard sequencing techniques. mtDNA deletions were observed in 54.1% [13/24] of patients [8.9 kb deletion in all samples, 5.0 kb in one and 7.5 kb in two patients], representing mtDNA damage which may be due to oxidative stress in mitochondria. Our results showed that there is no association between mtDNA haplogroups and AT. This data may indicate involvement of mitochondrial damage in the pathogenesis of AT

RNAi induced inhibition of MRP1 expression and reversal of drug resistance in human promyelocytic HL60 cell line

Multidrug resistance [MDR] is a complex phenomenon in which many different genes regulating drug transport, cellular repair, detoxification and drug metabolism are involved. Nevertheless, in most drug resistant cell lines and cancer patients up-regulation of ABC-transporter genes such as MDR associated Protein [MRP1] gene could be at the basis of the drug resistance phenotype. We aimed to decrease MRP1 expression at the mRNA level to modulate drug resistance phenotype in the methotrexate-resistant HL60 cell line. We designed a small interfering RNA [siRNA] molecule against MRP1 and applied it to HL60 cell line in a 0 to 72 hours time range. siRNA could specifically inhibit gene expression by 80% of the initial mRNA level with in 36 to 48 hours. The siRNA-treated cells demonstrated 100-fold reduction in methotrexate [MTX] resistance compared to untreated cells. The data indicate that this approach may be applicable to the study of MRP1 expression and development of future strategies to reverse the MRP1 dependent drug-resistance phenotype in tumors back to a drug-sensitive one

Investigation of polymorphisms in non-coding region of human mitochondrial DNA in 31 Iranian hypertrophic cardiomyopathy [HCM] patients

The D-loop region is a hot spot for mitochondrial DNA [mtDNA] alterations, containing two hypervariable segments, HVS-I and HVS-II. In order to identify polymorphic sites and potential genetic background accounting for Hypertrophic CardioMyopathy [HCM] disease, the complete non-coding region of mtDNA from 31 unrelated HCM patients and 45 normal controls were sequenced. The sequences were aligned upon the revised Cambridge Reference Sequence [rCRS] and any incompatibilities were recorded as numerical changes in homoPolymeric C Tract [PCT], single base substitutions, insertions and deletions [Indels]. Nucleotide substitutions were found to make up the majority of the mutations, rather than indels. We drew significantly high transition rate [81.8%] versus lower frequency of transversions [18.2%]. 12 polymorphisms were identified in this study which had not been published in the MitoMap database. PCT changes at position 303-309 were detected in 83% of our samples. Our results suggest that an increased level of HVS-I and HVS-II substitutions may be an indicator of mitochondrial DNA instability. Furthermore, mtDNA mutations may play an important role in pathogenesis of cardiac arrest which has remained unexplained for long

Mutation analysis of connexin 26 gene and del[GJB6-D13S1830] in patients with hereditary deafness from two provinces in Iran

Mutations in the connexin 26 [Cx26] gene at the DFNB1 locus on chromosome 13q12 are associated with autosomal recessive non-syndromic hearing loss [ARNSHL]. There are many known mutations in this gene that cause hearing loss. A single frameshift, at position 35 [35delG] accounts for 50% of mutations in the Caucasian population with carrier frequencies of 1.5-2.5%. In this study we investigated the prevalence of Cx26 gene mutations by directly sequencing the coding exon of this gene belonging to ARNSHL individuals from 53 families in Qom and Markazi provinces of Iran. Seven different Cx26 variants were identified. Five Cx26 mutations including 35delG, 233delC, 176del16, W24X, L90P were found in 10 of 53 families [18.87%]. One polymorphism V153I was also found. One variant A171T with unknown effects was also detected. Six of the 53 families were observed to have GJB2 mutations in both alleles [11.32%]. The most common mutation was 35delG. Three out of 10 families [30%] with GJB2 variants contained 35delG mutation in both alleles and the frequency of 35delG allele was 0.50 among 10 out of 53 families. Also screening for the 342-kb GJB6 deletion mutant did not reveal any large deletion among families studied. Thus, in the two provinces, contribution of GJB2 [Gap Junction Protein Beta 2] mutations to familial deafness appears to be less significant. This necessitates further assessment of the other known genes regions as well as a search for new genetic factors in hereditary deafness in the Iranian population

ATM gene mutations detection in Iranian Ataxia-Telangiectasia patients

Ataxia-Telangiectasia [AT] is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, radiation sensitivity and cancer predisposition. The ATM gene on human chromosome 11q22.3 has recently been identified as the gene responsible for ataxia-telangiectasia [AT]. The gene mutated in AT, which has been designated as the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. More than 100 mutations are broadly distributed throughout the ATM gene. The large size of the ATM gene [66 exons spanning approximate 150kb of genomic DNA] together with the diversity and broad distribution of mutations in AT patients, greatly limits the utility of direct mutation screening as a diagnostic tool. In this study, 20 families with at least one affected child clinically suspected to have ataxia-telagiectasia were examined and their DNA was extracted and amplified with standard methods. Sequencing methods were used to detect the new point mutation. Four exons which were hot spots for point mutations in ATM gene were detected by PCR-SSCP or PCR-RFLP

Identification of hitherto undefined B-cell epitopes by antibodies in the sera of vitillgo patients using phage-display peptide library

A random 12 mers phage library was used to screen a pool of immunoglobulin fractions obtained from vitiligo patients. Subsequent to panning experiments, a panel of affinity selected phage from vitiligo patients were obtained. This panel was tested using an ELISA for their reactivity with pooled sera from patients and normal controls. Among the 16 randomly selected clones, two of clones showed distinct positive reactivity with the patient's sera compared with controls. The peptides displayed by these phages expressed the following amino acid sequences: SHMPLANQYQWA and NHVQAWEQFWDS. Thus, screening with phagedisplayed random peptide library of vitiligo sera can reveal peptide sequences that mimic vitiligo-related self-antigen