title: Quantitative analysis of mRNA expression levels and DNA methylation profiles of three neighboring genes: FUS1, NPRL2/G21 and RASSF1A in non-small cell lung cancer patients.
abstract: BACKGROUND
  Tumor suppressor gene (TSG) inactivation plays a crucial role in carcinogenesis. FUS1, NPRL2/G21 and RASSF1A are TSGs from LUCA region at 3p21.3, a critical chromosomal region in lung cancer development. The aim of the study was to analyze and compare the expression levels of these 3 TSGs in NSCLC, as well as in macroscopically unchanged lung tissue surrounding the primary lesion, and to look for the possible epigenetic mechanism of TSG inactivation via gene promoter methylation.
  
  
  METHODS
  Expression levels of 3 TSGs and 2 DNA methyltransferases, DNMT1 and DNMT3B, were assessed using real-time PCR method (qPCR) in 59 primary non-small cell lung tumors and the matched macroscopically unchanged lung tissue samples. Promoter methylation status of TSGs was analyzed using methylation-specific PCRs (MSP method) and Methylation Index (MI) value was calculated for each gene.
  
  
  RESULTS
  The expression of all three TSGs were significantly different between NSCLC subtypes: RASSF1A and FUS1 expression levels were significantly lower in squamous cell carcinoma (SCC), and NPRL2/G21 in adenocarcinoma (AC). RASSF1A showed significantly lower expression in tumors vs macroscopically unchanged lung tissues. Methylation frequency was 38-76%, depending on the gene. The highest MI value was found for RASSF1A (52%) and the lowest for NPRL2/G21 (5%). The simultaneous decreased expression and methylation of at least one RASSF1A allele was observed in 71% tumor samples. Inverse correlation between gene expression and promoter methylation was found for FUS1 (rs = -0.41) in SCC subtype. Expression levels of DNMTs were significantly increased in 75-92% NSCLCs and were significantly higher in tumors than in normal lung tissue. However, no correlation between mRNA expression levels of DNMTs and DNA methylation status of the studied TSGs was found.
  
  
  CONCLUSIONS
  The results indicate the potential role of the studied TSGs in the differentiation of NSCLC histopathological subtypes. The significant differences in RASSF1A expression levels between NSCLC and macroscopically unchanged lung tissue highlight its possible diagnostic role in lung cancer in situ recognition. High percentage of lung tumor samples with simultaneous RASSF1A decreased expression and gene promoter methylation indicates its epigenetic silencing. However, DNMT overexpression doesn't seem to be a critical determinate of its promoter hypermethylation.
authors.affiliation: Department of Molecular Bases of Medicine, Medical University of Lodz, Pomorska 251, C-5, 92-213, Lodz, Poland. dorota.pastuszak-lewandoska@umed.lodz.pl.
authors.forename: Dorota
authors.initials: D
authors.lastname: Pastuszak-Lewandoska
authors.affiliation: Department of Chest Surgery, General and Oncological Surgery, University Hospital No. 2, Medical University of Lodz, Lodz, Poland. jacek.kordiak@umed.lodz.pl.
authors.forename: Jacek
authors.initials: J
authors.lastname: Kordiak
authors.affiliation: Department of Molecular Bases of Medicine, Medical University of Lodz, Pomorska 251, C-5, 92-213, Lodz, Poland. monika.migdalska-sek@umed.lodz.pl.
authors.forename: Monika
authors.initials: M
authors.lastname: Migdalska-Sęk
authors.affiliation: Department of Molecular Bases of Medicine, Medical University of Lodz, Pomorska 251, C-5, 92-213, Lodz, Poland. karolina.czarnecka@umed.lodz.pl.
authors.forename: Karolina H
authors.initials: KH
authors.lastname: Czarnecka
authors.affiliation: Department of General and Oncological Pulmonology, Medical University of Lodz, Lodz, Poland. adam.antczak@umed.lodz.pl.
authors.forename: Adam
authors.initials: A
authors.lastname: Antczak
authors.affiliation: Department of Pneumology and Allergology, Medical University of Lodz, Lodz, Poland. pawel.gorski@umed.lodz.pl.
authors.forename: Paweł
authors.initials: P
authors.lastname: Górski
authors.affiliation: Department of Molecular Bases of Medicine, Medical University of Lodz, Pomorska 251, C-5, 92-213, Lodz, Poland. ewa.nawrot@umed.lodz.pl.
authors.forename: Ewa
authors.initials: E
authors.lastname: Nawrot
authors.affiliation: Department of Molecular Bases of Medicine, Medical University of Lodz, Pomorska 251, C-5, 92-213, Lodz, Poland. justyna.kiszalkiewicz@gmail.com.
authors.forename: Justyna M
authors.initials: JM
authors.lastname: Kiszałkiewicz
authors.affiliation: Department of Molecular Bases of Medicine, Medical University of Lodz, Pomorska 251, C-5, 92-213, Lodz, Poland. daria.domanska@umed.lodz.pl.
authors.forename: Daria
authors.initials: D
authors.lastname: Domańska
authors.affiliation: Department of Molecular Bases of Medicine, Medical University of Lodz, Pomorska 251, C-5, 92-213, Lodz, Poland. ewa.brzezianska@umed.lodz.pl.
authors.forename: Ewa
authors.initials: E
authors.lastname: Brzeziańska-Lasota
mesh_terms: D000368:Aged; D014408:Biomarkers, Tumor / Q000096:biosynthesis / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D053487:DEAD-box RNA Helicases / Q000096:biosynthesis* / Q000235:genetics; D019175:DNA Methylation / Q000502:physiology*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; C492816:NPRL2 protein, human; C411762:RASSF1 protein, human; D012333:RNA, Messenger; C499993:TUSC2 protein, human; D025521:Tumor Suppressor Proteins; C086252:DDX1 protein, human; D053487:DEAD-box RNA Helicases