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Evaluation of ITGB2 (CD18) and SELL (CD62L) genes expression and methylation of ITGB2 promoter region in patients with systemic sclerosis

Rheumatology International volume 38, pages 489–498 (2018)Cite this article

Abstract

Systemic sclerosis (SSc), an autoimmune disease of connective tissue, is characterized by inflammation, fibrosis, and vessel endothelial damage. Products of Integrin subunit beta 2 (ITGB2) and selectin L (SELL) genes participate in several functional pathways of immune system. The aim of this investigation was to survey the transcript level of ITGB2 and SELL genes as well as methylation status of CpG sites in promoter region of differently expressed gene in PBMCs of SSc patients. PBMCs were isolated from whole blood of 50 SSc patients and 30 healthy controls. Total RNA and DNA contents of PBMCs were extracted. Gene expression was analyzed by real-time PCR using the SYBR Green PCR Master Mix. To investigate the methylation status of CpG sites, DNA samples were treated by bisulfite, amplified through nested PCR, and sequenced through Sanger difficult sequencing method. ITGB2 gene in PBMCs of SSc patients was overexpressed significantly in comparison to healthy controls. However, no altered SELL expression was observed. Three CpG sites of 12, 13 and 14 were significantly hypomethylated in patients group, despite overall methylation status of ITGB2 gene promoter revealed no significant difference between study groups. There was no statistically significant correlation between methylation status of ITGB2 promoter and the gene expression in patients. Regarding to lack of correlation of increased expression of ITGB2 with its promoter hypomethylation in SSc patients, our study suggests that upregulation of ITGB2 in PBMCs from SSc patients is probably due to another mechanism other than methylation alteration.

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Acknowledgements

Authors are deeply grateful of the individuals who contributed to the accomplishment of this study.

Funding

This study was funded by the Deputy of Research, Tehran University of Medical Sciences (Grant no. 95-01-30-31356).

Author information

Affiliations

  1. Rheumatology Research Center, Tehran University of Medical Sciences, Tehran, Iran

    Navid Dashti, Mahdi Mahmoudi, Farhad Gharibdoost, Hoda Kavosi, Ramazan Rezaei, Vahideh Imeni, Ahmadreza Jamshidi, Saeed Aslani & Shayan Mostafaei

  2. Department of Medical Immunology, School of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran

    Navid Dashti, Ramazan Rezaei & Mohammad Vodjgani

Authors
  1. Navid Dashti
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  2. Mahdi Mahmoudi
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  3. Farhad Gharibdoost
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  5. Ramazan Rezaei
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  6. Vahideh Imeni
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  7. Ahmadreza Jamshidi
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  8. Saeed Aslani
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  10. Mohammad Vodjgani
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Contributions

ND: email: naviddashtigoolahgoolah@yahoo.com. Performed the experiments and participated in manuscript drafting. MM: email: mahmoudim@tums.ac.ir. Developed the main idea and red the manuscript critically. FG: email: gharibdoost@sina.tums.ac.ir. Provided the financial support of the project. HK: email: h-kavosi@sina.tums.ac.ir. Examined the SSc patients. RR: email: ramin.rezaei25@gmail.com. Participated in performing the experiments. VI: email: vahide.amini@yahoo.com. Participated in questionnaire filling of the patients and conducting experiments. AJ: email: jamshidia@tums.ac.ir. Provided the financial support of the project. SA: email: s-aslani@razi.tums.ac.ir. Participated in manuscript writing. SM: email: mostafa.shayan@modares.ac.ir. Performed the statistical analysis. MV: email: vojganim@sina.tums.ac.ir. Provided the financial support of the project.

Corresponding authors

Correspondence to Farhad Gharibdoost or Mohammad Vodjgani.

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Ethical approval

All participants in the study were treated in agreement with the ethical standards of the Ethics Committee at Tehran University of Medical Sciences and with the revised Helsinki Declaration in 2000.

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Informed consent was obtained from all individual participants included in the study.

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296_2017_3915_MOESM1_ESM.tif

Correlation of mRNA expression level of ITGB2 with methylation percentage in (A) SSc patients, (B) dSSc patients, and (C) lSSc patients (rho; Spearman’s correlation coefficient) (TIF 51 KB)

296_2017_3915_MOESM2_ESM.tif

Scatter plots demonstrating the correlation of Rodnan scores with mRNA expression of ITGB2 (A) and methylation level of ITGB2 promoter in SSc patients. Rodnan score correlation with mRNA expression and promoter methylation of ITGB2 in dSSc patients is shown in plots C and D, respectively. Rodnan score correlation with mRNA expression and promoter methylation of ITGB2 in lSSc patients is shown in plots E and F, respectively (r; Pearson’s correlation coefficient) (TIF 186 KB)

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Dashti, N., Mahmoudi, M., Gharibdoost, F. et al. Evaluation of ITGB2 (CD18) and SELL (CD62L) genes expression and methylation of ITGB2 promoter region in patients with systemic sclerosis. Rheumatol Int 38, 489–498 (2018). https://doi.org/10.1007/s00296-017-3915-y

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Keywords

  • Systemic sclerosis
  • ITGB2
  • SELL
  • CpG site
  • DNA methylation