Alteration of Ogg1, Myh and Mth1 Genes Expression in Relapsing-remitting Multiple Sclerosis Patients

Content Provider	Semantic Scholar
Author	Roya, Amirinejad Ali, Sahraian Mohammad Bahram, Mohammad Soltani Mehrdad, Behmanesh
Copyright Year	2017
Abstract	Introduction: Previous studies revealed that oxidative stress is elevated in multiple sclerosis (MS). It can harm to biological macromolecules such as DNA. However, the molecular mechanism in protection of genetic information from DNA damages is not clear in MS disease. In this study the expression level of some important genes of OGG1 and MYH involved in base excision repair pathway and, MTH1 and ITPA as main cleaning genes of nucleotide pool from rough nucleotides are examined in MS patients in compared to healthy group. Methods: Peripheral blood mononuclear cells were isolated from relapsing-remittingMS patients and healthy subjects. After RNA extraction and cDNA synthesis, the expression levels of target genes were examined by RT-qPCR technique. Results: The level of the MTH1 and MYH genes expression were decreased, but the level of OGG1 mRNA was higher in patients in comparison to the control group. Obtained result did not shown any correlation between expression of examined genes and clinical features of patients such as MS severity and disease duration. Conclusion: These preliminary results provide more supportive evidences for involvement of oxidative damage and variation in expression of DNA repair genes in MS. Significant increase of OGG1 suggest that the development and progression of pathogenesis in Iranian MS can be related to chronic and direct oxidative damage of genomic DNA not nucleotide pools. D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 23 + 03 30 o n M on da y N ov em be r 4t h 20 19 DNA repair genes expression in multiple-sclerosis patients Physiol Pharmacol 21 (2017) 129-136 | 130 (Behmanesh et al, 2009, Adamczyk and AdamczykSowa, 2016). It has been shown that by establishing active demyelination process, ROS-mediated injury by peripheral immune cells and central counterparts promotes progressive tissue damage in MS (Kremer et al. 2004, Adamczyk and Adamczyk-Sowa, 2016). Also, microglia and infiltrated macrophages can generate proinflammatory mediators and oxidizing radicals (Colton and Gilbert, 1993). Free radicals are normally produced in physiological conditions but their level increased in oxidative stress conditions. In all of eukaryotic cells, adjusted levels of free radicals have a crucial role in regulating various cellular functions such as inflammatory response in which production of ROS and RNS attribute to monocyte interactions with brain endothelium (Tasset et al., 2012). Uncontrolled production of free radicals may cause oxidation of different types of biomolecules such as free nucleotides and genomic DNA. Oxidative modification of genetic material and accumulation of oxidized bases, cause different types of mutation in the DNA and instability in RNA molecules (Behmanesh et al., 2005). Among biomarkers of oxidative DNA damage, 8-oxo-guanine has the crucial role in DNA alteration. It has been shown that this rough base has different quantity in the genomic DNA of blood and brain patients with neurodegenerative diseases (Sliwinska et al., 2016). To protect the genetic integrity of the cells from endogenous and exogenous source of free radicals, all of living cells are equipped with DNA repair systems. Nucleotide pool cleaners and base excision repair (BER) pathway are among the major mechanisms which are responsible for cell protection from accumulation of oxidized lesions in genomic DNA or nucleotide pools (Nakabeppu et al., 2014). Several studies are shown that DNA damage and other oxidative stress biomarkers are increased in peripheral blood samples of MS patients (Grecchi et al., 2012; Ibitoye et al., 2016; Tasset et al., 2012). Satoh et al. (2005) identified an aberrant expression of some DNA repair genes in peripheral blood mononuclear cells (PBMC) of MS patients. The associations of some DNA pathway repair genes polymorphisms with MS have been verified (Briggs et al., 2010; Karahalil et al., 2015), but there is not enough information about the essence of DNA damages types which are involved in MS pathogenesis. To get this point the expression of 8-oxoguanine DNA glycosylase (OGG1) and MUTYH glycosylase (MYH) involved in BER pathway as well as nudix hydrolase 1 (MTH1) and inosine triphosphatase (ITPA) as the main nucleotide pool cleaner genes compared in PBMC samples from RR-MS patients and healthy subjects. We evaluated OGG1, MYH, ITPA and MTH1 genes expression based on previous information about the possible association of DNA repair system with the pathological features of disease, elevated levels of oxidative stress (Miller et al., 2011; Tasset et al., 2012) and abnormal oxidized base (Haider et al., 2011) in MS patients. Materials and methods Patients and PBMC collection Thirty one eligible patients were recruited for this study that diagnosed according to McDonald criteria (McDonald et al., 2001) and MRI test was performed for all of the patients. Patient’s age was 22-52 years and The Expanded Disability Status Scale (EDSS) values of patients were between 0-5.5. They had presented to the MS Research Center of Sina Hospital of Tehran University of Medical Sciences, Tehran, Iran from November 2012 to October 2013 (Naghavi Gargari et al., 2015). All of the patients had no familial relation and no other inflammatory or autoimmune disease. They were RR-MS subtype. Also, 27 healthy samples were collected from volunteers with no history of hospitalization for neurological and autoimmune diseases. All of precipitants were selected after interviewed by an experienced neurologist. Written informed consent was obtained from all participants prior to the blood sampling. The Ethics Committees of Tarbiat Modares University approved this study. Three milliliters of whole blood was taken and collected in the anticoagulant EDTA tubes from each participant. PBMC were isolated by density gradient centrifugation on Ficoll-Paque solution (lympholyte, Cedarlane, Netherlands) according to manufacturer’s instructions as described. RNA extraction and cDNA synthesis Total RNA of PBMC was extracted by acid D ow nl oa de d fr om p pj .p hy ph a. ir at 1 0: 23 + 03 30 o n M on da y N ov em be r 4t h 20 19 131 | Physiol Pharmacol 21 (2017) 129-136 Amirinejad et al. guanidinium-phenol-chloroform procedure using RNXTM-plus solution (SinaClon co., Iran) according to the manufacturer's instructions. The isolated RNAs were treated with DNAaseI (Fermentas, Lithuania) for 20 min at 37°C to eliminate any genomic DNA contamination. Integration, concentration and purity of RNAs were verified by agarose gel electrophoresis and spectrophotometry. Three micrograms of purified RNA was used for cDNA synthesis with random hexamer and oligo (dT)18 primers (MWG, Germany) through M-MuLV reverse transcriptase (Thermo scientific, USA) in total 20 μl reaction mixture according to manufacturer’s instructions. Real-time PCR analysis The quantification of OGG1, MTH1, MYH and ITPA mRNAs expression was performed with Real-time PCR and Hot FIREPol EvaGreen qPCR Mix plus ROX (Solis BioDyne, Estonia). Relative levels of genes expression were detected by specific primers designed by Oligo software version 6 (USA). The sequence of used primers were: 5'GTATATGGGCTGGCCTTGGAAG-3' and 5'CTGTTGGCCCTGATACACACG-3' for MYH, 5'ACCCTGGCTCAACTGTATCACCAC-3' and 5'CCGCTCCACCATGCCAGTGATG-3' for OGG1, 5'GGGCCAGATCGTGTTTGAGTTCGT-3' and 5'TCGTCGGGCCACATGTCCTTG-3' for MTH1 and 5'AAGAAGCTGGAGGAGGTCG-3' and 5'TCCAAGGGCATTGAAGCACA-3' for ITPA mRNAs amplification. The GAPDH primers sequences were forward: 5'-CCATGAGAAGTATGACAAC-3' and reverse: 5'-GAGTCCTTCCACGATACC-3' which was used as the internal control. Real-time PCR was carried out using Applied Biosystems StepOneTM Real-Time PCR Systems (Applied Biosystems/MDS SCIEX, Foster City, CA, USA) in a final reaction volume of 20 μl with 10 ng cDNA, 4 μl of EvaGreen 5X master mix and 200 nM of each forward and reverse primers. The PCR condition was as follows: an initial denaturation at 95oC for 5 min, followed by 40 cycles of denaturation at 95oC for 20 sec, annealing at 60oC for 15sec and extension at 72oC for 30sec. Specificity of PCR products were confirmed by running of qPCR products on 12% poly acrylamide gel electrophoresis and melting curve analysis. All experiments were done at least in duplicate. Each gene expression of the individual samples was normalized with the GAPDH gene expression and relative fold change was calculated by 2 -ΔΔCt formula (Livak and Schmittgen, 2001). Statistical analysis Data obtained from this study was tested for normal distribution by calculating Kolmogorov–Smirnov test and performing the Shapiro–Wilk normality test. Based on the normal distribution Pearson and spearman rank was carried out to analyze correlations between gene expression and clinical features. The expression level of genes were compared to controls via independent T-test and Mann–Whitney U test using SPSS software (Version 21; SPSS Inc, Chicago, IL) and GraphPad Prism Software (Version 5.0, Inc., San Diego, CA) to compare parametric and nonparametric continuous variables, respectively. Data are presented as mean ± standard deviation (SD) and p<0.05 was defined as the statistical significance.
Starting Page	129
Ending Page	136
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Volume Number	21
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Language	English
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